Growth hormone, IGF-I and insulin and their abuse in sport

There is widespread anecdotal evidence that growth hormone (GH) is used by athletes for its anabolic and lipolytic properties. Although there is little evidence that GH improves performance in young healthy adults, randomized controlled studies carried out so far are inadequately designed to demonstrate this, not least because GH is often abused in combination with anabolic steroids and insulin. Some of the anabolic actions of GH are mediated through the generation of insulin-like growth factor-I (IGF-I), and it is believed that this is also being abused. Athletes are exposing themselves to potential harm by self-administering large doses of GH, IGF-I and insulin. The effects of excess GH are exemplified by acromegaly. IGF-I may mediate and cause some of these changes, but in addition, IGF-I may lead to profound hypoglycaemia, as indeed can insulin. Although GH is on the World Anti-doping Agency list of banned substances, the detection of abuse with GH is challenging. Two approaches have been developed to detect GH abuse. The first is based on an assessment of the effect of exogenous recombinant human GH on pituitary GH isoforms and the second is based on the measurement of markers of GH action. As a result, GH abuse can be detected with reasonable sensitivity and specificity. Testing for IGF-I and insulin is in its infancy, but the measurement of markers of GH action may also detect IGF-I usage, while urine mass spectroscopy has begun to identify the use of insulin analogues.

Keywords: GH, IGF-I, insulin, sport, abuse

Introduction

It is widely believed that growth hormone (GH) has been used by sportsmen and women since the 1980s to improve their athletic performance (McHugh et al., 2005) despite being banned for many years and appearing on the World Anti-Doping Agency list of banned substances.

The actions of GH that interest athletes are anabolic and lipolytic, leading to an increase in lean body mass and reduction in fat mass. Some of the anabolic GH actions are mediated through the generation of insulin-like growth factor-I (IGF-I), and there is anecdotal evidence that this too is being abused by athletes either alone or in combination with GH. The regulation of protein synthesis involves the synergistic actions of GH and IGF-I stimulating protein synthesis, while insulin simultaneously inhibits protein breakdown (Russell Jones et al., 1993). GH stimulates protein synthesis through a mechanism that is separate and distinct from anabolic steroids and therefore it seems likely that their effects will be additive. This has led many athletes to combine GH with insulin and anabolic steroids (Sönksen, 2001).

The GH doses used by athletes are thought to be up to 10 times higher than those used by endocrinologists, and as such athletes are putting themselves at risk of harmful effects such as hypertension, diabetes and Creutzfeld–Jakob disease.

The detection of GH poses the greatest contemporary challenge to the anti-doping community. The detection of GH abuse has proved difficult for several reasons. Unlike many substances of abuse, such as synthetic anabolic steroids, GH is a naturally occurring substance. The demonstration of exogenous administration must therefore rely on finding concentrations exceeding normal physiological levels while excluding pathological causes such as acromegaly. This is made harder because GH is secreted in a pulsatile manner with exercise and stress being major stimulators of GH secretion (Prinz et al., 1983; Savine and Sönksen, 2000). Consequently, GH concentrations are often at their highest in the immediate post-competition setting when most dope testing occurs. Recombinant human GH is almost identical to pituitary GH, whereas cadaveric GH, which is in plentiful supply on the Internet, is indistinguishable from endogenously produced GH. Blood testing is needed for GH and IGF-I because less than 0.1% is excreted unchanged and even that is erratic, rendering urine testing unfeasible (Moreira-Andres et al., 1993).

The review will explore why athletes abuse GH, IGF-I and insulin and also the methodology that has been developed to catch the cheats.

GH abuse in sport

Growth hormone was first extracted from human pituitary glands in 1945 (Li et al., 1945). It was shown to promote growth in hypopituitary animals and was soon used to restore growth in children with hypopituitarism. How and where GH was first used as a doping agent is unknown, but the earliest publication to draw attention to it was Dan Duchaine’s ‘Underground Steroid handbook’, which emerged from California in 1982 (Duchaine, 1982). Although it contains some fundamental errors, such as the recommendation and advertisement of animal GH for use in humans, the description of the actions of GH in this article was remarkably accurate and pre-dated adult endocrinology experience by about a decade. GH was described as the ‘most expensive, most fashionable and least understood of the new athletic drugs. It has firmly established itself in power-lifting and within a few years will be a commonly used drug in all strength athletics.’

The most famous case of GH abuse in professional athletics came to light in 1988 after Ben Johnson won the 100m gold medal at the Olympic Games in Seoul. He was subsequently disqualified after stanazolol was detected in his urine, but at a later hearing, both he and his coach Charley Francis admitted under oath that he had taken human GH in addition to anabolic steroids.

It is impossible to determine the precise prevalence of GH abuse among sportsmen and women, as much of our evidence comes from anecdotal reports (McHugh et al., 2005). Although initially advocated for strength disciplines, endurance athletes are also attracted to GH’s lipolytic actions and reduced fat mass; in 1988, a large quantity of GH was found in a team car at the Tour de France.

There is evidence that adolescents are using GH. In a survey of two US high schools, 5% of male students admitted to having taken GH and nearly one-third knew someone who had taken GH (Rickert et al., 1992). Most GH users were unaware of its side effects and reported their first use between 14–15 years of age.

Growth hormone is an expensive drug and this has led some parents to sell GH prescribed to treat their child’s GH deficiency on the black market. Officials have also misappropriated GH for their athletes. At the 1998 World Swimming Championships, Yuan Yuan, a Chinese swimmer, was stopped on entry into Perth with a suitcase full of GH that had been exported to China for therapeutic reasons.

A few athletes have admitted to taking GH. In a death-bed confession, Lyle Alzado, an American football player, admitted that 80% of American footballers have taken GH. In 2000, Australian discus champion Walter Reiterer claimed institutional and supervised usage of GH. With this in mind, it is interesting to note that 6 months before the Sydney Olympic Games, 1575 vials of GH were stolen from an importer’s warehouse in Sydney.

More recently, Victor Conte, the owner of the Bay Area Laboratory Co-Operative, claimed that he had supplied GH to many high-profile American athletes including Tim Montgomery and Marion Jones. This admission came after the raid on the Bay Area Laboratory Co-Operative’s headquarters on 3 September 2003, when evidence of systematic doping was found and many of the top names in athletics, baseball and American football were implicated in the scandal. Although many have denied taking GH, Tim Montgomery allegedly admitted to taking GH before a US Federal grand jury and later faced a 2-year ban for doping offences. Conte was imprisoned for 4 months for his role in the scandal (Fainaru-Wada and Williams, 2006).

The recent conviction of Sylvester Stallone, who was caught with GH in his possession on entering Australia, suggests that GH is readily available in athletic and body-building circles.

IGF-I abuse in sport

The prevalence of IGF-I abuse is probably much lower than for GH because, unlike GH, there is no readily available natural source, and therefore all IGF-I is obtained through recombinant DNA technology. Two companies currently market IGF-I, and these preparations have only recently received approval for use in humans to treat growth failure in children with severe primary IGF-I deficiency or with GH gene deletion who have developed neutralizing GH antibodies. The first product is Increlex or recombinant human IGF-I, manufactured by Tercica, and the second product manufactured by Insmed is Iplex, which differs from Increlex in that the recombinant human IGF-I is supplied bound to its major binding protein, IGFBP-3 (Kemp et al., 2006; Kemp, 2007).

Although in relatively short supply, other companies make IGF-I for cell culture and other uses, and this material could also become available to athletes. The wider availability of IGF-I, together with an appreciation of the efforts to detect GH abuse, is likely to increase its illicit use by athletes, despite being prohibited by the World Anti-Doping Agency.

Abuse of insulin in sport

We have only sketchy details about the use of insulin by professional athletes. It is alleged that short-acting insulin is being used in a haphazard way to increase muscle bulk in body builders, weight lifters and power lifters (Sönksen, 2001). After concerns raised by the Russian medical officer at the Nagano Olympic games, the International Olympic Committee immediately banned its use in those without diabetes. Athletes with insulin-requiring diabetes may use insulin with a medical exemption.

Physiology of the GH–IGF axis

Growth hormone is the most abundant pituitary hormone and is secreted in a pulsatile manner under the control of the hypothalamic hormones, GH-releasing hormone, somatostatin and ghrelin (Figure 1).

Figure 1 The growth hormone (GH)–insulin-like growth factor (IGF)-I axis. GH is secreted from the pituitary gland under the control of the hypothalamic hormones, somatostatin, Ghrelin and GH-releasing hormone (GHRH). GH circulates bound to its binding (more …)

Regulation of GH secretion

Growth hormone-releasing hormone and ghrelin stimulate the synthesis and release of GH, whereas somatostatin is inhibitory in action (Veldhuis, 2003). Although ghrelin is secreted by the hypothalamus, the major source of ghrelin is the stomach and it is thought to be one mechanism that controls the GH response to eating (van der Lely et al., 2004). Circulating IGF-I reduces GH secretion through classical negative endocrine feedback (Carroll et al., 1997).

There are a number of physiological stimuli that increase or inhibit GH release, the most important of which are exercise and sleep (Savine and Sönksen, 2000). The highest GH peaks occur at night within the first hour of sleep during slow-wave sleep (Takahashi et al., 1968). Nutritional status regulates GH secretion both acutely and chronically; hypoglycaemia, reduced circulating free fatty acids (FFA) and higher amino-acid concentrations all increase GH secretion (Ho et al., 1988; Pombo et al., 1999), whereas in the longer term, GH secretion is increased in anorexia nervosa and decreased with obesity (Veldhuis et al., 1991; Argente et al., 1997).

Age and gender are important determinants of GH secretion (Savine and Sönksen, 2000). Secretion is highest during the pubertal growth spurt, and after the mid-20s, GH secretion decreases by 14% every decade (Toogood, 2003), and this decrease may be responsible for some of the age-related body composition changes (Holt et al., 2001). Women have higher baseline GH secretion but the pulses are not as high and are less erratic than in men (van den Berg et al., 1996).

The actions of GH and IGF-I

Growth hormone exerts its multiple metabolic and anabolic actions through binding to specific GH receptors that are found on every cell of the body (Holt, 2004). Following binding to the GH receptor, the tyrosine kinase Janus kinase 2 is activated and multitude of signalling cascades are initiated that result in the wide variety of biological responses, including cellular proliferation, differentiation and migration, prevention of apoptosis, cytoskeletal reorganization and regulation of metabolic pathways (Lanning and Carter-Su, 2006). Although a detailed description of these signalling cascades is beyond the scope of this review, it is worth remarking on the number of signalling proteins and pathways activated by GH, which include JAKs, signal transducers and activators of transcription, the mitogen-activated protein kinase pathway, and the phosphatidylinositol 3′-kinase pathway. Although these pathways are well described, the inter-relationship between the different pathways is not fully understood.

Growth hormone exerts most of its anabolic actions through the generation of circulating IGF-I (the somatomedin hypothesis) (Le Roith et al., 2001), the majority of which is produced in the liver. IGF-I may also act in a paracrine or autocrine fashion in response to GH action at other target tissues. Transgenic animals, in which the IGF-I gene has been selectively deleted in the liver and whose serum IGF-I is marked reduced, have led some to question the somatomedin hypothesis as these animals appear to grow normally (Sjogren et al., 1999; Yakar et al., 1999), even though with the development of insulin resistance (Sjogren et al., 2001; Yakar et al., 2001). It is known, however, that the total amount of circulating IGF-I in an adult is not required for normal growth, as neonates have much lower circulating levels but grow very rapidly. The precise mechanisms regulating IGF-I bioavailability to the tissues are not yet understood but certainly involve IGF-binding proteins (IGFBPs). As IGFBP distribution in liver IGF-I knockout mice, as in newborn humans, is quite different from that in healthy adults, it has been argued that these data do not really challenge the view that endocrine IGF-I has a role in growth regulation.

Effects on whole body physiology

The physiological effects of GH are best examined by considering the condition of adult GH deficiency. Studies of hypopituitary individuals with appropriate replacement of thyroid, steroid and sex steroid replacement have shown that GH plays a pivotal role in body composition, well-being, physical performance and cardiovascular health (Cuneo et al., 1992; Carroll et al., 2000). In the absence of GH, lean tissue is lost and fat accumulates, and correspondingly waist-to-hip ratio increases as visceral fat increases (Table 1).

Table 1 Clinical features of GH deficiency and effect of GH replacement

Effects on intermediate metabolism

Protein metabolism

Protein synthesis and degradation are each regulated by multiple hormonal and nutritional factors, and protein turnover in individual tissues and the whole body is in a state of constant flux. Insulin, GH and IGF-I have synergistic anabolic effects on protein metabolism (Figure 2) (Sönksen, 2001).

Figure 2 The synergistic action between insulin, IGF-I and GH in regulating protein synthesis. Without insulin, GH loses much (if not all) of its anabolic action. GH and IGF-I stimulate protein synthesis directly, whereas insulin is anabolic through inhibition (more …)

Growth hormone causes nitrogen retention, as shown by decreased urinary excretion rates of urea, creatinine and ammonium. In healthy humans, acute administration of GH modestly stimulates muscle and whole body protein synthesis (Fryburg et al., 1991). When GH is infused locally into the brachial artery, forearm muscle protein synthesis increases, whereas systemic IGF-I concentrations and whole body protein turnover are unchanged, indicating that GH stimulates protein synthesis directly as well as indirectly through IGF-I (Fryburg et al., 1991).

Insulin-like growth factor-I has an anabolic effect on protein metabolism, inhibiting whole body protein breakdown and stimulating protein synthesis (Fryburg, 1994). This effect is dependent on serum insulin and an adequate supply of amino acids. After IGF-I is administered systemically, serum insulin and amino acids decrease, the former through decreased production and the latter as a result of increased clearance from the blood. This attenuates the stimulation of whole body protein synthesis, but if the amino acids and insulin are replaced during the administration, the effect on protein synthesis by IGF-I is clearly seen (Russell Jones et al., 1994; Jacob et al., 1996).

Protein breakdown is inhibited by physiological concentrations of plasma insulin, the ‘Chalonic’ action of insulin (Umpleby and Sönksen, 1985; Tessari et al., 1986). In contrast, the rates of protein degradation are increased in cardiac and skeletal muscles in situations where insulin concentrations are low such as type 1 diabetes or starvation (Charlton and Nair, 1998). Although the rate of protein synthesis is reduced by 40–50% in young diabetic rats, a physiological anabolic effect of insulin on protein synthesis has not been confirmed in humans (Liu and Barrett, 2002).

The similarities between IGF-I and insulin suggest that these proteins act in a coordinated manner to regulate protein turnover. There are interesting differences, however, in their respective dose–response curves. Low physiological insulin concentrations inhibit protein breakdown and increase glucose disposal into skeletal muscle, whereas higher, non-physiological concentrations are required to stimulate protein synthesis (Louard et al., 1992). In contrast, increases in IGF-I that have no effect on glucose uptake stimulate protein synthesis, and higher concentrations are required to inhibit protein breakdown (Fryburg, 1994). During the last decade, there have been advances in the understanding of the intracellular signalling mechanisms of insulin and IGF-I, many of which are shared, such as insulin receptor substrate 1. The precise mechanism by which these similar but divergent pathways interact is not fully understood, but IGF-I, similar to GH, most likely acts through stimulating amino-acid uptake.

Lipolysis

The administration of GH to humans, either by continuous infusion or by a bolus injection, leads to stimulation of lipolysis and increased fasting FFA concentrations with the peak effect around 2–3h after the injection (Hansen, 2002). These findings are in keeping with the changes in FFAs following physiological stimuli of GH secretion. In young healthy subjects, the nocturnal or exercise-induced peak of GH precedes the peak of FFAs by 2h (Moller et al., 1995). Furthermore, during times of fasting or energy restriction, the lipolytic effect of GH is enhanced, although the effect is suppressed by co-administration of food or glucose (Moller et al., 2003). Acromegaly is associated with increased circulating FFAs, increased muscle uptake of FFAs and increased lipid oxidation.

Although GH receptors are abundantly expressed on adipocytes, it has been suggested that GH has a permissive effect on catecholamine-induced lipolysis (Hansen, 2002). In vitro studies have shown that GH has no direct lipolytic effect on human fat cells, but markedly increases the maximal lipolysis induced by catecholamines (Marcus et al., 1994).

Glucose homeostasis

The first observation that GH had an effect on glucose metabolism came in the 1930s when it was observed that hypophysectomy ameliorated the hyperglycaemia of experimental diabetes in dogs (Houssay and Biasotti, 1930). In both healthy subjects and those with type 1 diabetes, GH increases fasting hepatic glucose output, by increasing hepatic gluconeogenesis and glycogenolysis, and decreases peripheral glucose utilization through the inhibition of glycogen synthesis and glucose oxidation (Tamborlane et al., 1979; Bak et al., 1991; Fowelin et al., 1991, 1993, 1995). Patients with acromegaly develop insulin resistance and hyperinsulinaemia (Sönksen et al., 1967), and up to 40% become diabetic (Ezzat et al., 1994; Colao et al., 2000).

Although the effect on glucose homeostasis would appear to be disadvantageous for the athletes, it is worth bearing in mind that long-standing adult GH deficiency is associated with insulin resistance and that any acute effect on glucose homeostasis does not take into account changes in IGF-I, which also affects insulin sensitivity (Salomon et al., 1994).

Intravenous IGF-I causes hypoglycaemia in rats by stimulating peripheral glucose uptake, glycolysis and glycogen synthesis, although having only a minimal effect on hepatic glucose production (Jacob et al., 1989). In dogs, however, IGF-I has been shown to suppress hepatic glucose output, but to a lesser degree than insulin at the doses used, as well as increasing peripheral glucose utilization (Shojaee Moradie et al., 1995). As hepatic expression of the IGF-I receptor is reportedly low (Stefano et al., 2006), it is possible that this effect of IGF-I on hepatic glucose output is by an indirect mechanism. For example, IGF-I may bind the insulin receptor with low affinity (Holt et al., 2003). Alternatively, it may improve whole body insulin sensitivity by inhibiting the secretion of GH.

The effects of an intravenous IGF-I infusion in humans are similar to those described in animals and lead to hypoglycaemia (Zenobi et al., 1992). Insulin sensitivity increases with respect to glucose by IGF-I through increased peripheral glucose uptake and decreased hepatic glucose production (Boulware et al., 1994; Russell Jones et al., 1995). A subcutaneous infusion of IGF-I also causes hypoglycaemia, but the effect is slower in onset than insulin and decreases more slowly after the infusion was stopped, because of the presence of the IGF-binding proteins.

Bone metabolism

Growth hormone has profound effects on bone metabolism. GH deficiency is associated with osteopaenia, which is reversed by GH replacement (Gomez et al., 2000). Male subjects with osteoporosis have reduced GH peaks and low serum IGF-I concentrations, and furthermore, IGF-I concentrations correlate well with estimates of bone mineral density (Patel et al., 2005). In addition to a direct effect on bone stimulating the cycle of bone growth, there is evidence to suggest that GH and IGF-I may modify intestinal calcium absorption and serum 1,25 hydroxy vitamin D concentrations. Patients with acromegaly have increased intestinal calcium absorption (Lund et al., 1981), and GH therapy in pigs increases intestinal calcium absorption, probably through increased production of serum 1,25 hydroxy vitamin D (Chipman et al., 1980). GH replacement therapy in adult GHD leads to a short-term increase in serum 1,25 hydroxy vitamin D concentrations (Burstein et al., 1983). These data suggest that GH increases intestinal calcium absorption by its effects on renal 25 (OH)D 1-α-hydroxylase activity, but other mechanisms may also be involved (Halloran and Spencer, 1988).

Why do athletes abuse GH?

The importance of GH in adult physiology as well as in children was confirmed beyond doubt in 1989 by two independent studies undertaken in the UK and Denmark (Jorgensen et al., 1989; Salomon et al., 1989). Both groups undertook double-blind, placebo-controlled trials in adults with hypopituitarism given appropriate replacement therapy with everything except GH. The studies showed remarkably congruous results after 6 months of GH treatment.

The most impressive finding was a change in and normalization of body composition with an average 6kg increase in lean body mass, largely accounted by an increase in skeletal muscle, and a concomitant loss of fat. The body composition changes were accompanied by improvements in quality of life, particularly in the area of ‘increased energy’ and performance enhancements (McGauley et al., 1990; Cuneo et al., 1991a, 1991b). Longer studies over the first 3 years of GH replacement showed that exercise performance continued to improve (Jorgensen et al., 1994).

Does GH enhances performance in normal healthy young adults?

There has been considerable debate about the ability of GH to translate these body composition and other metabolic changes into improved performance (Rennie, 2003). Despite the theoretical benefits of GH, sceptics point to the condition of acromegaly and negative clinical studies as evidence of a lack of benefit.

Acromegaly—nature’s experiment of GH excess

It has been argued that acromegaly, where there is over-secretion of GH usually from a pituitary adenoma, provides evidence that excess GH is not performance enhancing, as it is not associated with athletic prowess. Indeed, acromegaly is usually associated with muscle weakness rather than excessive strength (Table 2) (McNab and Khandwala, 2005). It needs to be appreciated, however, that acromegaly frequently remains undiagnosed for many years and the clinical presentation at diagnosis may not reflect earlier stages of the disease. Many patients if questioned carefully will give a history of increased strength in the first few years of their condition. Indeed, we know a rower who competed at an elite level during the early stages of his acromegaly. Not only was he one of the strongest crew, but he could also tolerate harder training sessions than his colleagues and recovered more quickly afterwards (PHS, unpublished data).

Table 2 Clinical features of acromegaly

Although only a clinical case report, it illustrates that the timing and degree of GH excess are important for the physiological effect. Prolonged massive GH excess coupled frequently with deficiencies of other pituitary hormones, such as ACTH, may tell us little about the effects of lesser GH excess earlier in the natural history of the illness.

Can clinical trials provide us with the answer?

Although there have been many negative studies in this area, traditional randomized controlled trials that have the power to examine differences of 20–30% are ill suited to detect the much smaller differences that determine whether an individual wins a gold medal or not. Despite these difficulties, one trial showed that, in healthy, elderly men, the combination of GH and testosterone led to a 20% improvement in fitness as measured by maximal oxygen uptake (Giannoulis et al., 2006), which was larger than with either compound alone. Very recently, a well designed and executed study in past abusers of anabolic steroids showed for the first time an ergogenic effect of GH in healthy young athletes (Graham et al., 2008).

A further problem with the designs of our clinical trials is that they are designed to test one or at most two interventions. In reality, for the reasons described above, GH is frequently used by athletes in combination with insulin and anabolic steroids in varying concentrations during differing training and dietary regimens. It is impossible to control for all these variables within a single trial, and therefore it seems likely that the athletes using the ‘n=1′ design are probably best placed to address the question whether GH is performance enhancing. This certainly appeared to be a potent weapon when abused by the former East German coaches (Franke and Berendonk, 1997).

A definitive answer to this question will probably never be found, as it would be difficult to obtain ethics approval for a suitable study. It should be remembered, however, that there were similar arguments about anabolic steroids 20 years ago that have subsequently been shown to have performance benefits.

Pharmacology of GH, IGF-I and insulin administration

The half-life of endogenously secreted GH is around 13min (Sohmiya and Kato, 1992). It is rapidly cleared from the body by the liver, kidney and peripheral tissues through interaction with GH receptors. If recombinant human GH (rhGH) is administered intravenously, the half-life is similar to endogenously secreted GH (Refetoff and Sönksen, 1970; Haffner et al., 1994). In practice, however, the pharmacokinetics of exogenously administered GH differs, as it is given by intermittent, usually daily, subcutaneous injection. Following injection, GH concentrations increase and reach a maximum concentration after about 2–6h, depending on the age and gender of the recipient (Kearns et al., 1991; Janssen et al., 1999). The estimated bioavailability of rhGH is 50–70%, because of degradation at the site of injection. This may explain why intramuscular injection results in a higher maximal and area-under-the-curve GH concentration than subcutaneous injection (Keller et al., 2007). Thereafter, rhGH is rapidly cleared and GH is usually undetectable in women 12 hours after injection, even after high doses, while men have only low levels of GH. (Giannoulis et al., 2005). There is quicker clearance of GH in women, probably reflecting their extra body fat that contains a high density of GH receptors (Vahl et al., 1997). Longer-acting GH preparations are currently being developed.

The pharmacokinetics of IGF-I is complicated by the presence of a family of highly specific binding proteins (IGFBPs) that coordinate and regulate the biological functions of IGF-I. Less than 5% of serum IGF-I is free and most is bound in a ternary complex of IGF-I, IGFBP-3 and an acid-labile subunit (Figure 1). The half-life of free IGF-I is only a few minutes, whereas the half-lives of IGF-I bound in a binary and ternary complex are 20–30min and 12–15h, respectively (Guler et al., 1989). When subcutaneous IGF-I is administered to healthy volunteers, the maximum concentration is achieved at about 7h and half life is 20h (Grahnen et al., 1993). The half-life is prolonged when IGF-I is administered as a complex with IGFBP-3. In patients with severe GH insensitivity syndrome, IGF-I concentrations peaked between 15 and 19h after the injection of the complex, and a single injection was effective in increasing IGF-I concentrations in these patients for a 24h period (Camacho-Hubner et al., 2006).

The half-life of intravenous insulin is only 4min, but apart from the treatment of diabetic emergencies and possibly the replenishment of glycogen using an insulin clamp (Sönksen and Sönksen, 2000), pharmacologically administered insulin is through subcutaneous injection (Matthews et al., 1985). Insulin manufacturers have developed many preparations of insulin including insulin analogues in the attempt to provide insulin replacement to people with diabetes in the most physiological way (Peterson, 2006). Consequently, the shortest-acting insulin analogues appear in the circulation within 5–10min of injection and cleared within 4–6h, whereas longer-acting insulins are present for over 24h.

Potential adverse effects of GH administration

The side effects of GH administration to adults with GH deficiency are well documented, and any athlete receiving GH will potentially be at risk of these side effects (Powrie et al., 1995). It is believed, however, that many athletes are using doses that are up to 10 times higher than those used therapeutically. The effects of chronically administering this dose of GH are unknown, but it would be reasonable to expect that athletes may develop some of the features of acromegaly with prolonged use (Table 2).

Sodium and fluid retention

Growth hormone causes fluid retention through its action on the kidney to promote sodium reabsorption (Powrie et al., 1995; Moller et al., 1999). This may be manifest as ankle swelling, hypertension and headache.

Diabetes

Patients with acromegaly develop insulin resistance and hyperinsulinaemia, and up to 40% become diabetic (Sönksen et al., 1967; Ezzat et al., 1994; Colao et al., 2000).

Cardiomyopathy

Cardiovascular complications are a major cause of morbidity and mortality in patients with acromegaly (Colao et al., 2001). The excess of GH and IGF-I causes a specific derangement of cardiomyocytes, leading to abnormalities in cardiac muscle structure and function, inducing a specific cardiomyopathy. In the early phase, there is a hyperkinetic syndrome, characterized by increased heart rate and systolic output. Two-thirds of patients have concentric cardiac hypertrophy and this is commonly associated with diastolic dysfunction and eventually with impaired systolic function leading to heart failure, if the acromegaly is left untreated. In addition, abnormalities of cardiac rhythm and those of heart valves have also been described. The coexistence of arterial hypertension and diabetes may further aggravate acromegalic cardiomyopathy. It is alleged that the American sprinter, Florence Griffith-Joyner (Flo Jo), purchased GH from fellow sprinter Darrell Robinson. When Flo Jo died at the age of 38 years, her heart was enlarged consistent with cardiomyopathy (Sullivan, 1998).

Cancer

Although controversial, the consensus statement of the Growth Hormone Research Society (2001) was that there is no increased risk of cancer when GH is given at physiological replacement doses (2001). There is evidence to suggest that acromegaly, where GH levels have been much higher than physiological doses for many years, may be associated with increased rates of colorectal, thyroid, breast and prostate cancers (Jenkins et al., 2006).

Creutzfeld–Jakob disease

Initially, the only source of GH came from extracts of human pituitary glands, and tragically, this was discovered to be a source for the prion-induced Creutzfelt–Jacob disease (Brown et al., 1985). As a result, pituitary-derived GH was withdrawal from the market place in 1985 and was replaced with recombinant human GH in 1987. Despite the dangers, supplies of pituitary-derived GH continue to be available on the black market to this day, and athletes continue to use this and thus a case of Creutzfelt–Jacob disease may emerge in an elite athlete at some time in the future.

Potential adverse effects of IGF-I and insulin administration

We have only limited experience with the use of exogenous IGF-I, and so most of the known side effects relate to short-term usage only. It seems reasonable to hypothesize, however, that many of the longer-term effects of GH administration would also occur with IGF-I, as the anabolic effects of GH are closely related to the production of IGF-I in different tissues.

In the clinical trials, the commonest short-term side effects are oedema, headache, arthralgia, jaw pain and hypoglycaemia (Kemp et al., 2006; Kemp, 2007). These appear to be more marked when IGF-I is used alone as the recombinant human IGFBP-3 seems to buffer the acute effects of IGF-I.

The side effects of insulin are well documented from our experience in treating people with diabetes. The most commonly experienced side effect is hypoglycaemia. Weight gain is also a problem in people with diabetes, but this is probably less of an issue for athletes whose diet and training regimens are closely monitored.

Detection of GH abuse

Two complementary approaches have been investigated to detect GH abuse: the first is based on the detection of different pituitary GH isoforms, whereas the second relies on measurement of GH-dependent markers. The very different approaches are viewed as a major strength, as their different properties mean that they are useful in different situations.

The isoform or differential immunoassay method

Growth hormone exists as multiple isoforms; 70% of circulating GH is in the form of a 22-kilo Dalton (kDa) polypeptide, whereas 5–10% occurs as a 20kDa isoform, from mRNA splicing. Dimers and oligomers of GH exist as do acidic, desaminated, acylated and fragmented forms (Baumann, 1999). The differential immunoassay approach is based on the principle that endogenous GH occurs as a number of isoforms, whereas rhGH contains only the 22kDa isoform (Figure 3). When rhGH is administered in sufficiently high doses, there is a suppression of endogenous GH secretion through negative feedback to the pituitary, and therefore the ratio between 22kDa GH and non-22kDa GH is increased (Bidlingmaier et al., 2003). This change in ratio can be detected with specific immunoassays that distinguish the different isoforms.

Figure 3 Principle of the isoform method. rhGH contains 22kDa and this is specifically recognized by assay 1. Pituitary GH contains multiple isoforms and these are recognized by assay 2. When rhGH is administered, endogenous production of pituitary GH (more …)

The isoform method was first established by Christian Strasburger and Martin Bidlingmaier in Germany by employing one assay that specifically measured 22kDa GH and another permissive assay that measured all GH isoforms (Figure 3). A slightly different approach has been adopted by an Australian Japanese Consortium that developed assays that specifically measure either 22 or 20kDa GH (Momomura et al., 2000).

When the German method is applied to a normal population, the ratio between 22kDa and total GH is less than 1 with a normal distribution of values, whereas individuals receiving GH have values that are greater than one (Figure 4) (Wu et al., 1999). Age, sex, sporting discipline, ethnicity and pathological states do not seem to affect the relative proportions of GH isoforms (Holt, 2007), but exercise causes a transient relative increase in the 22kDa isoform, thereby lowering the sensitivity of the test if samples are taken immediately after competition (Wallace et al., 2001a, 2001b).

Figure 4 Ratio between 22kDa-hGH assay and total hGH assay in serum samples obtained from 125 controls and 30 individuals treated with rhGH. Mean values are 1.43±0.21 for treated individuals and 0.50±0.12 for controls (P<0.0001). (more …)

The short half-life and rapid clearance of rhGH, even when injected subcutaneously, means that the ‘window of opportunity’ for detection of GH doping with this test is less than 36h (Keller et al., 2007). As GH is usually administered in the evening, GH is frequently undetectable in a blood sample taken the following day (Giannoulis et al., 2005). The 20kDa GH remains suppressed for 14–30h in women depending on the dose used, whereas in men, 20kDa GH remains undetectable for up to 36h (Keller et al., 2007). Spontaneous GH secretion returns 48h after the last dose of rhGH treatment (Wu et al., 1990). Consequently, any athlete who stops administering GH several days before the competition will not be detected. Thus the isoform method is unlikely to catch a GH abuser in the classical ‘post competition’ dope testing scenario, and the optimal use of this method must be in unannounced ‘out of competition’ testing. This method does not detect individuals receiving cadaveric GH, IGF-I or GH secretagogues.

This test was introduced at the Athens and Turin Olympic Games and no positives were detected in samples taken ‘post competition’. One of the World Anti-Doping Agency rules for the use of immunoassays is that two antibodies recognizing different epitopes are needed for each analyte. The German group had carefully characterized their assays and antibodies, and this may have been an overriding factor in the decision to implement the test.

The GH-dependent marker method

Growth hormone administration leads to the alteration of the concentrations or ratios of several serum proteins, and this change may be used as a means of detecting exogenous GH. An ideal marker or combination of markers would have well-defined reference ranges, would change in response to GH administration and would remain altered after GH has been discontinued (Holt, 2007). The marker should be largely unaffected by other regulators of GH secretion such as exercise or injury and should be validated across populations.

This approach was pioneered by the large multi-centre GH-2000 project coordinated by Peter Sönksen with funding from the European Union under their BIOMED 2 initiative, with additional funding from the International Olympic Committee and the rhGH manufacturers Novo Nordisk and Pharmacia. The aim was to develop a test in time for the Sydney Olympic Games. It had three main components, the first of which was a cross-sectional study of elite athletes at National or International events to establish a reference range of selected markers of GH action (Healy et al., 2005). Blood samples taken within 2h of competitions showed that the markers were dependent on age as is the case in the general population, but in contrast, sporting discipline, gender and body shape had little effect. The findings of this study were subsequently confirmed by the Australian Japanese consortium led by Ken Ho. In a study of 1103 elite athletes sampled out of competition, less than 10% of the total variance of the markers was explained by gender, sporting discipline, ethnicity and body mass index, whereas age contributed to between 20 and 35% of the variance (Nelson et al., 2006).

The second component of the GH-2000 project was the ‘wash-out’ study (Wallace et al., 1999, 2000). When the project was conceived, 25 potential markers of GH action were considered. The aim of the ‘wash-out’ study was to narrow this number down to the most suitable markers for a more in-depth analysis. rhGH was administered to recreational male athletes for 1 week, with blood samples collected during and after the GH administration. Subjects also undertook exercise tests to assess the potential effect of ‘competition’ on the markers. Nine markers, either members of the IGF–IGF-binding protein axis or markers of bone and soft tissue turnover, were then selected for analysis in the third component of the GH-2000 project. This was a 28-day GH administration study involving self-administered rhGH at two doses to 102 recreational athletes under double-blind, placebo-controlled conditions to evaluate the potential markers for their ability to discriminate active drug from placebo and to assess the ‘window of opportunity’ when the test remained positive after rhGH was stopped (Dall et al., 2000; Longobardi et al., 2000).

From these studies, the GH-2000 project proposed a test based on IGF-I and type 3 pro-collagen (P-III-P) (Powrie et al., 2007) (Figure 5). These markers were chosen because they provided the best discrimination between individuals receiving GH or placebo during the randomized controlled trial. They exhibit little diurnal or day-to-day variation and are largely unaffected by exercise or gender (McHugh et al., 2005). In the wash-out study, IGF-I and P-III-P increased 20 and 10.2%, respectively, following exercise, but this increase was small in comparison with the larger 300% increase in the markers with GH (Wallace et al., 1999, 2000). Although discrimination was the prime reason for the selection, it is important to note that these proteins are produced by different tissues, thereby reducing the number of pathological conditions that could lead to an elevation in both markers and potential false-positives.

Figure 5 Change in IGF-I (a) and P-III-P (b) following the administration of GH or placebo for 28 days to 50 healthy male volunteers.

It is known that there is sexual dimorphism in the GH–IGF axis. There are small differences in IGF-I and P-III-P concentrations in elite male and female athletes (Healy et al., 2005), although gender explained around 1% of the overall variance in these markers (Nelson et al., 2006). Women are known to be inherently more resistant to the actions of GH and so the increase in markers is less pronounced in women than in men (Dall et al., 2000; Longobardi et al., 2000). This potential disadvantage may be offset because women may need to receive higher doses to obtain a performance-enhancing benefit.

Although a single marker could be used, by combining markers in conjunction with gender-specific equations, ‘discriminant functions’, the sensitivity and specificity of the ability to detect GH abuse can be improved compared with single-marker analysis (Powrie et al., 2007).

The procedure used to generate the discriminant functions involves splitting the available data into two: a ‘training’ set of data is used to calculate the discriminant function and a ‘confirmatory’ set is then used to validate the sensitivity and specificity of the discriminant function. The confirmatory set required to ensure the model is applicable to the general population and not just the ‘training’ set.

The sensitivity of any test is dependent on the specificity. Standard medical practice accepts as ‘normal’ values those being within two standard deviations of the mean, but by definition, 5% of the population lie outside the ‘normal range’. This creates an unacceptably high false-positive rate if applied to athletes. The specificity to be used has not been determined by the anti-doping authorities, but nevertheless the GH-2000 formulae show reasonable sensitivity even up to false-positive rates of 1 in 10000 and beyond (Figure 6). The formula has been modified more recently to take into account the effect of age to prevent younger athletes from being placed at a disadvantage (Powrie et al., 2007).

Figure 6 Change in GH-2000 score in men (a) and women (b) following 28 days of GH administration. Dotplot of the standardized scores for each visit day of the studies by group and data set. The mean of a normal population is 0 and the standard deviation is 1. (more …)

The results of the GH-2000 project were presented at an International Olympic Committee workshop in Rome in March 1999 to review critically and assure the quality of the results. The conclusion of the workshop was strong support for the methodology, but it was felt that several issues needed to be addressed before the test could be fully implemented at an Olympic games. The biggest issue was related to potential ethnic effects of GH, as the vast majority of volunteers in the GH-2000 study were white Europeans. It was felt that injury could confound the test and further work was needed to develop immunoassays owned by International Olympic Committee and subsequently World Anti-Doping Agency to prevent arbitrary changes being made to commercially owned immunoassays.

Apart from the assay development, these issues have largely been addressed by the GH-2004 study. A further cross-sectional study of elite athletes has shown that although there are small differences in the mean values between ethnic groups—for example, the IGF-I concentrations in Afro-Caribbean men are approximately 8.2% lower than white European men—nearly all the values lie within the 99% prediction intervals for white European athletes, regardless of ethnic background. A further double-blind GH administration study suggests that the response to GH in other ethnic groups is similar to white European amateur athletes. The effect of injury was systemically examined by the GH-2004 team who followed 143 men and 40 women following a sporting injury. There was no change in IGF-I over the 12-week follow-up, but P-III-P increased by approximately 20%, reaching a peak 2–3 weeks after injury. This, however, did not cause any false-positive readings in the proposed test combining IGF-I with P-III-P.

Several other groups have also examined the use of GH-dependent markers, the first of which pre-dated the GH-2000 study. In this first study, the ratio of IGFBP-2 to IGFBP-3 was found to discriminate between those taking GH or placebo (Kicman et al., 1997). These findings were not supported by the GH-2000 study, although several other groups have confirmed the utility of IGF-I and P-III-P. The Institut für Dopinganalytik und Sportbiochemie in Kreischa, Germany, undertook a 14-day GH administration study in amateur male athletes and derived a discriminant function based on IGF-I, P-III-P and IGFBP-3 (Kniess et al., 2003). Most recently, the Australian Japanese Consortium presented the results of an 8-week GH administration study at the American Endocrine Society meeting in Toronto in June 2007. This also confirmed the value of IGF-I and P-III-P, although this suggested that an alternative bone marker (carboxyterminal cross-linked telopeptide of type I collagen) might provide better discrimination during the wash-out phase. This study also examined the effect of co-administration of anabolic steroids in males and showed that there were additive effects on P-III-P.

These confirmatory studies are important because it is unknown how well these GH-2000 formulae will perform in ‘real life’, where the patterns and doses of GH abused by athletes are unclear. When the male GH-2000 formula was applied to an independent data set obtained from the Institut für Dopinganalytik und Sportbiochemie Kreischa, 90% of the individuals who had received GH were correctly identified and there were no false-positives, findings that were identical to those of the GH-2000 data when the formula was used (Erotokritou-Mulligan et al., 2007).

Although this methodology has been rigorously tested, the development of World Anti-Doping Agency-owned immunoassays has lagged behind the science underpinning the method, despite the International Olympic Committee having been made aware of the need for these assays before the worldwide introduction of this test.

Future technologies to detect GH

Surface plasmon resonance

Surface plasmon technology is a non-labelled optical methodology that measures the refractive index of small quantities of a material absorbed onto a metal surface allowing measurement of mass. This technology is being applied for the detection of GH and dependent markers by the Barcelona anti-doping laboratory, but at present does not yield the same sensitivity as conventional immunoassays.

Mass spectrometry

Surface-enhanced laser desorption/ionization–time-of-flight mass spectrometry is a proteomic technique in which proteins are bound to proprietary protein chips with different types of adsorptive surfaces. It can be used to analyse peptide and protein expression patterns in a variety of clinical and biological samples, and biomarker discovery can be achieved by comparing the protein profiles obtained from control and patient groups to elucidate differences in protein expression. This technique has been applied to the detection of GH abuse to find potential new markers of GH abuse, such as the haemoglobin α-chain (Chung et al., 2006), but the sensitivity is insufficient to deal with analyses of IGF-I and P-III-P.

The detection of IGF-I

At present, there are no technologies to detect IGF-I abuse, but it is reasonable to adopt a similar approach as the GH-dependent marker test, and this is currently being evaluated by the GH-2004 team. Similarly, this approach should detect athletes abusing GH secretagogues.

The detection of insulin

The challenges of detecting insulin are in many ways similar to GH in that insulin is a naturally occurring pulsatile peptide hormone. At present, there are no methods to detect endogenous insulin abuse but urinary mass spectroscopy may be useful to detect the presence of analogue insulin. This technique involves concentration of the urine and followed by isolation by immunoaffinity chromatography. The eluate may then be analysed using microbore liquid chromatography/tandem mass spectrometry that produces characteristic product spectra obtained from the analogues that are distinguishable from human insulin (Thevis et al., 2006; Thomas et al., 2007). Some insulin analogues are handled differently from insulin and excreted in much higher quantities, which may facilitate this approach (Tompkins et al., 1981).

Future challenges

A major challenge for the future is the use of gene doping, where DNA is incorporated into target tissues, such as skeletal muscle, with or without the aid of a vector, such as an adenovirus. Expression of the gene may then lead to enhanced local production of an anabolic substance such as IGF-I.

Proof-of-concept experiments have been undertaken in animals in which injection of a recombinant adeno-associated virus genetically manipulated to induce myocyte overexpression of IGF-I in young mice induced a 15% increase in muscle mass and a 14% increase in muscle strength without inducing a systemic increase in IGF-I (Barton-Davis et al., 1998).

It is unclear whether this technology is being used by athletes; certainly, it has not been used in clinical practice, where it is highly needed despite significant investment. Anecdotal evidence, however, suggests that athletes are considering its use and this would provide new challenges to the anti-doping community. Traditional blood and urine testing may be of no benefit if gene doping causes no change in serum concentrations of the relevant proteins. Although the detection of vectors may be possible and changes in blood markers may occur, new technologies will be needed to catch this new form of doping.

Conclusion

Doping with GH and its related protein IGF-I remains a major challenge for those working in anti-doping. Anecdotal evidence suggests that abuse with GH and insulin is common, whereas the abuse of IGF-I is set to increase. There are compelling physiological reasons to explain why GH may have performance benefits. The athletes are risking long-term harm by using these drugs. Over the last decade, there have been major advances in methodologies to detect GH, and this should mean that once World Anti-Doping Agency has established suitable assays for IGF-I and P-III-P in its worldwide network of laboratories, athletes will no longer be able to cheat by taking GH without being caught.

Acknowledgments

The GH-2004 project is funded by the United States Anti-Doping Agency and the World Anti-Doping Agency. Our thanks go to the rest of the GH-2004 team: Eryl Bassett, Ioulietta Erotokritou-Mulligan, David Cowan, Christiaan Bartlett and Cathy McHugh. The GH-2004 study was undertaken in the Wellcome Trust Clinical Research Facility (WT-CRF) at Southampton General Hospital and we acknowledge the support of the WT-CRF nurses and Southampton medical students who have supported the study. We also pay tribute to our scientific collaborators, Astrid Kniess, Ken Ho, Anne Nelson, Christian Strasburger and Martin Bidlingmaier.

Abbreviations

FFA	free fatty acid
GH	growth hormone
IGF-I	insulin-like growth factor-I
IGFBP	IGF binding protein
P-III-P	type 3 pro-collagen
rhGH	recombinant human growth hormone

Regulation of Wound Healing by Growth Factors and Cytokines IGF, TGF, leptin, BMP

ABSTRACT

Werner, Sabine, and Richard Grose. Regulation of Wound Healing by Growth Factors and Cytokines. Physiol Rev 83: 835–870, 2003; 10.1152/physrev.00032.2002.—Cutaneous wound healing is a complex process involving blood clotting, inflammation, new tissue formation, and finally tissue remodeling. It is well described at the histological level, but the genes that regulate skin repair have only partially been identified. Many experimental and clinical studies have demonstrated varied, but in most cases beneficial, effects of exogenous growth factors on the healing process. However, the roles played by endogenous growth factors have remained largely unclear. Initial approaches at addressing this question focused on the expression analysis of various growth factors, cytokines, and their receptors in different wound models, with first functional data being obtained by applying neutralizing antibodies to wounds. During the past few years, the availability of genetically modified mice has allowed elucidation of the function of various genes in the healing process, and these studies have shed light onto the role of growth factors, cytokines, and their downstream effectors in wound repair. This review summarizes the results of expression studies that have been performed in rodents, pigs, and humans to localize growth factors and their receptors in skin wounds. Most importantly, we also report on genetic studies addressing the functions of endogenous growth factors in the wound repair process.

I. INTRODUCTION

Injury to the skin initiates a cascade of events including inflammation, new tissue formation, and tissue remodeling, which finally lead to at least partial reconstruction of the wounded area (57, 176; Fig. 1). The repair process is initiated immediately after injury by the release of various growth factors, cytokines, and low-molecular-weight compounds from the serum of injured blood vessels and from degranulating platelets. Disruption of blood vessels also leads to the formation of the blood clot, which is composed of cross-linked fibrin, and of extracellular matrix proteins such as fibronectin, vitronectin, and thrombospondin (56, 57, 176). Apart from providing a barrier against invading microorganisms, the blood clot also serves as a matrix for invading cells and as a reservoir of growth factors required during the later stages of the healing process. Within a few hours after injury, inflammatory cells invade the wound tissue. Neutrophils arrive first within a few minutes, followed by monocytes and lymphocytes. They produce a wide variety of proteinases and reactive oxygen species as a defense against contaminating microorganisms, and they are involved in the phagocytosis of cell debris. In addition to these defense functions, inflammatory cells are also an important source of growth factors and cytokines, which initiate the proliferative phase of wound repair. The latter starts with the migration and proliferation of keratinocytes at the wound edge and is followed by proliferation of dermal fibroblasts in the neighborhood of the wound. These cells subsequently migrate into the provisional matrix and deposit large amounts of extracellular matrix. Furthermore, wound fibroblasts acquire a contractile phenotype and transform into myofibroblasts, a cell type which plays a major role in wound contraction. Massive angiogenesis leads to the formation of new blood vessels, and nerve sprouting occurs at the wound edge. The resulting wound connective tissue is known as granulation tissue because of the granular appearance of the numerous capillaries. Finally, a transition from granulation tissue to mature scar occurs, characterized by continued collagen synthesis and collagen catabolism. The scar tissue is mechanically insufficient and lacks appendages, including hair follicles, sebaceous glands, and sweat glands. Scarring can also be excessive, leading to hypertrophic scars and keloids. In contrast, wound healing in mammalian embryos until the beginning of the third trimester results in essentially perfect repair, suggesting fundamental differences in the healing process between embryonic and adult mammals (57, 168, 176).

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FIG. 1. Schematic representation of different stages of wound repair. A: 12–24 h after injury the wounded area is filled with a blood clot. Neutrophils have invaded into the clot. B: at days 3–7 after injury, the majority of neutrophils have undergone apoptosis. Instead, macrophages are abundant in the wound tissue at this stage of repair. Endothelial cells migrate into the clot; they proliferate and form new blood vessels. Fibroblasts migrate into the wound tissue, where they proliferate and deposit extracellular matrix. The new tissue is called granulation tissue. Keratinocytes proliferate at the wound edge and migrate down the injured dermis and above the provisional matrix. C: 1–2 wk after injury the wound is completely filled with granulation tissue. Fibroblasts have transformed into myofibroblasts, leading to wound contraction and collagen deposition. The wound is completely covered with a neoepidermis.

 

In addition to the importance of cell-cell and cell-matrix interactions, all stages of the repair process are controlled by a wide variety of different growth factors and cytokines. Multiple studies have demonstrated a beneficial effect of many of these growth factors, e.g., platelet-derived growth factors (PDGFs), fibroblast growth factors (FGFs), and granulocyte-macrophage colony stimulating factor (GM-CSF) on the healing process, both in animal models and also in patients suffering from different types of wound healing disorders (1, 79, 107, 115, 196). However, the roles of endogenous growth factors in the healing response have been only partially elucidated, and in most cases, the suggested function of these molecules is based on descriptive expression studies and/or functional cell culture data. However, in vivo functions of many growth factors remain largely unconfirmed.

The development of transgenic and knock-out mouse technologies has provided new insights into the function of many different genes during embryonic development. These technologies allow gain of function experiments (overexpression of genes) as well as loss of function experiments (gene knock-outs by homologous recombination in embryonic stem cells or overexpression of dominant-negative mutants). Most importantly, spatial and temporal control of gene ablation or overexpression, using both inducible and cre-lox technologies, makes it possible to determine the functions of proteins formerly precluded due to embryonic lethality. A large number of viable genetically modified mice are now available that can be used to elucidate the role of the deleted, mutated, or overexpressed genes in different types of repair processes. Indeed, the past years have seen an exponential growth in the number of genetically modified mice that were used for wound healing experiments, and these studies have provided interesting, and often unexpected, results concerning the in vivo function of growth factors in wound repair (see http://icbxs.biol.ethz.ch/members/grose/woundtransgenic/home.html). In this review, we summarize the reported expression and function of endogenous growth factors and cytokines in cutaneous wound repair. Results of experiments with exogenous growth factors for the treatment of wound repair are only mentioned briefly, and reviews are cited wherever possible. In addition, we focus on those growth factors and cytokines for which results from functional in vivo studies are available.

II. PLATELET-DERIVED GROWTH FACTOR FAMILY

PDGFs comprise a family of homo- or heterodimeric growth factors, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD (reviewed in Ref. 120). They exert their functions by binding to three different transmembrane tyrosine kinase receptors, which are homo- or heterodimers of an α- and a -chain (120, 121).

PDGF was the first growth factor shown to be chemotactic for cells migrating into the healing skin wound, such as neutrophils, monocytes, and fibroblasts. In addition, it enhances proliferation of fibroblasts and production of extracellular matrix by these cells. Finally, it stimulates fibroblasts to contract collagen matrices and induces the myofibroblast phenotype in these cells (56, 121). Thus it has long been suggested to be a major player in wound healing. Indeed, a series of experimental and clinical studies have demonstrated a beneficial effect of PDGF for the treatment of wound healing disorders (121). Furthermore, PDGF was the first growth factor to be approved for the treatment of human ulcers (80, 169).

A. Expression of PDGF at the Wound Site

In addition to its therapeutical potency, a series of studies suggest an important role of endogenous PDGF in the repair process. Upon injury, PDGF is released in large amounts from degranulating platelets (233), and it is present in wound fluid, particularly early after injury (35, 116, 173, 183, 204, 255, 275, 281). Furthermore, expression of PDGFs and their receptors has been demonstrated in various cells of murine, pig, and human wounds using in situ hybridization and immunohistochemistry (5, 6, 21, 223, 294). The patterns of PDGF and PDGF receptor expression suggest a paracrine mechanism of action, since the ligands are predominantly expressed in the epidermis, whereas the receptors are found in the dermis and the granulation tissue. Interestingly, expression of PDGFs and their receptors was reduced in wounds of healing-impaired genetically diabetic db/db mice and glucocorticoid-treated mice (19, 21), indicating that a certain expression level of PDGFs and their receptors is essential for normal repair. This hypothesis was supported by the finding that impaired wound healing in aged mice is associated with a delay in appearance of PDGF A and B isoforms, and α- and -receptors (10). Finally, the levels of PDGF in nonhealing human dermal ulcers were strongly reduced compared with surgically created acute wounds (216), further supporting an important role of PDGF for normal healing.

On the other hand, augmented PDGF production might be involved in the pathogenesis of hypertrophic scars and keloids as suggested by the potent effect of PDGF on fibroblast proliferation and extracellular matrix production by these cells (see above), the presence of enhanced levels of this growth factor in hypertrophic scar tissue (198), and the increased responsiveness of keloid fibroblasts to PDGF (114).

B. Inhibition of PDGF Action in Healing Skin Wounds

Based on its expression pattern in the healing wound and its known in vitro activities, PDGF has been suggested to have two major but distinct roles in wound repair: an early function to stimulate fibroblast proliferation and a later function to induce the myofibroblast phenotype (56). This hypothesis was supported by the finding that addition of neutralizing PDGF antibodies to human wound fluid caused a 45% reduction in the mitogenic effect of the wound fluid for cultured fibroblasts (143). However, a recent study demonstrated that the PDGF-B chain of hematopoietic origin is not necessary for granulation tissue formation and that its absence even enhances vascularization (42). In this study, the authors prepared hematopoietic chimeras, in which the hematopoietic system of a normal adult mouse was replaced by that of a PDGF B-chain -/- donor. In these chimeras the extent of local granulation tissue was not affected, and vascularization was increased. These findings suggest that the production of PDGF by other cell types in the wound is sufficient for normal healing. The use of neutralizing antibodies for wound healing studies or analysis of tissue-specific PDGF or PDGF receptor knock-out mice will help to further clarify the role of endogenous PDGF in wound repair.

III. FIBROBLAST GROWTH FACTOR FAMILY

FGFs comprise a growing family of structurally related polypeptide growth factors, currently consisting of 22 members (206). They transduce their signals through four high-affinity transmembrane protein tyrosine kinases, FGF receptors 1–4 (FGFR1–4) (138), which bind the different FGFs with different affinities. Additional complexity is achieved by alternative splicing in the extracellular domains of FGFR1–3, which dramatically affects their ligand binding specificities. Most FGFs bind to a specific subset of FGF receptors. FGF1, however, binds to all known receptors, and FGF7 specifically interacts with a splice variant of FGFR2, designated FGFR2IIIb (207). A chararacteristic feature of FGFs is their interaction with heparin or heparan sulfate proteoglycans, which stabilizes FGFs to thermal denaturation and proteolysis, and which strongly limits their diffusibility. Most importantly, the interaction with heparin or heparan sulfate proteoglycans is essential for the activation of the signaling receptors (205).

Most members of the FGF family have a broad mitogenic spectrum. They stimulate proliferation of various cells of mesodermal, ectodermal, and also endodermal origin. The only exception is FGF7 (keratinocyte growth factor, KGF), which seems to be specific for epithelial cells, at least in the adult organism (289). In addition to their mitogenic effects, FGFs also regulate migration and differentiation of their target cells, and some FGFs have been shown to be cytoprotective and to support cell survival under stress conditions (17, 206, 289).

Numerous in vivo effects of FGFs have been demonstrated, which suggest a role of these growth factors in wound repair. In particular, FGF1 and FGF2 were shown to stimulate angiogenesis in various assay systems (226). Furthermore, FGFs are mitogenic for several cell types present at the wound site, including fibroblasts and keratinocytes (1). Thus FGFs are clear candidates for contributing to the wound healing response, and this hypothesis has been corroborated by a number of studies where local application of FGF1, FGF2, FGF4, FGF7, or FGF10 stimulated tissue repair (1, 289).

A. Expression of FGFs in Healing Skin Wounds

Some FGFs have been detected at the wound site, indicating that the endogenous proteins are also regulators of wound healing. FGF2 was found in human and porcine wound fluid, particularly at early stages after injury (35, 50, 61, 106, 199, 281). Using immunohistochemistry, this FGF has been localized in injured skin. In a mouse incisional wound model, FGF2 was found extracellularly at the surface of the wound and within the dermis adjacent to the wound. Interestingly, this staining pattern was only seen in wounds of adult mice but not in fetal wounds where FGF2 immunoreactivity was undetectable. It was suggested that this difference could explain at least in part the reduced amount of capillary formation seen in the fetal versus adult wounds (294). In a full-thickness excisional wound model in mice, FGF2 was associated with hair bulbs at the wound edge and with basal keratinocytes of the normal and hyperproliferative wound epidermis (152). In rat burn wounds, FGF2 immunoreactivity was detected in the regenerated epidermis, in a bandlike zone near the regenerated epidermis, in renewed capillaries, and in cells infiltrating in the granulation tissue (145). Finally, a diffuse extracellular staining was seen at the edge of human burn wounds (101). The observed differences are probably due to species-specific differences or to different cross-reactivities of the antibodies with other members of the FGF family. To overcome this problem, two groups determined the expression of FGFs during wound healing at the mRNA level. Using in situ hybridization, Antoniades et al. (7) found upregulation of FGF1 and FGF2 expression in keratinocytes of porcine wound epidermis. Werner et al. (291) determined the mRNA levels of different FGFs in full-thickness excisional mouse wounds by RNase protection assay. Expression of FGF1, FGF2, FGF5, and FGF7 was found in normal and wounded skin, and expression of all these FGFs increased after skin injury. The most dramatic effect was seen with FGF7, which was more than 100-fold upregulated within 24 h after wounding. The strong upregulation of FGF7 expression was subsequently also confirmed for acute human wounds (172). In both mouse and human wounds, FGF7 mRNA was predominantly detected in dermal fibroblasts adjacent to the wound and in fibroblasts of the granulation tissue (172, 291). In addition, γδT cell receptor-bearing dendritic epidermal T cells (DETCs) were recently identified as a major source of FGF7 in murine skin wounds (134). Finally, FGF10 (KGF-2) was also shown to be expressed in mouse wounds (20, 134, 267), although upregulation of this type of FGF was only found in one study using RT-PCR (267), but not in another study where expression was determined by RNase protection assay (20). Similar to FGF7, FGF10 was predominantly expressed by DETCs (134) and fibroblasts (unpublished data).

In addition to the ligands, all FGF receptors are expressed in normal and wounded mouse skin (291; Werner, unpublished data). FGFR2IIIb, the only high-affinity receptor for FGF7, is expressed in keratinocytes of the normal and wounded epidermis as well as in hair follicles of murine, porcine, and human wounds (69, 172; Werner, unpublished data), and FGFR1 was found in the regenerating epidermis as well as in blood vessels of rat burn wounds (269).

Three different studies demonstrated a correlation between reduced FGF expression/responsiveness and wound healing disorders. Thus the mRNA levels of FGF1, FGF2, and FGF7 were reduced during wound healing in healing-impaired genetically diabetic mice compared with control mice (290). Furthermore, impaired would healing was seen in aged mice, and this impairment was associated with reduced levels of FGF2 and with a reduced angiogenic response in the skin of these mice upon addition of FGF2 (265). Finally, a member of the FGF family, most likely FGF2, was identified in a search for woundregulated proteins (250). Expression of this FGF was found to be upregulated after injury in normal but not in diabetic rats.

B. A Role for FGF2 in Wound Repair

To provide functional evidence for a role of FGF2 in wound repair, Broadley et al. (37) used a neutralizing polyclonal antibody that was raised against human FGF2. They incorporated the purified IgG into pellets, which were placed in the center of a polyvinyl alcohol sponge disk, and the disks were then implanted subcutaneously under ventral panniculus carnosus of rats. The continuous release of the antibody caused a striking reduction in cellularity and vascularization compared with the granulation tissue formed in the control IgG sponges. In addition, DNA, protein, and collagen levels in the anti-FGF2 sponges were reduced by ∼25–35% relative to control at day 7 after implantation. This study strongly suggested an important role of endogenous FGF2 in wound repair, although cross-reactivity of this antibody with other members of the FGF family could not be excluded. The role of FGF2 in wound repair was finally clarified when FGF2 null mice were used for wound healing studies. Interestingly, these mice appeared superficially indistinguishable from wild-type littermates. However, when they were challenged by full-thickness excisional wounding, they showed delayed healing (208). In addition to a retardation in the rate of reepithelialization, mice null for FGF2 showed reduced collagen deposition at the wound site, and they had thicker scabs. In contrast, no wound healing abnormalities were observed in FGF1 knock-out mice, and in FGF1/FGF2 double knock-out mice, the defects were similar in extent to those seen in the FGF2 null animals (188). These results demonstrate that FGF1 is dispensable for wound healing in mice.

C. FGF Receptor Signaling Is Important for Reepithelialization

In addition to FGF2, several studies have provided evidence for an important role of FGF7 and its receptor (FGFR2IIIb) in cutaneous wound repair. The strong upregulation of this FGF in fibroblasts and DETCs after skin injury and the expression of its receptor in keratinocytes (see above) suggested that FGF7 stimulates wound reepithelialization in a paracrine manner. To test this hypothesis, transgenic mice were generated that express a dominant-negative FGFR2IIIb mutant in the epidermis (292). The mutant receptor lacks a functional tyrosine kinase domain and, upon ligand binding, forms nonfunctional heterodimers with full-length wild-type receptors, thereby blocking signal transduction (278, 279). The truncated FGFR2IIIb is known to bind FGF7, FGF10, FGF1, FGF3, and, although with lower affinity, also FGF2 (130, 207). Therefore, it should inhibit the action of all these ligands. The skin of the animals expressing the dominant-negative receptor mutant was characterized by epidermal atrophy, disorganization of the epidermis, hair follicle abnormalities, and dermal hyperthickening (292). Histological analysis of full-thickness excisional wounds revealed a severe delay in wound reepithelialization in the transgenic mice compared with control littermates. At day 5 after injury, the number of proliferating keratinocytes in the hyperproliferative epithelium was 80–90% reduced compared with control mice. These results demonstrated an important role for FGF receptor signaling in wound repair, although the type of FGF that is responsible for this defect was not defined by this study.

D. FGF7-Deficient Mice Show No Defect in Wound Healing

To further determine the role of FGF7 in development and repair, Guo et al. (113) used embryonic stem cell technology to generate mice lacking FGF7. Their knock-out mice revealed no obvious defects, with the exception of the fur, which appeared matted and greasy, especially in male animals. Most surprisingly, the healing process of full-thickness incisional wounds was not obviously affected by the lack of FGF7, and the proliferation rate of the keratinocytes at the wound edge was not altered. These data demonstrate that incisional wounds can heal in the absence of FGF7. It would, however, be interesting to study the healing process of excisional wounds in these animals, since the extent of reepithelialization is much higher in excisional than in incisional wounds.

The lack of obvious phenotypic abnormalities in the FGF7 null mice is contradictory to the results obtained with the dominant-negative FGFR2IIIb mutant (see above). Although it might be possible that FGF7 is indeed not involved in reepithelialization of skin wounds, this seems unlikely, since the pattern of FGF7 expression correlates well with its postulated functions in normal and wounded skin. The most likely explanation for the discrepancies between the knock-out and the dominant-negative receptor results is a redundancy in ligand signaling. Although FGF7 might normally be the most important ligand of FGFR2IIIb in the skin, the lack of this gene could be compensated for by other known ligands of this receptor. More recent data suggest that FGF10 is the principal candidate for effecting this compensation, since it is also expressed in normal and wounded skin (20, 134, 267). Furthermore, mice lacking DETCs have a significant delay in wound reepithelialization, most likely due to the lack of DETC-derived FGF7 and FGF10 in the healing wound (134). Studies using neutralizing FGF7 and/or FGF10 antibodies during wound repair should help to further clarify the roles of FGF7 and FGF10 in the healing process. The tissue-specific knockout of FGFR2IIIb, as well as double knock-outs of different ligands of this receptor, will shed more light on the role of FGFR2IIIb and the various types of FGF in normal and wounded skin.

IV. EPIDERMAL GROWTH FACTOR FAMILY

The epidermal growth factor (EGF) family of mitogens comprises several members, including EGF, transforming growth factor-α (TGF-α), heparin-binding EGF (HB-EGF), amphiregulin, epiregulin, betacellulin, neuregulins, the recently discovered epigen, as well as proteins encoded by Vaccinia virus and other poxviruses (263, 276, 303). In addition, more distantly related proteins known as neuregulins (heregulins, neu differentiation factors, NDF 1–4) can also bind to some EGF receptor family members (303). All these growth factors exert their functions by binding to four different high-affinity receptors, EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4 (Fig. 2A). Upon ligand binding, these receptors form homo- or heterodimers (303). Overexpression of these receptors, in particular of HER2, is often found in human cancers and is likely to have a causative role in tumorigenesis. In addition, a series of experimental and clinical studies have demonstrated a positive effect of EGF, TGF-α, and HB-EGF on wound repair, suggesting that the endogenous growth factors are also involved in the healing process (107, 240, 259).

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FIG. 2. Epidermal growth factor (EGF; A) and vascular endothelial growth factor (VEGF; B) family members and their receptors. Upon ligand binding, receptors form homo- or heterodimers. Note the lack of a ligand for HER2 homodimers. However, this receptor binds the ligand of a partner upon heterodimerization.

 

A. Expression of EGF, TGF-α, and HB-EGF at the Wound Site

First evidence for a role of EGF receptor ligands in wound healing came from the analysis of wound fluid. Grotendorst et al. (111) detected EGF-like factors in wound fluid collected from rats. Acid extracts from this type of wound fluid contained a chemotactic activity for endothelial cells that was neutralized with anti-EGF antisera (111). In addition, substantial levels of EGF and TGF-α were found in wound fluid from skin graft donor site wounds in patients with small to moderatesized burn injuries (106). The result was confirmed for TGF-α in another study (204). However, this group detected only very low levels of EGF in the same type of wound fluid. Several publications report on the presence of HB-EGF in wound fluid. Thus this growth factor was shown to be present at high levels in human burn wound fluid (184). In addition, HB-EGF was identified as the major heparin-binding growth factor in wound fluid of porcine partial-thickness excisional wounds (173). Because HB-EGF is mitogenic for fibroblasts and keratinocytes, it was suggested to play an important role in reepithelialization and granulation tissue formation. Interestingly, it was shown to act synergistically with insulin-like growth factor (IGF) I, another growth factor present in wound fluid, in stimulating keratinocyte proliferation in vitro (174).

In a search for the cellular source of these EGFR ligands in wounds, Rappolee et al. (219) detected TGF-α mRNA in isolated wound macrophages. With the use of in situ hybridization and immunohistochemistry, this growth factor was also detected in eosinophils in a rabbit cutaneous open wound model and also in hamster wounds (272, 297). In addition, epidermal keratinocytes at the wound edge as well as hair follicle epithelial cells were identified as a source of TGF-α in partial-thickness murine burn wounds, in particular during the phase of keratinocyte proliferation (65). EGF immunoreactivity was found to be associated with the presence of wound inflammatory cells and wound fibroblasts in early rat CO₂ laser wounds (304). Finally, HB-EGF was localized in the advancing epithelial margin, islands of regenerating epithelium within human burn wounds, and in eccrine sweat glands (184). In another study, the same growth factor was detected in marginal surface keratinocytes and hair follicle epithelial cells of murine partial-thickness burn wounds, with maximal levels being found during the period of keratinocyte proliferation (65).

B. Expression of EGF Receptors at the Wound Site

EGF, TGF-α, and HB-EGF exert their function via binding to the EGFR, a transmembrane protein tyrosine kinase that is expressed on many different cell types. Consistent with the expression of the ligands at the wound site, EGFR mRNA and protein were also detected in healing wounds. With the use of enzyme-linked immunosorbent assay and histological methods, an increase in the number of immunoreactive receptors was found in a tape stripping wound model before an increase in epidermal thickness. This early increase was followed by a decline in EGFR levels, which was followed by a decline in epidermal thickness (262). This expression pattern suggested a role of the EGFR in reepithelialization of skin wounds. In early human full- and partial-thickness burn wounds, EGFR was detected in undifferentiated, marginal keratinocytes, in keratinocytes of the hyperproliferative wound epidermis and hair follicles, as well as in sweat ducts and sebaceous glands (288). At later stages after injury, immunoreactive EGFR was still detected in the hyperthickened wound epidermis and in all appendages, but was absent from leading epithelial margins (288). This expression pattern of the EGFR in human burn wounds provided further evidence for a role of EGFR signaling in reepithelialization. In addition, the observed delayed appearance of EGF and EGF receptors in incisional wounds of aged mice compared with young mice (10) further suggests a functional role of these proteins in the healing process.

C. Ectodomain Shedding of EGF Receptor Ligands Is Required for Keratinocyte Migration During Wound Healing

In addition to these correlative data, recent functional studies revealed an important role of EGFR ligands in wound repair. All EGFR ligands are synthesized as membrane-anchored forms, which are proteolytically processed to the bioactive soluble forms (180). Interestingly, the transmembrane forms are also able to stimulate the growth of adjacent cells in a juxtacrine manner, indicating that both transmembrane and soluble forms might play a role in wound healing. However, processed HB-EGF was detected in wound fluid (173), suggesting that ligand shedding could play an important role in wound healing. Indeed, in vitro scratch wounding of a keratinocyte monolayer induced shedding of EGFR ligands, particularly of HB-EGF. Shedding was inhibited by the compound OSU8–1, and this in turn suppressed keratinocyte migration. Most interestingly, the application of this compound to full-thickness mouse wounds caused a strong retardation of reepithelialization as a result of impaired keratinocyte migration. This inhibition was reversed by addition of recombinant soluble HB-EGF along with OSU8–1 (273). These results indicate an important role of EGFR ligand shedding for keratinocyte migration in vitro and in vivo.

D. Wound Healing in Mice Deficient in TGF-α

Based on the presence of TGF-α in wound fluid (see above), its strong upregulation early after injury (113), and the beneficial effect of exogenous TGF-α for wound healing, TGF-α was expected to play an important role in the repair process. To test this possibility, two groups generated mice lacking this growth factor (164, 171). Surprisingly, these mice appeared normal with the exception of eye abnormalities and waviness of whiskers and fur. The epidermis of these animals was indistinguishable from that of control mice. Most interestingly, no significant wound healing abnormalities were observed in these mice, whereby two different wound models (full-thickness back skin excisions and tail amputation) were used. However, one group observed more variability in the rate of wound closure in TGF-α-deficient mice (164), suggesting that the lack of this mitogen can be compensated for to a variable extent by other growth factors. Such compensation could be achieved by other EGFR ligands, in particular HB-EGF. This hypothesis is supported by the severe phenotypic abnormalities of mice lacking the EGF receptor (187, 252) and of transgenic mice expressing a dominant-negative EGF receptor in the epidermis (193), although the wound-healing process in these animals has not been analyzed yet. In contrast, the lack of TGF-α is unlikely to be compensated for exclusively by FGF7, since incisional wound healing also appeared normal in mice lacking both TGF-α and FGF7 (113).

Although these initial studies suggested that TGF-α is dispensable for wound healing, a more detailed analysis revealed a role of this factor in the early phase of reepithelialization (146). These investigators generated full-thickness head wounds and partial-thickness ear wounds in the TGF-α knock-out mice. In the ear model, where healing is mainly achieved by reepithelialization, the knock-out mice had significantly larger epithelial gaps compared with control animals at days 3 and 5 after injury, and the epithelial thickness was reduced at these time points. However, wounds of both genotypes were completely reepithelialized at day 8 postwounding. In contrast, head wounds that heal by reepithelialization and granulation tissue formation were indistinguishable in TGF-α null mice and control animals. These data suggest a role of TGF-α in the early phase of reepithelialization, but the lack of this factor is compensated if healing is accompanied by granulation tissue formation. These results demonstrate the importance of the chosen wound model for the analysis of growth factor function in wound repair.

E. A Role of Neu Differentiation Factor in Wound Repair?

In addition to the EGF receptor ligands, Neu differentiation factor (NDF) might also play a role in the regulation of wound repair. Thus recombinant NDF-α2 stimulated epidermal migration, epidermal thickness, and keratinocyte differentiation in a rabbit ear model of excisional wound repair (68). Endogenous NDF was found to be upregulated during the healing process of full-thickness excisional wounds, possibly as a response to increased levels of FGF7 and HGF which were found to be potent inducers of NDF expression in cultured keratinocytes (46). With the use of in situ hybridization, NDF α-isoforms were found to be expressed in dermal fibroblasts of wounded and unwounded rabbit ear skin. HER2 and HER3 receptors, which mediate the function of NDF, were expressed in unwounded epidermis and dermal adnexa. After injury, expression of HER2 decreased in the wound neoepidermis, while neoepidermal HER3 expression was strongly upregulated (68). These results suggest that NDF stimulates keratinocyte migration during cutaneous wound repair in a paracrine manner.

V. VASCULAR ENDOTHELIAL GROWTH FACTOR FAMILY

The VEGF family currently includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta growth factor (PLGF). They exert their biological functions by binding to three different transmembrane tyrosine kinase receptors, designated VEGFR-1, VEGFR-2, and VEGFR-3 (95; Fig. 2B). The biological functions of VEGF-A and its receptors VEGFR-1 and VEGFR-2 have been characterized in most detail. Based on a series of in vitro and in vivo studies, VEGF-A has been identified as a major regulator of vasculogenesis and angiogenesis during development (95), indicating that it might also be involved in the regulation of angiogenesis during wound healing.

A. Expression of VEGF-A and Its Receptors in Skin Wounds

In support for a role of VEGF-A in wound repair, expression of this gene was shown to be strongly induced after cutaneous injury, with keratinocytes and macrophages being the major producers (40, 90). In addition, its receptors were detected on blood vessels of the granulation tissue (153, 213). This expression pattern suggested that VEGF-A stimulates wound angiogenesis in a paracrine manner. The important role of VEGF-A for the healing process was supported in several studies where reduced expression of VEGF-A or its accelerated degradation were found to be associated with wound healing defects (90, 140, 153, 265). Furthermore, treatment of ischemic wounds with VEGF-A or VEGF-A-overexpressing fibroblasts accelerated the healing process (33, 55), and adenovirus-mediated VEGF-165 gene transfer enhanced wound healing in diabetic mice by promoting angiogenesis (232).

B. A Role for VEGF-A in Wound Angiogenesis

The important role of VEGF-A in wound healing was recently revealed in a study where application of neutralizing VEGF-A antibodies caused a striking reduction in wound angiogenesis, fluid accumulation, and granulation tissue formation in a pig wound model (124). Furthermore, the angiogenic activity present in human wound fluid from later time points after injury was strongly inhibited by VEGF neutralization (200). Finally, retroviral delivery of a dominant-negative VEGFR-2 to murine skin wounds caused a strong reduction in angiogenesis and granulation tissue formation (277). However, wound closure was not affected in these animals due to increased wound contraction. These findings support the important role of endogenous VEGF in wound angiogenesis, although functional VEGFR2 signaling is obviously not critical for normal closure of acute excisional wounds.

C. Lack of PLGF Results in Impaired Wound Angiogenesis

In addition to VEGF-A, PLGF was recently identified as a regulator of wound angiogenesis. Expression of PLGF mRNA and protein was strongly upregulated in migrating keratinocytes of acute human skin wounds. Furthermore, endothelial cells of capillaries adjacent to the wound expressed PLGF (84). This upregulation appears to be of functional importance, since PLGF knockout mice were characterized by impaired wound healing as a result of a defect in angiogenesis (45). Interestingly, a synergy between VEGF-A and PLGF was detected in these studies, indicating that the presence of both growth factors is important for normal wound angiogenesis.

D. Expression of VEGF-C and Its Receptor in Healing Skin Wounds

Besides the formation of new blood vessels, lymphangiogenesis occurs during the healing of skin wounds. Several groups have shown the formation of lymphatic vessels to be regulated via VEGFR-3 and its ligands VEGF-C and VEGF-D (142). In a recent study using a pig wound model, VEGFR-3-positive lymphatic vessels were found in the wound granulation tissue (209). These vessels appeared in the wound concurrently with blood vessels but regressed earlier. The responsible ligand is probably VEGF-C, which is expressed in normal and wounded mouse skin (unpublished data). Interestingly, a relative absence of lymphatic vessels was found in chronic human wounds (209), which might be one of the reasons for their impaired healing. Taken together, members of the VEGF family are likely to be major regulators of angiogenesis and lymphangiogenesis not only during development but also during cutaneous wound repair.

VI. ANGIOPOIETINS

In addition to the VEGFs, angiopoietins comprise a second family of growth factors acting on the vascular endothelium. Up to now, four different angiopoietins have been discovered that bind to a transmembrane tyrosine kinase receptor, Tie2, that is exclusively present on endothelial cells. Interestingly, angiopoietins-1 and -4 were identified as activators of this receptor, whereas angiopoietins-2 and -3 are likely to block the activity of this receptor under most circumstances. Unlike VEGFs, angiopoietins do not regulate endothelial cell proliferation; rather, angiopoietin-1 is responsible for the stabilization of blood vessels, whereas angiopoietin-2 causes vessel destabilization and remodeling (95).

A. Expression of Angiopoietins and Their Receptor in Healing Skin Wounds

First evidence for a role of angiopoietins in wound healing came from studies by Wong et al. (296), who demonstrated upregulation of Tie2 protein and mRNA in rat and mouse skin wounds, respectively. Moreover, Tie2 was found to be tyrosine-phosphorylated in the healing wound, indicating active downstream signaling. In addition to the receptor, two groups demonstrated expression of angiopoietins-1 and -2 in normal and wounded mouse skin. Whereas angiopoietin-1 expression was not affected by skin injury, angiopoietin-2 expression was transiently upregulated during the period of granulation tissue formation in normal mice (30, 140). In healing-impaired genetically diabetic mice, the period of angiopoietin-2 upregulation was extended (140). Thus wounds in diabetic mice are characterized by high levels of angiopoietin-2 but low levels of VEGF-A (90), a situation that has been suggested to lead to blood vessel regression during tumorigenesis (123). These findings suggest that the strongly impaired angiogenic response in diabetic animals could result from an imbalance in the levels of VEGF-A and angiopoietins.

VII. INSULIN-LIKE GROWTH FACTORS

IGF-I and IGF-II are potent stimulators of mitogenesis and survival of many different cells types, and they exert their functions in an autocrine, paracrine, or endocrine manner. Their actions are mediated through the type I IGF receptor, a tyrosine kinase that resembles the insulin receptor. In addition, IGF-II also binds to the IGF type II/mannose-6-phosphate receptor, which results in internalization and degradation of IGF-II (201). The availability of free IGF for interaction with the IGF-I receptor is modulated by six IGF-binding proteins (IGFBPs). In addition, IGFBPs have also been shown to have IGF-independent effects on cell growth (54). Several studies have revealed a beneficial effect of exogenous IGF-I on wound healing, in particular in combination with other growth factors (167). In addition, liposome-mediated IGF-I gene transfer improved the pathophysiology of a thermal injury (136). These findings suggested important activities of IGFs in the healing wound.

A. Expression of IGFs and Their Receptors in Skin Wounds

Several groups demonstrated expression of IGF-I and IGF-II in wounds of different species. Thus IGF-I was found in rat and porcine wound fluid (174, 230, 260), and minimal degradation of this protein was observed (231). In an attempt to localize IGFs and their receptors at the wound site, one group used a rat ear freeze-thaw injury model to study IGF-I expression by immunohistochemistry (135). In normal skin, only a few cells in the dermis and epidermis expressed this protein. However, all epidermal cells as well as macrophages and some other inflammatory cells were positive within 1–3 days after wounding. Others used an incisional wound model as well as a subcutaneous sponge implant model to determine expression of IGF-I and IGF-II in the wound (97). Interestingly, the mRNA levels of both IGFs increased significantly after injury in both models. Increased IGF-I mRNA levels but unaltered IGF-I receptor expression were observed in a rat wound model where steel wire mesh cylinders were implanted in the subcutaneous tissue of the back (260). Finally, in situ hybridization studies on porcine wounds revealed expression of IGF-I, IGF-I receptor, and IGF-II receptor mRNAs in epithelial cells of normal and wounded skin. In this study, however, no major differences between nonwounded and wounded skin were observed (7).

B. Impaired Wound Healing Is Associated With Abnormal Expression of IGFs and Their Receptors

Several studies suggest a role of the IGF system in the wound healing abnormalities associated with diabetes and glucocorticoid treatment. Thus one group found that streptozotocin-induced diabetes in rats caused a 42% reduction in wound fluid IGF-I levels (27). Others analyzed the expression of IGF-I and IGF-II during wound healing in normal and genetically diabetic mice (39). The normal induction of IGF-I mRNA expression was severely delayed and reduced in diabetic mice. Delayed induction was also seen for IGF-II, although peak concentrations of IGF-II mRNA were higher in diabetic compared with control mice. Consistent with the RNA data, a delayed appearance of the proteins was noted in diabetic animals. In another study, subcutaneously implanted polyvinyl sponges and stainless steel mesh chamber models were used to analyze the levels of IGF-I, IGF-I receptor, and IGFBP3 mRNAs in wound tissue of healing-impaired diabetic and glucocorticoid-treated rats (26). Interestingly, expression of all these genes was strongly reduced in the healing-impaired animals, further supporting the importance of the IGF system for normal healing. These findings are likely to be important for the pathogenesis of chronic human wounds, since IGF-I protein was absent in the basal layer of the epidermis and in fibroblasts of diabetic patients but not of healthy control patients. Furthermore, it was absent in the basal keratinocyte layer at the edge of human diabetic foot ulcers (28). Taken together, these studies suggest that reduced expression of IGFs and/or their receptors leads to impaired wound healing, although this hypothesis has yet to be confirmed by functional studies.

On the other hand, enhanced expression of IGF-I might lead to excessive scarring as suggested by the observed overexpression of IGF-I in postburn hypertrophic scar tissue compared with control skin. Because IGF-I was shown to increase the expression of the pro alpha 1(I) chain of type I procollagen and the pro alpha 1 (III) chain of type III procollagen in cultured dermal fibroblasts, these findings indicate a causative role of elevated IGF-I levels in the pathogenesis of hypertrophic scars (98).

VIII. SCATTER FACTORS

The family of scatter factors (SF), also known as plasminogen-related growth factors (PRGF), encompasses two members to date: hepatocyte growth factor (HGF)/SF, also called PRGF-1, and macrophage-stimulating protein (MSP), also called hepatocyte growth factor-like protein (HGFL) or SF2 or PRGF-2. They are both secreted as large inactive precursors, which are proteolytically cleaved to produce active, disulfide-linked heterodimers (59).

HGF was independently discovered as a powerful mitogen for hepatocytes and as a stimulator of dissociation of epithelial cells. Due to these features it was designated HGF or SF. It is predominantly produced by cells of mesenchymal origin and acts via a high-affinity transmembrane tyrosine kinase receptor (MET) on various cell types. In addition, heparan sulfate proteoglycans act as low-affinity receptors for HGF and allow accumulation of the ligand in the proximity of its target cells (59). Because HGF stimulates migration, proliferation, and matrix metalloproteinase production of keratinocytes (78, 182), as well as new blood vessel formation (43), it has been suggested to play a role in cutaneous wound repair.

MSP is a liver-derived serum protein that regulates proliferation and differentiation of various cell types. In the serum MSP is predominantly present in the inactive precursor form, whereas active MSP is only generated at the surface of its target cells. The latter express RON, the only known high-affinity receptor for this protein. It is present on many different cell types, including macrophages and keratinocytes, suggesting a function of MSP in wound repair (155, 253).

A. Overexpression of HGF Enhances Granulation Tissue Formation and Wound Angiogenesis

Expression of HGF and its receptor MET was found to be strongly upregulated in keratinocytes of the wound epidermis as well as in several cell types in the granulation tissue during the healing of excisional wounds in rats (63). This upregulation is likely to be of functional importance, since transgenic mice overexpressing HGF under the control of the metallothionein promoter were characterized by enhanced granulation tissue formation after full-thickness excisional wounding, and the number of blood vessels in the granulation tissue was strongly increased. This effect on wound angiogenesis seems to be at least partially mediated via VEGF-A, since the latter was overexpressed in these transgenic mice (274). In contrast, reepithelialization was obviously not affected by overexpression of HGF. These results revealed important activities of HGF during wound healing, although the role of the endogenous protein in the healing process remains to be determined.

B. Expression of MSP at the Wound Site

First evidence for a role of MSP in wound healing came from studies by Nanney et al. (195), who demonstrated the presence of MSP in wound exudates of burn patients. Interestingly, a large percentage of the wound exudate-derived MSP was found to be in the active form, and MSP was shown to be responsible for the stimulatory effect of wound exudate on macrophages. In the same study, a marked upregulation of RON expression was demonstrated in burn wound epidermis and accessory structure as well as on macrophages and capillaries of the granulation tissue (195). Because MSP stimulates macrophage pinocytosis and phagocytosis in vitro (253), this study suggested that MSP may enhance macrophage-dependent wound debridement. In another study, the localization of MSP and RON was determined in full-thickness excisional wounds in rats (63). MSP-positive cells were identified by immunofluorescence at the wound edge as well as in cells within the wounds, and some of them were shown to be monocytes. In addition, RON was detected in the granulation tissue, but not in the wound epidermis.

C. MSP Is Dispensable for Wound Repair

To determine the role of MSP in cutaneous wound repair, mice lacking the msp gene were generated (24). Although these animals were characterized by delayed macrophage activation, no macroscopic differences in the healing of incisional wounds were observed. However, it is still possible that these mice have subtle wound healing abnormalities that are only detectable upon histological and/or molecular analysis.

IX. NERVE GROWTH FACTOR

Nerve growth factor (NGF) is the prototype for the neurotrophin family of polypeptides, which are essential for the development and survival of certain sympathetic and sensory neurons in both the central and peripheral nervous systems (158). In addition, it plays a key role in the initiation and maintenance of inflammation in various organs. Thus it has been suggested that NGF is also involved in cutaneous wound repair. This hypothesis was supported by the observation that removal of the submandibular glands of mice retards the rate of contraction of skin wounds and that licking of wounds enhances contraction (128). Because NGF is present at high levels in saliva, this growth factor was thought to be responsible for this effect. Indeed, exogenous NGF was shown to accelerate wound healing in normal and healing-impaired diabetic mice (159, 181) and to promote the healing of pressure ulcers in humans (23).

A. Expression of NGF in Skin Wounds

A role of endogenous NGF in wound healing was further supported by studies of Constantinou et al. (60), who found a marked increase in NGF levels after wounding of neonatal but not of adult rats. Subsequently, a rise in serum NGF levels after generation of full-thickness wounds in mice was demonstrated, which was shown to be due to release of NGF from the salivary gland (181). In addition, NGF levels also increased at the wound site in the same wound model, and NGF mRNA was detected in newly formed epithelial cells at the wound edge and in granulation tissue fibroblasts (181). A particular high expression of NGF was found in myofibroblasts within the granulation tissue of rat wounds, with much higher levels being found in myofibroblasts of neonatal compared with adult animals (118).

B. Multiple Roles for NGF in Wound Healing?

Due to its potent effects on sensory nerves, the major function of NGF in the wound tissue appears to be the stimulation of nerve ingrowth. This hypothesis is supported by results obtained in an in vitro coculture model, which demonstrated a potent effect of adult rat dorsal foot skin on dorsal root ganglia neurite outgrowth. This function was blocked by neutralizing antibodies to NGF (224). However, the activity in wounded neonatal skin was not blocked by these antibodies, suggesting the presence of other factors in neonatal wounds that induce neurite outgrowth (224).

Because innervation has been shown to be essential for normal wound healing (117 and references therein), the stimulatory effect of NGF on the wound repair process is likely to be at least partially due to its effect on nerves. This might be of particular importance in diabetic patients who suffer from peripheral neuropathy, which often results in impaired wound healing. Indeed, NGF administration was shown to protect against experimental diabetic sensory neuropathy (8), and NGF depletion was found in keratinocytes in diabetic human skin (4), suggesting that NGF might be helpful for the treatment of diabetic foot ulcers.

In addition to its effect on nerves, NGF affects other cell types present in the healing skin wound. Thus NGF stimulates proliferation and inhibits apoptosis of keratinocytes in vitro (217), and enhances proliferation and adherence molecule expression on human dermal microvascular endothelial cells (220). Finally, a recent report demonstrated that NGF has a potent effect on fibroblast migration and increases α-smooth muscle actin expression and collagen gel contraction by these cells (186), indicating that NGF regulates various processes during cutaneous wound repair. Independent from its mechanisms of action, the presence of higher levels of NGF in neonatal compared with adult wounds suggests that this growth factor is at least partially responsible for the faster healing observed in neonatal animals.

X. TRANSFORMING GROWTH FACTOR-

The TGF- superfamily encompasses a diverse range of proteins, many of which play important roles during development, homeostasis, disease, and repair. The structurally related but functionally distinct mammalian members include TGF-1–3, bone morphogenetic proteins (BMPs), Mullerian inhibiting substance, nodals, inhibins, and activins (178). Their biological effects are mediated by heteromeric receptor complexes, which activate intracellular signaling cascades (282).

The three mammalian TGF- isoforms (TGF-1, –2, and –3) are synthesized as latent precursors, usually being secreted as a complex with latent TGF--binding protein, which is then removed extracellularly via proteolytic cleavage (227; Fig. 3). Active TGF-s then exert their biological functions via binding to a heteromeric receptor complex, consisting of one type I and one type II receptor, both of which are serine-threonine kinases. In addition, they bind with high affinity to a nonsignaling type III receptor, which functions mainly to present TGF- to the type II receptor (227; Fig. 3). The three TGF- isoforms have both distinct and overlapping functions. In vitro, they have been shown to be mitogenic for fibroblasts, but they inhibit proliferation of most other cells, including keratinocytes. Furthermore, TGF-s are very potent stimulators of the expression of extracellular matrix proteins and integrins (178, 179, 228). Thus they possess the properties expected of wound cytokines and indeed are among the most studied molecules in the wound healing scenario.

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FIG. 3. Activation of Smad proteins by transforming growth factor (TGF)- receptors. TGF- is first produced as an inactive precursor that binds to latency-associated protein (LAP). The latter is covalently bound to latent TGF- binding protein (LTBP). Upon activation, TGF- is either sequestered by extracellular binding proteins (decorin, fibromodulin) or it binds to a type III receptor that presents it to the signal-transducing receptors (type II and type I). Upon ligand binding, TGF- type II receptor recruits and phosphorylates the type I receptor. The latter subsequently binds and phosphorylates Smad2 and Smad3. Phosphorylated Smad2 and Smad3 bind to Smad4 and translocate to the nucleus where they bind to other transcription factors that confer specificity, leading to activation of target genes. Other signaling pathways that are also used by the TGF- receptor (282) are not included in the figure.

 

A. Expression of TGF- at the Wound Site

Immediately after wounding, TGF-1 is released in large amounts from platelets (13) (Fig. 4). This initial kick-start of active TGF-1 from platelets serves as a chemoattractant for neutrophils, macrophages, and fibroblasts, and these cell types further enhance TGF-1 levels in various cell types (Fig. 4). As well as active forms, latent TGF-s are also produced and sequestered within the wound matrix, allowing sustained release by proteolytic enzymes. This combination of different cellular sources and temporary storage ensures a continuous supply of TGF- throughout the repair process (228). Several publications report on the presence of TGF-s in wound fluid of different species (27, 35, 204, 281). Furthermore, expression of all three isoforms was detected in many different cell types during repair, with each isoform having a characteristic distribution in the wound tissue (91, 141, 157, 238, 270, 271). In most studies, a rapid induction of TGF-1 and –2 was observed, whereas an increase in TGF-3 expression was seen at later stages of repair. These results have been reviewed in detail (203). Interestingly, at least some of the TGF- present at the wound site was shown to be active as determined by a new in situ activity assay (301). In addition to the ligands, the type I and the type II TGF- receptors are present in various cell types within the healing wound (104, 238, 239).

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FIG. 4. Multiple functions of TGF- during wound healing. Upon local hemorrhage, TGF- is released in large amounts from platelets. In the healing wound, it is produced by leukocytes, macrophages, fibroblasts, and keratinocytes and acts on these cells to stimulate infiltration of inflammatory cells, fibroplasia, matrix deposition, and angiogenesis. In contrast, endogenous TGF- has been shown to inhibit reepithelialization.

 

On the basis of the expression pattern of TGF-s and their receptors in the healing skin wound and on the observed effects of exogenous TGF-, it has been suggested that TGF-s stimulate reepithelialization and granulation tissue formation. The effect of TGF- on reepithelialization appears paradoxical; its expression by keratinocytes after wounding together with the inhibitory effect of TGF- on keratinocyte proliferation in vitro and in vivo (58, 243) suggests TGF- as a negative regulator of reepithelialization. On the other hand, it also induces the expression of integrins necessary for keratinocyte migration across the fibronectin-rich provisional wound matrix (94, 305), and exogenous TGF- was shown to stimulate keratinocyte migration and wound reepithelialization (119, 305). However, treatment of hairless mouse ear wounds with neutralizing antibodies to TGF-1 and –2 suggested that the endogenous growth factors are not essential for reepithelialization and neovascularization in this healing model (82). Furthermore, treatment of porcine burn wounds with a synthetic TGF- antagonist accelerated wound reepithelialization (125). Most importantly, several results obtained with transgenic and knock-out mouse models revealed an inhibitory role of endogenous TGF-1 in wound reepithelialization (see below).

The presence of TGF- in the granulation tissue was expected to be important for efficient healing, since TGF- was shown to stimulate angiogenesis, fibroblast proliferation, myofibroblast differentiation, and matrix deposition (71, 228, 229). This hypothesis is supported by a series of studies in several animal models that demonstrated a beneficial effect of exogenous TGF- for wound repair, in terms of both the rate of healing and the strength of the healed wound (228). Complementary to these data are findings suggesting that aberrant expression of TGF-s is associated with the wound healing defect seen in glucocorticoid-treated (91) and aged mice (10) as well as in diabetic rats (27).

B. Neutralizing Antibodies to TGF-1 and –2 Reduce Scarring

Several studies support an important role of TGF-s in cutaneous scarring. First of all, a reduced and/or more transient expression of TGF-s and their receptors was observed in nonscarring fetal wounds compared with adult wounds (62, 177, 197, 264, 294). In addition, a strong and persistent expression of TGF-s and their receptors was detected in fibroblasts of human postburn hypertrophic scars (99, 239, 241, 283, 306), and overexpression of TGF-1 and –2 was found in keloid tissues and keloid-derived fibroblasts (154, 211). Finally, the activity of TGF- appears to be increased in scar tissue. Thus the expression of decorin, an extracellular matrix proteoglycan that inhibits TGF- bioactivity (Fig. 3), was downregulated in postburn hypertrophic scars (241). Fibromodulin, another TGF- binding protein (Fig. 3), was expressed at significantly higher levels in nonscarring fetal wounds compared with scarring wounds at later stages of gestation (256). On the other hand, decorin was shown to be downregulated in scar-free healing embryonic rat wounds compared with later wounds that develop a scar (18). Thus the role of decorin in the scarring response remains to be determined.

Interestingly, treatment of fetal wounds with different concentrations of TGF-1 caused marked scarring of these wounds, demonstrating a direct involvement of TGF-1 in cutaneous scarring (264). This finding was further supported by studies from Shah et al. (244, 245). In these experiments, incisional rat wounds were treated with neutralizing antibodies to TGF-1 or to a combination of TGF-1 and –2. This treatment caused a significant reduction in extracellular matrix deposition and subsequent scarring, suggesting that endogenous TGF-1 and –2 induce cutaneous scarring in adult animals. A reduced scarring response was also observed in mouse wounds that were topically treated with antisense TGF-1 oligodeoxynucleotides (51). Finally, topical application of a synthetic TGF- antagonist reduced scarring in porcine burn and excisional wounds as well as in rabbit skin excisions (125).

On the other hand, treatment of the same type of wounds with recombinant TGF-3 also inhibited scarring, indicating that this type of TGF- antagonizes the effect of the other TGF- isoforms (245). However, studies from other authors have yielded contradictory results concerning the effect of TGF-3 on connective tissue deposition. They demonstrated an increase in new dermal matrix by exogenous application of TGF-3 to wounds in age-impaired animal models (64). Independent of the effect of exogenous TGF-3 on the scarring response, the role of the endogenous protein in wound healing and scar formation remains to be determined.

Reflecting the importance of TGF- in wound repair, several groups have conducted wound healing studies on mice genetically modified such that they have either deficiency or gain of function at various levels of the TGF- signaling pathway.

C. TGF-1-Deficient Mice Show Severely Impaired Late-Stage Wound Repair

In the first study to use a knock-out approach to further clarify the role of the TGF-1 isoform in wound repair, Brown et al. (41) wounded transgenic mice deficient in TGF-1 due to a targeted disruption of the tgf-1 gene (151). These mice exhibit no obvious developmental abnormalities and appear phenotypically normal until, at ∼3 wk of age, they develop a severe wasting syndrome accompanied by a pronounced multifocal inflammatory response and tissue necrosis, resulting in multisystem organ failure and death. To overcome this problem, the animals were wounded at day 10 after birth.

Full-thickness excisional wounds healed almost normally for the first few days in the TGF-1-deficient mice. However, histological analysis of the wounds at day 10 after injury revealed a thinner, less vascular granulation tissue in the knock-out mice, which was dominated by a marked inflammatory cell infiltrate. Furthermore, decreased reepithelialization and collagen deposition were observed in mutant animals when compared with control mice (41). Superficially, this suggests that other TGF- isoforms or even different growth factors can compensate for the lack of TGF-1 in early wounds, but implies that TGF-1 plays a crucial role later in the repair process. Alternatively, maternal rescue of TGF-1 by transmission in the milk (156) might explain the lack of abnormalities in early wounds, with differences only becoming apparent as the mice are fully weaned and lack any maternal TGF-1. The lack of TGF-1 ultimately caused a severe inflammatory response in the wound, but since this was also seen in many other tissues it may not be of great significance to understanding the function of TGF-1 at the wound site. The defects in wound repair are likely to be a secondary effect, perhaps due also to the severe wasting syndrome observed in these mice. Malnutrition and weight loss have been associated with impaired wound healing (108), and the weight loss which accompanies the inflammatory response is also likely to exert an adverse effect on repair.

D. Immunosuppressive Approaches Allow the Study of TGF-1 Function in Adult Wounds

Two independent groups have used different approaches to dissect the TGF-1-dependent wound healing defects from the effects of severe inflammation. Although the studies used independently generated knock-out mice, the unwounded mice were reported to have essentially identical phenotypes (151, 251).

In a pharmacological approach, Koch et al. (149) used the immunosuppressive drug rapamycin to subdue the multifocal inflammatory disease phenotype seen in their TGF-1 null mice (251), extending their lifespan from <30 days to up to 60 days. This drug has no effect on repair in wild-type mice. Based on the observation that maternal TGF-1 protein is still present in wounds made to 10-day-old knock-out mice, they studied incisional wound repair in 30-day-old mice. At this age, immunohistochemistry revealed TGF-1 protein to be markedly reduced in wounds of knock-out mice, but the mice did not show any of the inflammatory foci characteristic of untreated TGF-1-null littermates. Wounds to TGF-1-null mice showed enhanced healing, with narrower, scabless wounds, less granulation tissue, and reduced collagen deposition. The rate of reepithelialization increased such that, 3 days postwounding, wounds in the knock-out mice were 90% covered with the neoepidermis. In contrast, only 22% of the wound surface was reepithelialized in control wounds, suggesting that endogenous TGF-1 is inhibitory to reepithelialization. However, these differences in repair come with the caveat that the unwounded skin of these TGF-1-null mice already shows clear differences compared with skin of wild-type littermates. Histological analysis revealed the epidermis, dermis, and panniculus carnosus of control mice to be 52, 58, and 48% thicker, respectively, compared with TGF-1 knock-out mice. Such differences could be at least partially responsible for the observed wound healing phenotype.

In a genetic approach, Crowe et al. (66) crossed TGF-1 null mice onto the immunodeficient Scid -/- background (66). Scid -/- mice lack T and B cells and, therefore, do not have the machinery to mount the large inflammatory response seen in nonimmunocompromised TGF-1-null mice (41). This enabled excisional wound healing experiments to be performed on mice of 8–10 wk of age. In contrast to what was predicted, the absence of inflammation in TGF-1 -/- Scid -/- mice resulted in a remarkable delay in repair, delaying all of the major phases by at least a week compared with TGF-1 +/+ Scid -/- controls. The wounds of TGF-1 -/- Scid -/- mice had still not fully repaired by 21 days postwounding, in contrast to the controls, 100% of which were fully healed by 16 days. This delay was not singly due to either the lack of TGF-1 or the lack of lymphocytes, but to the combination of the two. This suggests that TGF-1 and lymphocytes may affect compensatory pathways during repair. Alternatively, the delay may be a side effect of absence of TGF-1 in wounds leading to delayed expression of the other two TGF- isoforms, TGF-2 and –3. Although unable to distinguish between which of these hypotheses may be true, this study presents a valuable method for bypassing a knock-out phenotype that would otherwise mask a defect in wound repair.

E. TGF-1 Overexpression Studies Yield Contrasting Results, Dependent on the Transgenic Strategy

In contrast to the knock-out approaches described above, two groups have investigated the effect of excess levels of TGF-1 on wound repair. Shah et al. (246) began with the hypothesis that elevated levels of circulating TGF-1 would accelerate healing but also enhance scarring. Mice with elevated plasma levels of active TGF-1 were generated by overexpressing a constitutively active porcine TGF-1 mutant in the liver under the control of the mouse albumin promoter. Using a dorsal incisional wound model, complemented by ventral subcutaneous implantation of PVA sponges, they were able to study both normal cutaneous wound repair and cellular infiltration as a model of granulation tissue formation.

Surprisingly, they found that, while the PVA sponges yielded the expected results, with increased cellularity, granulation tissue formation and collagen deposition in transgenic animals, local TGF-1 levels were lower in the incisional wounds of transgenic mice than in their control littermates. As such, the data show that increased circulating levels of TGF-1 do not necessarily lead to increased levels of TGF-1 at the wound site. Concomitant with the decreased TGF-1 level in the wounds of transgenic mice, they observed an increase in levels of TGF-3 and type II TGF- receptor at the wound site, and this might be the reason for the improved neodermal architecture in the healed wounds of the transgenic mice.

In a different approach, Yang et al. (300) generated mice constitutively overexpressing latent human TGF-1 in the epidermis under the control of the human keratin 14 promoter (300). They showed increased levels of latent TGF-1 protein in unwounded keratinocytes, as well as a dramatic increase in both latent and active TGF-1 following wounding. A CO₂ laser wounding model was used to generate partial thickness dorsal burns, ablating cells down to the adipose tissue but not damaging the underlying musculature. In wild-type mice, such wounds normally (92%) complete reepithelialization within 12 days, but in hemizygous and homozygous TGF-1 overexpressing mice only 42 and 25% of wounds, respectively, had healed at this time point. Quantitative studies revealed transgenic mice to have significantly higher levels of active TGF-1 at the wound site, in contrast to the systemically overexpressing mice discussed above (246). The major effect of the excess TGF-1 was to inhibit keratinocyte proliferation, hence the delayed reepithelialization, although it also acted in a paracrine fashion to increase the expression of type I collagen mRNA and hydroxyproline in transgenic wounds. Similar findings were obtained when these mice were subjected to full-thickness excisional wounding (47). The different results obtained in the Yang/Chan studies on the one hand and the Shah study on the other hand may be due in part to the stability of latent TGF-1 (half-life 100 min) relative to active TGF-1 (half-life 2–3 min), as well as to the more specific targeting of transgene expression to the site of injury.

F. Mice Expressing a Dominant-Negative Type II TGF- Receptor in the Epidermis Show Accelerated Reepithelialization and Reduced Keratinocyte Apoptosis

Rather than adopting a ligand-based approach to understanding the role of TGF- at the wound site, Amendt and colleagues (2, 3) chose to target the type II TGF- receptor by overexpressing a dominant-negative human type II TGF- receptor in the basal layer of the epidermis of transgenic mice using a keratin 5 promoter. The dominant-negative receptor lacks most of the intracellular domain, including the kinase domain, and upon dimerization blocks signaling by wild-type receptors (2). This approach blocks the action of all TGF- isoforms in basal keratinocytes. Excisional wounds to hemizygous transgenic mice showed an enhanced rate of reepithelialization, characterized by increased proliferation (between 50–100% higher dependent on the transgenic line) and decreased apoptosis (∼50% lower) in keratinocytes at the wound edge. These data fit well with the study discussed below (12), where abrogation of the TGF- downstream signaling pathway led to enhanced cutaneous repair.

G. Impaired Wound Healing in Mice Lacking the TGF- Type II Receptor in Fibroblasts

To determine the role of TGF- receptor signaling in fibroblasts for cutaneous wound repair, Bhownick et al. (25) developed a mouse model that exhibits a conditional knock-out of the TGF- type II receptor in fibroblastic cells. Mice carrying two floxed TGF- receptor type II alleles were crossed with animals expressing Cre recombinase under the control of the promoter of the fibroblast specific protein 1 (FSP-1). The latter is expressed in the mesenchymal cells of fibroblastic origin beginning embryonic day 9. When these mice were challenged by excisional or incisional wounding, wound closure and keratinocyte organization were unaffected. However, the number of suprabasal keratinocytes was increased in the remodeled excisional wounds of the mutant mice compared with control littermates. Most importantly, the tensile strength of the wounds was severely reduced 7 days after wounding compared with control littermates, demonstrating the importance of TGF- for granulation tissue formation during wound healing (25).

H. Accelerated Cutaneous Wound Healing With an Increased Rate of Reepithelialization and Reduced Inflammation in Smad3-Null Mice

Downstream of receptor activation, TGF-s and activin, both of which regulate key cellular functions during cutaneous wound repair, are known to activate different signaling pathways (282). One of the major pathways uses the transcriptional regulators Smad2 and Smad3 (11, 70, 179; Fig. 3). These signaling proteins are recruited to ligand-bound TGF- and activin receptor complexes, where they are phosphorylated by the type I receptor. The phosphorylated Smads 2 and 3 undergo a conformational change, which allows them to bind to cytoplasmic Smad4, shuttle to the nucleus, and activate their downstream targets (52; Fig. 3).

In contrast to Smad2 null mice, which die during embryogenesis (287), mice lacking functional Smad3 survive into adulthood (302). Following full-thickness incisional wounding, Smad3-null mice show accelerated healing, characterized by an increased rate of reepithelialization and reduced inflammation (12). In addition to neutrophils and monocytes being almost absent in the Smad3 knock-out wounds, granulation tissue formation was dramatically reduced, and there was an overall decrease in the wound area. Wounds of Smad3 knock-out mice also showed significantly lower levels of TGF-1 expression, likely due to the decreased monocyte concentration, since these cells are a key source of TGF-1 in the early wound.

To determine whether the lack of TGF- was a cause of rather than effect of the lack of inflammatory response, exogenous TGF-1 was applied to the wounds of control and Smad3-null mice. While this treatment resulted in an augmented neutrophil infiltration into the wounds of control mice, it failed to rescue the inflammatory response in Smad3-null animals, indicating that Smad3 signaling may underpin TGF-1-mediated inflammatory cell chemotaxis. Contrastingly, exogenous TGF-1 did rescue the granulation tissue phenotype, resulting in a stimulation of matrix production in the wounds of Smad3-null mice, although the fibroblast numbers were not increased. Thus TGF-1-dependent matrix deposition seems to function in a Smad3-independent fashion in these mice, in agreement with previous studies that revealed an involvement of c-Jun in the TGF--mediated fibronectin expression (122).

In summary, these data suggest that Smad3 signaling plays an inhibitory role during wound repair, since its abrogation leads to enhanced reepithelialization and contraction of wounds, at least in an incisional wound healing scenario.

Using the same mice in a study to determine the role of TGF- signaling in the response to ionizing radiation, the same laboratory found that Smad3 signaling was responsible for the skin injury resulting from a single dose of 30–50 GΩ of γ-irradiation (89). Radiation-induced fibrosis shows several similarities to the fibrosis that results after repair of cutaneous wounds in the adult; there is an extensive infiltration of inflammatory cells, dermal fibroblasts misexpress α-smooth muscle actin, fibrous extracellular matrix is aberrantly deposited, and TGF- is implicated in its pathogenesis (175). Additionally, the epidermis becomes hyperthickened. Analysis of skin biopsies taken 6 wk postwounding revealed Smad3-null mice to have reduced inflammation (∼50% cell number), to express lower levels of TGF-, and to have 40% less blood vessels and myofibroblasts compared with wild-type mice. Thus Smad3-null animals seem to be largely protected from the cutaneous fibrosis caused by radiation injury.

XI. ACTIVINS

Activins are members of the TGF- superfamily, which regulate various aspects of cell growth and differentiation in many tissues and organs. They are dimeric proteins, consisting of two _A subunits (activin A), two _B subunits (activin B), or a _A and a _B subunit (activin AB). In addition, _C, _D, and _E subunits have been identified, although little is as yet known about the corresponding proteins. The biological functions of activins are mediated by two type I and two type II receptors that bind activin with high affinity. In addition to these transmembrane serine/threonine kinase signaling receptors, the biological activities of activin are also regulated by follistatin and follistatin-related protein, soluble activin-binding glyco-proteins, which inhibit activin function in vitro and in vivo (178, 215).

A. Increased Expression of Activin After Skin Injury

First evidence for a role of activin in wound healing came from studies of Hübner et al. (127). In these experiments, full-thickness excisional wounds on mouse back skin were analyzed for the expression of activins at different time points after injury. Most remarkably, expression of the activin _A and to a lesser extent of the _B subunit was strongly induced within 24 h after injury and remained high until the repair process was completed. Follistatin, follistatin-related protein, as well as the activin receptors were also expressed in normal and wounded skin, but their levels were not affected by skin injury (127, 285). In situ hybridization studies revealed that activin _A mRNA was predominantly expressed in the granulation tissue adjacent to the hyperproliferative epithelium and below the eschar, whereas highest levels of activin _B mRNA were detected in suprabasal keratinocytes of the hyperproliferative epithelium at the wound edge and in the migrating epithelial tongue (127). The upregulation of activin expression is likely to be important for normal wound repair, since the severe delay in wound healing observed after cyclosporin A treatment of rats was associated with a strong downregulation of activin A expression in granulation tissue fibroblasts (214).

B. Overexpression of Activin in the Epidermis of Trangenic Mice Enhances Wound Repair and Scarring

To gain insight into the function of activin in wound repair, transgenic mice that overexpress the activin A subunit specifically in the epidermis were generated (192). The skin of these animals was characterized by epidermal hyperthickening and dermal fibrosis. The latter effect is most likely due to diffusion of activin from the epidermis to the mesenchyme and suggests a role of the protein in fibrotic processes. The epidermal hyperthickening in transgenic mice was reminiscent of the phenotype seen in hyperproliferative human skin disease. Indeed, a two- to threefold increased proliferation rate of the epidermal keratinocytes of the transgenic mice was observed. This effect of activin on keratinocyte proliferation in vitro is probably indirect, since activin was shown to inhibit proliferation of human keratinocytes (242, 247). Thus activin might induce the expression of growth factors in dermal fibroblasts, which stimulate keratinocyte proliferation in a paracrine manner. In addition, the differentiation pattern of the epidermal keratinocytes was affected.

Analysis of full-thickness excisional wounds in these mice revealed a remarkable increase in granulation tissue, with a higher cell density and an enhanced deposition of extracellular matrix, compared with wild-type mice. The latter effect appears to be at least partially due to an earlier induction of fibronectin and tenascin-C expression in the wounds of activin overexpressing mice. In contrast, collagen type I expression was similar in normal and transgenic mice, indicating that the effects of activin on the synthesis of extracellular matrix proteins are selective, whereas TGF- seems to stimulate the synthesis of extracellular matrix in a more general manner (229).

C. Impaired Wound Healing in Transgenic Mice Overexpressing the Activin Antagonist Follistatin in the Epidermis

The results obtained with the activin-overexpressing mice demonstrated novel activities of activin in the regulation of the healing process. However, they do not allow conclusions regarding the roles of endogenous activin in wound healing. To address this question, Wankell et al. (286) overexpressed the soluble activin antagonist follistatin in the epidermis of transgenic mice (286). The skin of these animals was characterized by a mild dermal and epidermal atrophy. After injury, a severe delay in wound healing was observed. In particular, granulation tissue formation was strongly reduced, leading to a major reduction in wound breaking strength. The wounds, however, finally healed, and the resulting scar area was smaller compared with controls (286; Werner, unpublished data). These results are complementary to the results obtained with activin overexpressing mice and thus provide first evidence for an important function of endogenous activin in the control of wound repair and scar formation.

XII. BONE MORPHOGENETIC PROTEINS

In addition to TGF-s and activins, BMPs have also been suggested to play a role in wound repair. Fifteen BMPs have as yet been identified which exert their functions by binding to heteromeric receptor complexes of a type II receptor and two different type I receptors (189).

A. Expression of BMPs at the Wound Site

BMP-2, BMP-4, and BMP-7 are expressed in normal and wounded adult mouse skin, although their expression is not regulated by skin injury (286). The sites of expression of these proteins in wounded skin and their roles in wound repair have as yet not been determined, but exogenous BMP-2 induced massive dermal and epidermal growth in fetal wounds of lambs and an adultlike pattern of scar formation (261).

In contrast to other BMPs, the expression of BMP-6 in healing skin wounds has been well documented. It is highly expressed in the regenerating epidermis at the wound edge as well as in fibroblasts of the granulation tissue. After completion of wound closure, BMP-6 accumulated throughout the suprabasal layers of the newly formed epidermis (139). This localization suggested a role of BMP-6 in the inhibition of keratinocyte proliferation and/or induction of differentiation, a hypothesis which is supported by the finding that BMP-6 induces keratinocyte differentiation in vitro (77, 185).

B. Delayed Reepithelialization in Transgenic Mice Overexpressing BMP-6 in the Epidermis

To determine the activities of BMP-6 in the skin, Blessing et al. (29) generated transgenic mouse lines overexpressing this protein in the suprabasal layers of the epidermis. Interestingly, strong and uniform expression of the BMP-6 transgene inhibited cell proliferation but had little effect on differentiation, whereas weak and patchy expression resulted in keratinocyte hyperproliferation and in a psoriasis-like phenotype. Most importantly, reepithelialization was significantly delayed in the transgenic mice that overexpress low levels of BMP-6 in the epidermis (139), suggesting that this protein inhibits keratinocyte proliferation in wounded skin and is necessary for the reestablishment of a fully differentiated epidermis. Wound healing studies with BMP-6-deficient mice (254) will help to determine whether the endogenous protein indeed fulfills this function.

XIII. CONNECTIVE TISSUE GROWTH FACTOR/CYSTEINE-RICH 61/NEPHROBLASTOMA OVEREXPRESSED (CNN) FAMILY

The CNN family comprises as yet six different members, including connective tissue growth factor (CTGF), cysteine-rich 61 (cyr61), nephroblastoma overexpressed (nov), WISP-1, WISP-2, and WISP-3. They are secreted proteins that contain 38 conserved cysteine residues that are organized into 4 distinct structural modules. Members of this family appear to be involved in embryonic development, differentiation, as well as pathological processes (36). In addition, CTGF and cyr61 have been suggested to play a role in wound repair. CTGF is expressed in many different tissues and organs and stimulates proliferation and chemotaxis of fibroblasts directly (31). Most interestingly, it is a potent inducer of extracellular matrix proteins, such as collagen type I and fibronectin and their integrin receptors (93), and it acts as a mediator of TGF-1 in these processes (150). Due to this function and to the fact that it is overexpressed in various types of fibrotic disease, CTGF has been suggested to be a major player in the pathogenesis of fibrotic processes (36).

A. Expression of CTGF in Skin Wounds

First evidence for a role of CTGF in cutaneous wound repair came from studies by Igarashi et al. (129). Using a rat wound model consisting of a subcutaneously implanted stainless steel mesh chamber, they demonstrated the presence of CTGF mRNA in wounded but not in normal skin. Highest levels of CTGF transcripts were observed at day 9 after injury that coincides with the initial ingrowth of granulation tissue (129). In another study CTGF mRNA levels were analyzed in full-thickness excisional mouse wounds in mice. In this model, CTGF mRNA was most abundant at day 1 after injury and declined to basal levels within the next 5 days (67). Due to the potent effect of CTGF on fibroblast proliferation and matrix deposition by these cells, the upregulation of CTGF expression after injury is likely to be important for granulation tissue formation and subsequent scar formation. In addition, it was recently demonstrated that CTGF promotes endothelial proliferation, migration, survival, and adhesion in vitro and angiogenesis in vivo (14, 248), suggesting that this protein might also be involved in wound angiogenesis.

B. Expression of Cyr61 in Skin Wounds

In addition to CTGF, Cyr61 is likely to play a role in cutaneous wound healing. Cyr61 was shown to promote chemotaxis of fibroblasts and to enhance the mitogenic effect of other growth factors for these cells (147). Furthermore, it was identified as an angiogenic inducer in vivo (15). To determine the expression pattern of the cyr61 gene in healing skin wounds, the expression of this gene was examined in full-thickness incisional wounds of transgenic mice that express the bacterial lacZ gene encoding -galactosidase under the control of the endogenous cyr61 gene promoter (49). These studies revealed a strong expression of Cyr61 in dermal fibroblasts of the granulation tissue. In vitro, Cyr61 activated a genetic program for wound repair in cultured skin fibroblasts, indicating that Cyr61 regulates inflammation, angiogenesis, cell-matrix interactions, and matrix remodeling after skin injury (49).

XIV. CHEMOKINES

In addition to the “classical” growth factors, several cytokines have been shown to play important roles in wound repair. Cytokines are small, secreted proteins that affect the behavior of immune cells but also of other cells. They include the interleukins, lymphokines, and several related signaling molecules such as tumor necrosis factor-α (TNF-α) and interferons. Chemokines (chemotactic cytokines) are a subset of small cytokines that stimulate chemotaxis and extravasation of leukocytes. This large protein family with nearly 50 members in the human system is subdivided into four subfamilies, α-(CXC-) and -(CC-) chemokines, which include most of the chemokines, and two additional subfamilies, the CX3C chemokines and C-chemokines with only one or two members each. Chemokines exert their functions via binding to G protein-coupled receptors on the surface of target cells, the CXC-receptors and the CC-receptors that only recognize chemokines of the corresponding subfamily (16, 53, 234). Recent studies have provided evidence for an important role of chemokines in the recruitment of inflammatory cells to the wound site. In addition, the presence of chemokine receptors on resident cells suggests that chemokines also contribute to the regulation of reepithelialization, tissue remodeling, and angiogenesis. Expression of a wide variety of different chemokines has been detected at the wound site, and these results have been reviewed in detail (103). Here we only report on those chemokines for which functional wound healing data are available.

A. A Role for Macrophage Chemoattractant Protein in the Regulation of Inflammation, Granulation Tissue Formation, and Reepithelialization

The CC chemokine macrophage chemoattractant protein (MCP-1/CCL2) is one of the major chemoattractants for monocytes/macrophages, and it also acts on a subset of T cells and on mast cells carrying the CCR3 receptor (16, 53). In a murine excisional wound model, mRNA encoding the murine MCP-1/CCL2 homolog JE was found at high levels between 6 and 24 h after wounding and the levels subsequently declined (75, 133, 293). By in situ hybridization, JE transcripts were predominantly found in monocytic and macrophage-like cells, as well as in a few fibroblasts and other interstitial cells (75). In two other studies, keratinocytes of the hyperproliferative wound epidermis were identified as the major source of MCP-1/CCL2 mRNA or protein in the wound (133, 293). Consistent with the data obtained in the mouse, MCP-1/CCL2 mRNA was found in keratinocytes of early human burn wounds as well as in human excisional wounds. In addition, some endothelial cells and inflammatory cells in the granulation tissue expressed this chemokine (81, 100).

In all studies, the time course of MCP-1/CCL2 expression correlated well with macrophage infiltration, suggesting a role of MCP-1/CCL2 in the recruitment of these cells during wound healing (75). In addition, it might attract T cells and mast cells to the wound site. Most interestingly, the prolonged persistence of neutrophils and macrophages in the wounds of healing-impaired diabetic db/db mice correlated with a large and sustained induction of MCP-1/CCL2/JE. Treatment of wounds from these mice with neutralizing antibodies to MCP-1/CCL2/JE and macrophage inflammatory protein (MIP-2) caused a reduction in the number of neutrophils and macrophages at the wound site, suggesting a direct involvement of these chemokines in the late infiltration of inflammatory cells into db/db wounds (293).

To determine the role of MCP-1/CCL2 for normal repair, mouse wounds were treated with either MCP-1/CCL2 or neutralizing antibodies to this chemokine (76). Treatment with MCP-1/CCL2 resulted in a substantial increase in the number of macrophages that was accompanied by a slight increase in wound-breaking strength. On the other hand, treatment with neutralizing antibodies reduced the number of macrophages at the wound site. In contrast to these results, the number of wound macrophages was not altered in wounds of MCP-1/CCL2/JE knock-out mice (163). These differences between the neutralizing antibody studies and the data obtained with the knock-out mice might either be due to cross-reactivity of the antibody with other chemokines or to compensatory upregulation of other chemokines in the knock-out mice. Although the number of macrophages was not altered, MCP-1/CCL2/JE knock-out animals were characterized by significantly delayed wound reepithelialization and angiogenesis, and collagen synthesis was also reduced in these mice (163). These data revealed an important role of MCP-1/CCL2 in wound repair, probably by influencing gene expression/protein synthesis in murine macrophages. However, in humans, MCP-1/CCL2 seems to be mainly involved in macrophage trafficking rather than in the regulation of growth factor production by these cells, since stimulation of human macrophages with MCP-1/CCL2 did not induce expression of major growth factors (81).

B. Macrophage Inflammatory Protein 1α: A Chemoattractant for Macrophages in the Healing Wound?

Another important monocyte chemoattractant is MIP-1α/CCL3. Both MIP-1α/CCL3 mRNA and protein were detectable in mouse wounds between 12 h and 5 days after injury, with levels peaking at day 1 after wounding. This time point correlates with maximum macrophage infiltration. Treatment of mice with a neutralizing antiserum to this chemokine before injury resulted in a reduced number of macrophages at the wound site, followed by reduced collagen production (74). Whereas this study suggested an important role of MIP-1α/CCL3 in wound repair, analysis of wounds in MIP-1α/CCL3 knockout mice revealed no obvious defect in either reepithelialization, angiogenesis, or collagen production (163). In contrast to the murine situation, MIP-1α/CCL3 was not detected at significant levels in acute human incisional wounds (81), indicating species-specific differences in the expression and function of this chemokine in wound repair.

C. Growth-Related Oncogene-α Regulates Macrophage Infiltration Into Healing Wounds

GRO-α/CXCL1 and its possible murine homolog macrophage inflammatory protein 2 (MIP-2) are potent regulators of neutrophil chemotaxis (53). The involvement of GRO-α/CXCL1 in wound healing was first suggested by its detection in inflammatory cells at day 3 after injury in a model of wound healing using wound chambers (83). In acute human excisional wounds, GRO-α/CXCL1 mRNA was found at highest levels at day 1 after wounding in the superficial wound bed and the underlying provisional matrix, and its expression was spatially and temporally associated with neutrophil infiltration. Furthermore, its expression profile correlated with keratinocyte migration and with neovascularization (81). In human burn wounds, GRO-α/CXCL1 immunoreactivity was detected in suprabasal layers of the wound epidermis as well as in the granulation tissue and the overlying exudate (194). The presence of GRO-α/CXCL1 in human burn wounds was confirmed by Rennekampf et al. (222), who detected high levels of this protein in donor site wound fluids from days 1 to 5 after injury (222). In vitro experiments revealed a strong mitogenic activity of this chemokine for keratinocytes, suggesting its involvement in reepithelialization. This hypothesis was supported by the observed stimulatory effect of GRO-α/CXCL1 on reepithelialization of meshed split-thickness human skin grafts on athymic mice. In contrast, wound contraction was significantly reduced by GRO-α/CXCL1 (222).

In a mouse excisional wound model, expression of MIP-2 was found to be upregulated between days 1 and 5 after injury in normal mice. Immunohistochemistry revealed the presence of MIP-2 in keratinocytes of the outer root sheath of hair follicles adjacent to the wound, but not in the granulation tissue (293). Similar to MCP-1/CCL2, the upregulation of MIP-2 expression was prolonged in healing-impaired db/db mice. Treatment of wounds from these animals with neutralizing antibodies to MCP-1/CCL2 and MIP-2 caused a reduction in the number of neutrophils and macrophages at the wound site, suggesting a direct involvement of these chemokines in the late infiltration of inflammatory cells into db/db wounds (293).

D. Interleukin-8 Stimulates Inflammation but Inhibits Wound Contraction

Interleukin (IL)-8/CXCL8, for which a murine homolog has not yet been identified, is also expressed in healing skin wounds. In acute human excisional wounds, it is coexpressed with GRO-α/CXCL1 at day 1 after injury in the superficial wound bed, and its expression declines at day 4 after wounding (81). In addition, IL-8/CXCL8 was found to be the major bioactive chemoattractant for neutrophils in human blister and skin graft donor site wound fluids (221). When human adult or fetal skin was placed subcutaneously in the SCID mouse and subsequently wounded, expression of IL-8/CXCL8 increased within 4 h after injury in fetal and adult wounds. However, by 12 h, no IL-8/CXCL8 mRNA was detected in fetal wounds, whereas expression of this chemokine persisted for up to 72 h in adult wounds. These results suggest that a reduced inflammatory cytokine response in fetal tissue may be responsible for the lack of inflammation in fetal wound healing that may contribute to scarless wound repair (161).

Studies from Iocono et al. (132) suggest that high levels of IL-8/CXCL8 are associated with impaired wound repair. Thus the levels of this chemokine were increased significantly in nonhealing human thermal wounds compared with healing wounds or normal skin. In vitro studies demonstrated an inhibitory effect of IL-8/CXCL8 on keratinocyte proliferation and collagen lattice contraction by fibroblasts, suggesting that elevated levels of this chemokine may directly contribute to retarded wound repair (132). In contrast to these studies, others found a stimulatory effect of IL-8/CXCL8 on keratinocyte proliferation in vitro (221). In addition, in vivo topical application of this chemokine on human skin grafts in a chimeric mouse model stimulated reepithelialization as a result of increased keratinocyte proliferation. Consistent with the observed effect of IL-8/CXCL8 on collagen lattice contraction (see above), wound contraction was diminished by topical application of IL-8/CXCL8 (221).

E. Impaired Wound Healing in CXCR2 Knock-out Mice

The effects of GRO-α/CXCL1/MIP-2 and several other chemokines are mediated, at least in part, by CXCR2 receptors that are expressed on keratinocytes, neovascularizing endothelial cells, and neutrophils. To determine the role of this receptor in wound repair, full-thickness excisional punch biopsy wounds were made to mice lacking CXCR2 (73). After wounding, these mice exhibited a defective neutrophil recruitment, delayed monocyte recruitment, and decreased secretion of the proinflammatory cytokine IL-1. Histologically, they also showed a severe delay in reepithelialization. This effect is probably direct, since in vitro wounding studies with cultured keratinocytes from these animals revealed a retardation in wound closure compared with keratinocytes from wild-type mice (73). In addition, angiogenesis was severely impaired, most likely due to a diminished response to MIP-2 which is known to be angiogenic (22). These results demonstrate that CXCR2 and its ligands are not only involved in inflammatory cell recruitment, but also regulate the behavior of resident cells in the wound.

F. Overexpression of Interferon-γ-Inducible Protein 10 in the Epidermis of Transgenic Mice Stimulates Inflammation but Inhibits Reepithelialization

IP10/CXCL10 is a chemokine that is detected at high levels in several chronic inflammatory conditions, including psoriasis. It is a member of the CXC family of chemokines and acts primarily in the recruitment of lymphocytes carrying the receptor CXCR3 (53). Its expression was shown to be upregulated together with monokine induced by interferon-γ (MIG/CXCL9) following wounding, with an expression pattern that correlates well with recruitment of inflammatory cells to the wound site (81). To determine whether IP-10/CXCL10 could modulate an in vivo inflammatory response, Luster et al. (166) engineered mice that constitutively express IP-10/CXCL10 in keratinocytes. These mice showed no obvious abnormalities under normal laboratory conditions. After full-thickness injury, however, IP-10/CXCL10 overexpressing mice showed a more intense inflammatory phase, compared with control littermates, delayed reepithelialization, and a prolonged, disorganized granulation phase with impaired angiogenesis. The latter result was expected from the known inhibitory effect of IP-10 on angiogenesis (22). These data suggest that IP-10/CXCL10 is able to inhibit wound repair by disrupting the normal development of the granulation tissue. This adverse effect on wound healing could be at least partially achieved by the inhibitory effect of IP-10/CXCL10 on EGF-induced fibroblast motility (249).

G. Multiple Functions of Chemokines in Wound Repair

The results described above demonstrate the importance of chemokines for the recruitment of inflammatory cells into wounds. In addition, they have been shown to act directly or indirectly on resident cells, thereby regulating reepithelialization, angiogenesis (see above), and also myofibroblast differentiation as recently demonstrated for the chicken chemotactic and angiogenic factor (cCAF) (87). Finally, chemokines might also be involved in the regulation of skin homeostasis as suggested for stromal-derived factor 1 (SDF-1/CXCL12). This chemokine is constitutively expressed in dermal fibroblasts and blood vessels of human skin (210) but downregulated after injury in mice and humans (85; R. Gillitzer, personal communication). These results suggest that SDF-1/CXCL12 functions as a homeostatic regulator of tissue remodeling. The recent discovery of a plethora of additional chemokines (16) suggests that many of them might play a role in wound healing, and more exciting functions of these molecules in the repair process will undoubtedly be discovered.

XV. PROINFLAMMATORY CYTOKINES

It has long been thought that proinflammatory cytokines, including interleukins 1α and 1 (IL-1α and IL-1), IL-6, and TNF-α, play an important role in wound repair. They likely influence various processes at the wound site, including stimulation of keratinocyte and fibroblast proliferation, synthesis and breakdown of extracellular matrix proteins, fibroblast chemotaxis, and regulation of the immune response.

A. Expression of Proinflammatory Cytokines in Skin Wounds

In support of a role for proinflammatory cytokines in wound repair, expression of IL-1α, IL-1, IL-6, and TNF-α was shown to be strongly upregulated during the inflammatory phase of healing (109, 110, 126). Polymorphonuclear leukocytes and macrophages were shown to be the major source of these cytokines, but expression was also observed in some resident cell types (86, 126). The coordinated expression of these cytokines is likely to be important for normal repair, since expression of these genes was strongly reduced after wounding of healing-impaired glucocorticoid-treated mice (126) and prolonged in genetically diabetic db/db mice (293).

B. IL-6 Knock-out Mice Show Severe Deficits in Cutaneous Wound Repair

With the use of a full-thickness punch biopsy wounding model on IL-6 knock-out mice, it was shown that wounds to IL-6 knock-out animals took up to three times longer to heal than those of wild-type controls (96). As expected by the mitogenic effect of IL-6 for keratinocytes (236), wounds in these animals were characterized by a dramatic delay in reepithelialization. In addition, granulation tissue formation was impaired. These abnormalities were completely rescued by administration of recombinant murine IL-6 protein 1 h before wounding. Thus it appears that IL-6 is crucial for kick-starting the healing response, both via its mitogenic effects on wound edge keratinocytes and via its chemoattractive effect on neutrophils.

Whereas a complete lack of IL-6 caused impaired wound healing, excessive levels of IL-6 have been associated with cutaneous scarring. Thus the increase in IL-6 expression after injury was only transient in fetal wounds but prolonged in adult wounds. Most importantly, IL-6 administration to fetal wounds resulted in scar formation (160). These results suggest that the more transient upregulation of proinflammatory cytokines such as IL-6 as well as of chemokines (see above) in fetal wounds compared with adult wounds might at least partially underlie the different scarring response in fetal versus adult mammals.

C. STAT-3-Mediated Transduction of Cytokine Signals Is Important for Wound Repair

STATs (signal transducers and activators of transcription) are cytoplasmic proteins that transduce signals from a variety of growth factors, cytokines, and hormones. Once activated by tyrosine phosphorylation, they dimerize and translocate to the nucleus, where they bind to specific DNA elements and thus regulate target gene expression (38). STAT-3 is activated upon binding of IL-6 to its receptor and is thus a likely candidate for a role in wound repair. Because STAT-3 null mice die during embryogenesis (268), Sano et al. (235) used a Cre-lox approach to specifically delete the stat-3 gene in keratinocytes. They observed no effect on skin morphogenesis. However, following full-thickness excisional wounding, the healing process was severely compromised, with dramatically reduced reepithelialization. These abnormalities were remarkably similar to those seen in IL-6 knock-out mice (see above). However, the overall effect on repair was less dramatic than in the IL-6 null mice, since the cell types involved in both the inflammatory response and granulation tissue formation were unaffected by the keratinocyte-specific approach.

D. Accelerated Wound Healing in TNF Receptor p55-Deficient Mice

To determine the role of the TNF receptor p55 in cutaneous wound repair, Mori et al. (191) generated excisional wounds on the back of mice lacking this receptor. These animals showed an enhancement of angiogenesis, collagen content, and reepithelialization. These histological differences were accompanied by increased expression of TGF-1, CTGF, VEGF, VEGFR-1, and VEGFR-2 at the wound site. In contrast, leukocyte infiltration and mRNA expression of adhesion molecules and cytokines were reduced. Overall, these changes resulted in accelerated healing of wounds in TNF receptor p55-deficient mice.

XVI. GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR

GM-CSF is a pleiotropic cytokine that was shown to be mitogenic for keratinocytes (144) and to stimulate migration and proliferation of endothelial cells (44). Together with its potent effect on hematopoietic cells, it has been suggested to play an important role in cutaneous wound repair. Indeed, a series of animal experiments and clinical studies have demonstrated a beneficial effect of GM-CSF for the treatment of normal wounds and chronic ulcers (112). Recently, Mann et al. (170) demonstrated a strong increase in the levels of GM-CSF in skin extracts after generation of full-thickness excisional wounds in mice, although the cellular source has not been determined (170).

A. Overexpression of GM-CSF in the Epidermis of Transgenic Mice Accelerates Wound Reepithelialization

To gain further insight into the possible role of GMCSF in skin wound healing, the same group generated transgenic mice that overexpress this cytokine in the epidermis (34) and generated full-thickness excisional wounds in these animals (170). Interestingly, these animals displayed accelerated wound reepithelialization as a result of increased keratinocyte proliferation. Furthermore, neovascularization and granulation tissue formation were strongly enhanced. Interestingly, several cytokines that are known to be important for wound healing such as TGF-1 were elevated in the wounds of these animals, indicating that GM-CSF stimulates wound repair directly but also indirectly via induction of secondary cytokines (170).

XVII. LEPTIN

Leptin is a 16-kDa protein that is produced by adipocytes and induces weight loss in both normal and genetically obese ob/ob mice via binding to its high-affinity receptor (295). The phenotype of ob/ob mice is due to a defective ob gene, leading to a lack of circulating leptin (307). These animals are obese and they suffer from multiple metabolic abnormalities, mimicking those seen in patients with type 2 diabetes mellitus. Most interestingly, the wound healing process in these animals is severely delayed (105). However, it has remained unclear whether this abnormality is secondary to the systemic defects or whether leptin is directly involved in wound healing.

A. Systemic and Topical Application of Leptin Accelerates Wound Repair

Two groups demonstrated that systemic and topical application of leptin accelerates wound healing in ob/ob mice (92, 225). Furthermore, topical leptin administration also stimulated the wound healing process in wild-type mice (92). Reepithelialization was particularly accelerated (92), whereas angiogenesis was not improved (225, 258). The stimulatory effect of leptin on wound healing was obviously mediated via the leptin receptor, since wound healing in leptin receptor-deficient, genetically diabetic db/db mice was not affected by leptin treatment. The finding that topical administration of leptin accelerates wound repair both in ob/ob and also in wild-type mice strongly suggests a direct stimulatory effect of leptin on wound repair.

B. Expression of Leptin Receptors at the Wound Site

The hypothesis of a direct effect of leptin on wound repair was supported by the detection of leptin receptor mRNA at the wound site. With the use of RNase protection assay, mRNAs encoding all forms of leptin receptor, including the signal-transducing long form, were found in normal and wounded skin (92, 225), although expression of this gene was downregulated after wounding (92). In situ hybridization revealed expression of leptin receptors in subcutaneous and dermal blood vessels (225) as well as in keratinocytes of the normal and wounded epidermis (92). Interestingly, the signal-transducing receptor was restricted to proliferation-competent keratinocytes (92). This finding together with the increase in keratinocyte proliferation seen in the wounds of leptin-treated animals suggested a mitogenic effect of leptin for these cells. Indeed, proliferation of human and murine keratinocytes was stimulated by leptin in vitro (92, 257), demonstrating that leptin is indeed a keratinocyte mitogen and a mediator of wound reepithelialization in vivo.

XVIII. INTERLEUKIN-10

In addition to proinflammatory cytokines, anti-inflammatory cytokines have also been shown to be important regulators of wound repair. In particular, the role of IL-10 in the healing response has been studied in some detail. This cytokine is thought to play a major role in the limitation and termination of inflammatory responses. In addition, it regulates growth and/or differentiation of various immune cells, but also of keratinocytes and endothelial cells (190). Based on these activities, a role of IL-10 in wound healing appeared likely.

A. Expression of IL-10 at the Wound Site

Two groups determined the expression pattern of this cytokine in the healing wound. Increased levels of IL-10 mRNA were observed after incisional wounding of mice, with a peak at 60 min after injury (202). Using the same wound model, Sato et al. (237) found two peaks of IL-10 expression after injury: an early peak with a maximum at 3 h after wounding and a second peak at day 3 after wounding. Keratinocytes of the wound epidermis and infiltrating mononuclear cells were identified as the major producers of IL-10 mRNA and protein (237). Interestingly, increased expression of IL-10 was shown to be associated with impaired healing, since the levels of this cytokine were strongly increased in human chronic venous insufficiency ulcers compared with autologous donor wound tissue (165).

B. IL-10 Inhibits Inflammation and Scar Formation

To determine the function of IL-10 in wound repair, neutralizing antibodies to this cytokine were applied to incisional mouse wounds. This treatment demonstrated that endogenous IL-10 inhibits the infiltration of neutrophils and macrophages toward the site of injury as well as expression of several chemokines and proinflammatory cytokines (237). In another study, the role of IL-10 in fetal scarless healing was investigated (162). These investigators wounded embryonic skin from IL-10 null mice that had been grafted to strain-matched adult mice. Wounds to control embryonic skin grafts showed little inflammation and normal restoration of the dermal architecture. However, wounded IL-10 null grafts were characterized by a significantly higher inflammatory cell infiltration and collagen deposition and an adultlike scarring response. These results suggest an important role of IL-10 in regulating the expression of proinflammatory cytokines in fetal wounds, leading to reduced matrix deposition and scar-free healing.

XIX. TEMPORAL AND SPATIAL INTERACTION OF DIFFERENT GROWTH FACTORS AT THE WOUND SITE)

The results described in this review demonstrate that a multitude of growth factors and cytokines are present at the wound site. Their expression dynamics show characteristic temporal and spatial regulation, and changes in the expression pattern of growth factors are associated with impaired wound healing. Most importantly, alterations in the levels of one factor are likely to affect the production of other growth factors and cytokines. Thus it has been shown that proinflammatory cytokines and serum growth factors that are released during the early phase of wound healing are potent stimulators of the expression of various growth factors. One example is the regulation of FGF7, a growth factor produced by fibroblasts at the site of injury (see sect. III). In vitro studies using cultured fibroblasts or organotypic cocultures of fibroblasts and keratinocytes revealed that the proinflammatory cytokines IL-1 and TNF-α as well as the growth factor PDGF are potent inducers of FGF7 expression (32, 48, 266). This suggests that PDGF released from platelets might be responsible for the early induction of FGF7. IL-1 and TNF-α, which are predominantly produced by invading neutrophils and fibroblasts, are likely to maintain the high levels of this growth factor within the first few days after injury (Fig. 5A). Another example is the regulation of VEGF, a major regulator of angiogenesis (see sect. V), which is produced by keratinocytes and macrophages at the wound site (40). It has been demonstrated that proinflammatory cytokines can induce expression of VEGF in both cell types (90, 212). In addition, several growth factors, including FGF7, EGF, TGF-α, and HGF, have been shown to stimulate the production of VEGF by cultured keratinocytes (72, 90, 102) (Fig. 5B). Recent studies revealed that these regulatory interactions are not only occurring in vitro but also in wounded skin. Thus nonviral liposomal FGF7 gene transfer increased both VEGF production at the wound site and neovascularization of wounded skin. The latter finding can explain how FGF7, an epithelial-specific mitogen, can indirectly affect angiogenesis. An even more complex interaction was shown for HGF and VEGF. Toyoda et al. (274) demonstrated that overexpression of HGF in transgenic mice caused a strong increase in VEGF expression in nonwounded and particularly in wounded skin. In this case, both growth factors are likely to act synergistically to stimulate wound angiogenesis, since HGF was recently shown to enhance VEGF-induced angiogenesis in vitro and in vivo (299).

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FIG. 5. Growth factor interactions at the wound site. A: regulation of fibroblast growth factor (FGF) 7 at the wound site—hypothetical model. Local hemorrhage causes extravasation of platelets and their release of PDGF and EGF. These mitogens stimulate FGF7 expression in fibroblasts. In addition, invading neutrophils and macrophages secrete the proinflammatory cytokines IL-1 and TNF-α which then cause a further induction of FGF7 expression in fibroblasts. Finally, IL-1 and TGF-α derived from keratinocytes also stimulate FGF7 expression in fibroblasts. B: regulation of VEGF expression at the wound site—hypothetical model. Local hemorrhage causes extravasation of platelets and their release of TGF-. Invading macrophages also secrete this growth factor, together with the proinflammatory cytokines IL-1 and TNF-α. These factors stimulate VEGF expression in keratinocytes and macrophages. In addition, FGF7 and hepatocyte growth factor (HGF) derived from fibroblasts as well as keratinocyte-derived TGF-α stimulate VEGF expression in the epidermis.

 

Such examples highlight the complex growth factor interactions that occur during wound healing. These interactions have to be considered in the interpretation of results obtained by overexpression or elimination of a single growth factor at the wound site. It will be a major challenge in the future to study the interactions of different factors in vitro and particularly in wounded skin. Results from such studies are likely to be important for the development of novel strategies for the treatment of impaired wound healing, since they will facilitate the formulation of temporally and combinatorially optimized therapeutic approaches.

XX. CONCLUSIONS

The beneficial effect of exogenous growth factors in the treatment of wound repair as well as the identification of the in vitro activities of many growth factors and cytokines have implicated these proteins as key regulators of the wound healing process (Table 1). This hypothesis is strongly supported by the expression of multiple growth factors and their receptors in different cell types of healing skin wounds. In addition, upregulation of growth factor expression after injury is frequently observed, suggesting a need for high growth factor levels during the repair process. Finally, there are many examples where abnormal growth factor expression is associated with impaired wound healing or excessive scarring, indicating that a correct temporal and spatial expression of these genes is essential for normal repair.

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TABLE 1. Growth factors and cytokines and their effects on wound repair

 

To determine the in vivo function of endogenous growth factors and cytokines, several investigators have applied neutralizing antibodies to skin wounds or to wound fluid (Tables 2 and 3). In most cases, this treatment strongly affected the healing process and provided insight into the roles of growth factors in repair. However, these results have to be interpreted with care, since cross-reactivity with related growth factors cannot be excluded. Furthermore, it is unclear whether neutralizing antibodies get access to the complete wound area.

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TABLE 2. Use of neutralizing antibodies to block growth factor function in wound fluid

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TABLE 3. Use of neutralizing antibodies to block growth factor function in cutaneous wounds

 

 

Given these limitations, a genetic approach to identify growth factor function in wound repair appears desirable. Indeed, the use of genetically modified mice for wound healing studies recently revealed crucial roles for several growth factors and cytokines in the repair process (Table 4). For example, these studies have demonstrated an inhibitory effect of TGF- on wound reepithelialization (2, 12, 66, 149), an important role of FGF receptor signaling in this process (292), and a role of activin in granulation tissue formation and scarring (192, 286). In the latter case, the phenotype was more pronounced in a CD1 background compared with a B6D2F2 background (Werner, unpublished data), demonstrating the importance of the genetic background for the outcome of the repair process.

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TABLE 4. Summary of genetically modified mouse studies of growth factor and cytokine function in wound repair

 

In spite of the potency of the genetic approach for the study of gene function in wound repair, some of the normal functions of targeted genes might not be revealed due to redundancy or compensation. Indeed, lack of wound healing abnormalities was often observed in mice deficient in a particular growth factor that belongs to a multiprotein family, e.g., in mice lacking FGF7 or TGF-α (113, 164, 171). In the latter case, however, a more detailed analysis of the healing process and the use of another wound model did finally reveal a function of TGF-α in epithelial repair (146). Nevertheless, the observed phenotype was more subtle than expected from its known in vitro activities. Although it cannot be excluded that FGF7 and TGF-α are indeed not important for wound repair, their strong induction in healing skin wounds supports their functional significance. In both cases other growth factors, which bind to the same receptor and which are also expressed in wounded skin, might compensate for the lack of these mitogens. Wound healing studies using animals, which are deficient in two or more homologous molecules, as well as studies with dominant-negative receptor mutants or with soluble inhibitors that block the function of several members of a protein family, will be very useful in answering these questions. For example, the overexpression of a dominant-negative FGFR2IIIb in the epidermis of transgenic mice demonstrated an important role of FGFR signaling in wound reepithelialization (292), whereas the knock-out of individual ligands did not reveal this function (113, 188, 208).

At the other extreme, systemic abnormalities caused by the transgene or by the general loss of a gene might obscure the normal function of a gene in wound repair. Thus it has long been known that systemic defects such as malnutrition and weight loss can strongly impair the healing process (108). This was observed for the TGF-1 knock-out mice, which developed a strong inflammatory response in various tissues and organs, followed by severe weight loss at ∼3 wk of age. These systemic defects appear to be responsible for the impaired wound healing seen in these animals (41), making it impossible to study the local effects of the lack of TGF-1 on wound repair in this model. Suitable approaches to circumvent this problem, as mentioned above, were to cross the mice onto an immunodeficient background or to treat them with immunosuppressive drugs (66, 149). These studies serve to alert us to the difficulties of determining the functions of specific proteins in complex in vivo situations, but provide some elegant methods for circumventing these problems.

Another way to solve this problem is the generation of mice that have a tissue-specific knock-out or tissue-specific overexpression of a transgene (218). For example, overexpression of a dominant-negative TGF- receptor in the epidermis of transgenic mice did not cause any systemic abnormalities, but nevertheless allowed the identification of an inhibitory effect of TGF- on wound reepithelialization (2). An even more elegant approach will be the use of inducible systems, which allow the induction of a transgene or the deletion of an endogenous gene in a time- and tissue-specific manner. The first successful results with inducible systems in the skin have recently been published. Two have adopted an estrogen receptor-based approach, where Cre recombinase was fused in frame with the tamoxifen-responsive hormone-binding domain of the estrogen receptor [CreER(tam)]. This fusion protein was expressed under the control of the keratin 5 (131) or keratin 14 promoters (280) that target transgenes to the basal layer of the epidermis and to the outer root sheath of hair follicles. Upon systemic or local application of tamoxifen, the latter binds to the hormone-binding domain of the fusion protein and causes activation of Cre recombinase. The activated enzyme then allows the deletion of the targeted gene in keratinocytes.

In an analogous approach, activation of other intracellular proteins can be achieved by fusing the cDNA encoding the protein of interest to the hormone-binding domain of the estrogen receptor. In this case, tamoxifen can be used to activate the protein as recently demonstrated for c-Myc (9).

Another group has used topical application of anti-progestin to induce expression of TGF-1 in the epidermis (284). In this system, a fusion protein of a truncated progesterone receptor and the yeast GAL4 transcription factor was expressed under the control of the loricrin promoter. Thus by engineering a GAL4 binding domain, normally absent in mammalian cells, upstream of the target gene, transcription can be activated in a tissue-specific and temporally controlled manner. Finally, doxycycline-mediated gene expression (148) was recently used to inducibly express the erbB2 oncogene in the epidermis of transgenic mice (298).

Use of these types of systems will circumvent the problem of systemic defects and will also prevent abnormal skin development. The latter is important, since a wound healing phenotype might be secondary to a defect already present in nonwounded skin. Thus by induction of a transgene or deletion of an endogenous gene before wounding, the role of this particular gene in the healing response can be studied in the absence of secondary abnormalities. This type of study will undoubtedly unravel many exciting functions of growth factors and cytokines in normal wound repair as well as in impaired healing and scar formation.

IGF-1 use in wound healing and tissue reparation

Systemic administration of IGF-I enhances healing in collagenous extracellular matrices: evaluation of loaded and unloaded ligaments

Paolo P Provenzano,¹ Adriana L Alejandro-Osorio,² Kelley W Grorud,^1,3 Daniel A Martinez,^4,5 Arthur C Vailas,⁵ Richard E Grindeland,⁶ and Ray Vanderby, Jr^1,3

Background

Insulin-like growth factor-I (IGF-I) plays a crucial role in muscle regeneration, can reduce age-related loss of muscle function, and cause muscle hypertrophy when over-expressed [1–5]. These effects appear to be largely mediated by promoting proliferation and differentiation of satellite cells [3] as well as promoting recruitment of proliferating bone marrow stem cells to regions of muscle tissue damage [6]. Furthermore IGF-I and growth hormone (GH) are involved in a large variety of physiologic functions and are reported to promote healing and repair in bone [7,8], cartilage [9–11], gastric ulcers [12], muscle [13,14], skin [15–17], and tendon [18,19]. This action is largely mediated by the fact that GH and IGF-I directly affect cells involved in the healing response [20–30], with IGF-I having endocrine action, as well as local expression, resulting in autocrine and/or paracrine signaling that plays a role in proliferation, apoptosis, cellular differentiation, and cell migration [31–36]. Insulin-like growth factor-I also stimulates fibroblast synthesis of extracellular matrix (ECM) molecules such as proteoglycans and type I collagen [18,30,37,38], and IGF-I mRNA and protein levels are increased in healing ligaments [39] and tendons [40], respectively. As such, IGF-I is of particular interest in tissue regeneration due to its influence on cell behavior and role in type I collagen expression.

Fibrous connective tissues, such as ligament and tendon, are composed primarily of type I collagen with type III collagen levels increased during healing [41]. During development, collagen molecules organize into immature collagen fibrils that fuse to form longer fibrils [42–45]. In mature tendon and ligament these fibrils appear to be continuous and transfer force directly through the matrix [46]. In ligament, groups of fibrils form fibers and it is these fiber bundles that form fascicles; the primary structural component of the tissue. Previous studies in healing ligament have shown that disruption of the medial collateral ligament (MCL) results in substantial reduction in mechanical properties which does not return to normal after long periods of healing [47]. Such tissue behavior is likely associated with matrix flaws, reduced microstructural organization, and small diameter collagen fibrils in the scar region of the ECM [48–50]. Additionally, during normal ligament healing collagen fibrils from residual tissue fuse with collagen fibrils formed in the scar region [51]. However, in tissues which are exposed to a reduced stress environment such as joint immobilization [52] or hindlimb unloading [48] collagen fibers contain discontinuities and voids [48] which likely account for the substantial decrease in tissue strength when compared to ligaments experiencing physiologic stress during healing. Since soft tissue injuries are common and do not heal properly in a stress-reduced environment [48,52], such as is present during prolonged bed rest or spaceflight, methods to further understand tissue healing and promote tissue healing require study.

The purpose of this study is to test the hypotheses that systemic administration of IGF-I, GH, or GH+IGF-I will improve healing in a collagenous ECM. Furthermore, since the addition of GH has been shown to up-regulate IGF-I receptor [53], levels of IGF-I receptor in healing tissues were examined in order to begin to elucidate the molecular mechanism by which GH and/or IGF-I may be acting to locally to stimulate tissue repair. Since IGF-I and GH are feasible for clinical use, identifying benefits from short-term systemic administration, such as improved connective tissue healing, have great potential to improve the human condition. The hypotheses are examined in animals that are allowed normal ambulation after injury and in animals that are subjected to disuse through hindlimb unloading. The MCL was chosen as a model system since this ligament, unlike tendons, has no muscular attachment and therefore possible alterations in muscle strength after IGF-I and/or GH treatment do not impose substantial differential loads on the ligament during hindlimb unloading. Furthermore, since MCLs have two attachments/insertions into bone, and hindlimb unloading/disuse is known to reduce the mechanical properties of bone [48,54], failure location was recorded for all mechanical testing. Results indicate improved mechanical properties and collagen organization and composition of the collagenous extracellular matrix following treatment with IGF-I in both ambulatory and hindlimb unloaded animals or IGF-I+GH in hindlimb unloaded animals.

Results

Initial body weights were not different between groups and no wound infections or other apparent complications associated with surgery were observed. All of the ambulatory animals returned to normal cage activity shortly after surgery, and no treatment complications were observed in the suspended animals. The hindlimb unloaded (HU) animals were not able to gain weight as rapidly as the ambulatory animals, thus significant differences (p < 0.0001) in body weight were observed between groups after 10 days of healing. The GH, IGF-I, and GH+IGF-I treatment increased body weight in HU animals (compared to HU animals receiving only saline) but this difference was not statistically significant (p = 0.49, 0.49, and 0.26, respectively). No significant differences in body weight were present between the ambulatory animals (all p values > 0.38). At tissue harvest all healing ligaments showed a bridging of the injury gap with translucent scar tissue. In the ambulatory animals receiving GH, three animals had hematomas and tissue adhesions in the surgical site. In all other animals no gross differences in the tissues or the surgical wound site were observed.

The location of structural failure in each ligament was examined, revealing that the majority of the femur-MCL-tibia complexes failed in the MCL proper, not at the ligament to bone insertion sites, nor by bone avulsion (Table ​(Table1).1). Sham control ligaments failed in the tibial third of the ligament during 100% of the tests. For all other groups, the primary location of failure was the scar region of the ligament. The only exceptions to this were one failure in the tibial third of the ligament in the Amb + IGF group, one tibial avulsion in the HU + Sal group, and one tibial avulsion in the HU + IGF group, indicating that a significant portion of the failure locations was in the scar region (all p values < 0.01). These results indicate the dominant effects in the measured mechanical properties are due to changes in ligamentous tissue and not in the insertions.

Table 1 Failure location of the bone-ligament-bone complex at 3 weeks post-injury.

Substantial differences in tissue mechanical properties were present between groups. Hindlimb unloading adversely affected ligament healing while IGF-I or GH+IGF-I had a substantial effect on ligament healing in either ambulatory or hindlimb unloaded animals. Maximum force (Fig. ​(Fig.1)1) was significantly different between tissues from Sham and both Amb + Sal and HU + Sal groups (p = 0.0001 and p = 0.0001, respectively). Hindlimb unloaded animals had significantly decreased maximum force in the MCLs when compared to ambulatory healing animals (Amb + Sal; p = 0.03). In ambulatory animals the addition of IGF-I significantly improved maximum force values by approximately 60% when compared to ambulatory healing animals that received saline (p = 0.0002). Addition of IGF-I and GH+IGF-I in hindlimb unloaded animals significantly increased maximum force values (60% and 74%, respectively) when compared to HU + Sal animals (p = 0.0074 and p = 0.0013, respectively). In fact, addition of IGF-I or GH+IGF-I to hindlimb unloaded animals increased force to be comparable with Amb + Sal animals. Growth hormone alone did not have a significant effect within either the ambulatory or unloaded groups. However, in the unloaded group, GH increased maximum force and brought it closer to values in the Amb + Sal group, yet this increase was not statistically significant (p = 0.16). Ultimate stress (Fig. ​(Fig.2)2) was significantly decreased in tissues from both ambulatory healing and hindlimb unloaded healing animals when compared to sham control tissues (p = 0.0001 and p = 0.0001, respectively). Ligaments from HU + Sal animals had ultimate stress values that were significantly lower than saline receiving ambulatory animals (p = 0.022). Addition of only IGF-I to ambulatory animals significantly increased ultimate stress when compared to Amb + Sal animals (p = 0.0077). Delivery of IGF-I and GH+IGF-I significantly increased ultimate stress in tissues from the hindlimb unloaded animals when compared to hindlimb unloaded (plus saline) animals (p = 0.0236 and p = 0.0202, respectively). In fact, ultimate stress values after the addition of IGF-I or GH+IGF-I to hindlimb unloaded animals was comparable to ultimate stress values in ambulatory animals receiving saline. In ambulatory animals, no statistically significant effect on ultimate stress values were seen after GH or GH+IGF-I was administered. No significant differences in strain at failure were present between groups. Elastic modulus (Fig. ​(Fig.3)3) was statistically different between IGF-I and saline treated ambulatory healing animals. Addition of IGF-I in ambulatory animals resulted in an elastic modulus that had an ~49% greater mean value (p = 0.049). In hindlimb unloaded animals the addition of IGF-I or GH+IGF-I resulted in a significant increase in modulus when compared to unloaded animals receiving saline (p = 0.049 and p = 0.014, respectively). Growth hormone alone had no significant effect on elastic modulus in either group.

Figure 1 Maximum force values at 3 weeks (mean ± S.E.M.). Maximum force was significantly decreased in tissues from hindlimb unloaded (HU) animals receiving saline when compared to tissues from Amb + Sal animals (p = 0.03). The addition of IGF-I significantly (more …)

Figure 2 Ultimate stress values at 3 weeks (mean ± S.E.M.). Ultimate stress was significantly decreased in tissues from unloaded animals receiving saline when compared to tissues from Amb + Sal (p = 0.022). The additional of IGF-I significantly improved (more …)

Figure 3 Elastic modulus values at 3 weeks (mean ± S.E.M.). The additional of IGF-I significantly improved elastic modulus levels in tissues from ambulatory healing animals when compared to tissues from ambulatory healing animals which received saline (more …)

Representative H&E stained sections (Fig. ​(Fig.4)4) revealed hypercellularity and extracellular matrix disorganization in injured tissues from both ambulatory and suspended animals. Ligaments from Sham animals demonstrated the characteristic crimp pattern and aligned fibroblasts associated with normal ligament (Fig. ​(Fig.4).4). However, while Amb + Sal animals revealed normal scar morphology, tissues from HU + Sal animals had pockets of cell clusters, cellular misalignment, and misaligned collagen bundles oriented in separate directions creating discontinuities and voids within the matrix confirming a previous report [48]. Furthermore, while matrices from animals treated with GH had abnormal cell clusters and no gross improvement in matrix organization in both ambulatory and unloaded tissues, tissues from animals treated with IGF-I showed substantially increased matrix density and collagen alignment (Fig. ​(Fig.4);4); a morphology also present in tissues from HU + GH + IGF-I animals.

Figure 4 Representative tissue sections taken from the midsubstance region of control normal tissue (Sham) and scar tissues (Saline, GH, IGF-I, GH+IGF-I). The longitudinal axis of the ligament is from the top left to bottom right of each image (200X). Tissues (more …)

To further examine extracellular matrix organization in histology sections, multiphoton laser scanning microscopy (MPLSM), which allows greatly enhanced imaging of collagen structure, was employed (Fig. ​(Fig.5).5). Examination of sham, ambulatory, and hindlimb unloaded tissues supports morphological information obtained with classical histology (Fig. ​(Fig.4)4) and scanning electron microscopy [48], with tissues from HU animals showing good fiber bundle formation but fiber misalignment creating matrix discontinuities and voids (Fig. ​(Fig.5).5). Confirming results obtained from viewing H&E section with light microscopy, MPLSM analysis showed improved matrix organization in tissues from Amb + IGF-I, HU + IGF, and HU + GH + IGF-I animals (Fig. ​(Fig.5).5). In accordance with data showing increased matrix deposition and organization (Figs. ​(Figs.44 and ​and5),5), expression of type-I collagen was increased in tissues from IGF-I treated ambulatory (p = 0.0018) and unloaded (p = 0.0006) animals and GH+IGF-I treated unloaded tissue (p = 0.0005; Figs. ​Figs.6A6A and ​and6B).6B). Densitometry analysis further confirmed an increase in type-I collagen since the ratio of type-I to type-III collagen was significantly increased in tissues from ambulatory animals treated with IGF-I (p = 0.0129) and in unloaded tissues from GH+IGF-I treated animals (p = 0.0131), with a trend of increased levels in ambulatory GH+IGF and HU + IGF tissues (Fig. ​(Fig.7).7). Since normal ligaments are primarily composed of type I collagen and the scar region of normal healing ligaments contain an increase in type III collagen [41] that is remodeled to transition to more type I collagen rich region over the healing period, changes in the type I to III ratio are a measure of healing and may indicate part of the structural mechanism resulting in the observed differences in mechanical properties following treatment.

Figure 5 Multiphoton Laser Scanning Microscopy (MPLSM) was performed on hematoxylin and eosin sections in order to evaluate the organization and structure of the collagen matrix in more detail then allowed by conventional brightfield light microscopy. The longitudinal (more …)

Figure 6 Significantly increased expression of type-I collagen in tissues from animals treated with IGF-I. (A: Left) Immunohistochemistry shows increased staining for type I collagen in ambulatory tissues when compared to Sham, while hindlimb unloaded tissues (more …)

Figure 7 Densitometry analysis of type I and III collagen expression. Quantification of Western blots for type I and III collagen indicated that the ratio of type I to type III collagen was significantly increased in tissues from ambulatory animals treated with (more …)

Lastly, GH has been reported to increase levels of IGF receptors [53]. Herein, ambulatory IGF-I and GH+IGF-I treated animals showed a strong trend of increased IGF-I receptor expression, although this trend was not significant (Fig. ​(Fig.8).8). However, in hindlimb unloaded animals treated with IGF-I or GH+IGF-I, IGF-I receptor expression was significantly increased (Fig. ​(Fig.8).8). Hence, increased levels of IGF-I receptor in healing tissues may be part of the molecular mechanism by which systemic administration of IGF-I and GH+IHG-I act to locally to stimulate tissue repair.

Figure 8 Densitometry analysis of IGF-I receptor expression. Ambulatory IGF-I and GH+IGF-I treated animals showed a strong trend of increased IGF-I receptor expression, however, this trend was not significant. In hindlimb unloaded animals treated with IGF-I or (more …)

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Discussion

Previous work in our laboratory has shown that mechanical properties and matrix organization of MCLs are substantially reduced after injury and that this impairment is significantly compounded by stress reduction through hindlimb unloading [48]. This result is confirmed by examination of the mechanical properties in MCLs from the saline receiving control groups in this study (Sham, Amb + Sal, HU + Sal) which are not significantly different from values obtained in our previous work in tissues from sham, ambulatory healing, and hindlimb unloaded animals after three weeks of healing [48]. Furthermore, structural analysis of the matrix with MPLSM confirmed previous morphological information obtained with electron microscopy [48], elucidating changes in the structure-function relationship, such as collagen fiber misalignment and matrix voids, that help explain the reduced mechanical properties associated with tissue unloading (i.e. disuse).

Results of this study demonstrate a substantial increase in healing with the systemic application of IGF-I in ambulatory and hindlimb unloaded animals and with GH + IGF-I in hindlimb unloaded animals. Since the strong majority of tissues failed in the ligament and not by avulsion, it is clear that the mechanical properties of the ligament are being evaluated and that systemic treatment with IGF-I or GH+IGF-I improves the integrity of the collagenous matrix. This finding is in contrast with results from healing tendons following local injections of IGF-I that showed no significant improvement in the mechanical properties of treated tendons [18], indicating that local and systemic administration of IGF-I may act through different mechanisms. Furthermore, it is interesting that IGF-I alone positively influenced healing in tissues from both ambulatory and hindlimb unloaded animals, while combined GH and IGF-I only had a positive affect on tissues from HU animals. Since IGF-I increased tissue strength measures by ~60% in both ambulatory and hindlimb unloaded animals, it appears that IGF-I improves healing regardless of mechanical loading and that the affects of mechanical loading and IGF-I may be additive. The GH+IGF-I associated improvement observed only in hindlimb unloaded animals is more difficult to interpret. Nonetheless, this difference between ambulatory and HU animals after GH+IGF-I treatment may result from changes in the endocrine system due to unloading [55–57] or an inhibitory role of exogenously elevated GH on IGF-I function. Decreased levels of growth hormone, and changes in other endocrine factors, with unloading have been reported [55–60], and the addition of GH in unloaded rats may be simply re-establishing basal GH behavior in unloaded animals allowing the influence of increased IGF-I to proceed. In contrast, elevated levels of GH in ambulatory animals inhibit the positive affect of IGF-I, indicating that in fact elevated GH in ambulatory animals may not be permissive to elevated IGF-I function, but that improved healing from IGF-I addition is not dependent on exogenously elevated GH levels. Interestingly, addition of GH alone showed a trend of decreasing the mechanical properties of healing ligaments and hematomas in the healing site, further supporting the concept of a negative role for GH in ligament healing, even though combined supplementation of GH+IGF-I has been reported to increase serum IGF-I levels more than adding IGF-I alone [61,62]. Hence, the behavior reported herein is a complex phenomena superimposing the influence of mechanotransduction during tissue loading, local growth factor signaling, and endocrine hormone levels, yet implies a positive role for IGF-I and a negative role for GH in connective tissue healing through mechanisms that are, to date, not well understood.

Connective tissue atrophy and diminished levels of healing after disuse (e.g. spaceflight, hindlimb unloading, immobilization, etc.) is associated with reduced physical stimuli, however local growth factor signaling, and endocrine factors also play an important role. For instance, it is well established that microgravity or simulated microgravity disrupts pituitary GH function [58–60] and alters IGF-I expression and plasma concentration [60,63]. Interestingly, in this study the addition of GH alone, which can bind to cells via specific surface receptors activating numerous signaling pathways that direct changes in gene expression [64–67], did not offer any significant increase in mechanical properties. In contrast, the addition of IGF-I significantly improved the mechanical properties of tissues in treated animals, likely through structural improvements in the extracellular matrix as seen in Figures ​Figures44 and ​and5.5. In ambulatory animals treated with IGF-I and unloaded animals treated with IGF-I or GH+IGF-I, MCL matrix organization was greatly improved. This may be due to increased type-I collagen expression resulting in increased the matrix density, and altered cell behavior resulting in a more organized collagen matrix. Given the clear increases in matrix alignment, it appears probable that either collagen organization is initially improved during post-fibrillogenesis deposition or better organized during continued matrix remodeling, or both. Since collagen alignment is achieved before substantial tissue loading during fetal development [46,68], which is not reproduced during tissue repair in unloaded mature tissue [48], and it appears that matrix repair in adult tissue may be an imperfect reversion to processes seen in fetal development [46], addition of IGF-I may be stimulating signaling pathways similar to those seen during development. Moreover, one possible link in the molecular mechanism playing a role in increased matrix organization and collagen expression may be signals associated with IGF-I receptor signaling as indicated by increased IGFR levels in treated animals. However, IGFR activation was not examined in this study and multiple pathways associated with IGFR and matrix adhesion signaling (i.e. integrin signaling) are likely acting in concert to produce the profound improvements seen after IGF-I treatment. Hence, it is clear from these data that further work studying the systemic effects IGF-I need to be performed in order to better understand the mechanism of IGF-I administration on local tissue behavior.

It is known that soft tissue injuries do not fully recover even after long periods of healing [47] and stress reduction has a negative effect on healing in collagenous tissue, [48,52] which does not return to normal after re-establishing physiologic stress (i.e. remobilization) [69]. This pattern of healing is problematic since the injured joint often need immobilization, the growing aged population often experiences decreased activity levels or prolonged bed rest, and since prolonged space flight is becoming more feasible. Therefore, methods to improve tissue healing and counteract this negative decline during injury and/or disuse are increasing in need. The reported application of IGF-I clearly has a positive effect on tissue repair from a mechanical (functional) viewpoint, and therefore shows promise to improving normal tissue healing and to improving healing under normal or disuse conditions. Of particular note is the increase in force, stress, and elastic modulus in tissues from unloaded animals with IGF-I or GH+IGF-I, which become comparable to or surpass levels in ambulatory (Amb + Sal) animals. This improvement in tissue properties compares well with other methods to improve tissue repair, but may be more clinically feasible. For instance, application of PDGF-BB has also shown promising improvements in fibrous connective tissue healing. In two studies allowing unrestricted cage movement, Batten and co-workers [70] reported increases in maximal force up to 90% of controls, and Hildebrand and co-workers [71] reported increases in force of ~50% after PDGF-BB was administered locally immediately following injury. However, administering PDGF-BB 48 hrs post-injury resulted in decreased force values [70], while administering IGF-I post-injury herein improved tissue healing. Therefore, the results presented in this paper showing an increase in maximal force of ~60% in ambulatory animals after 3 weeks of systemic IGF-I compares favorably to the levels of improvement previously reported in the literature but treatment can begin post-injury. Yet, although short term systemic application of IGF-I or GH+IGF-I provide compelling evidence for improved healing in a collagenous matrix, potential side effects of altering GH and IGF-I levels for long periods of time have not yet been fully explored in conjunction with these data and therefore further study is required before long term use is warranted in humans.

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Conclusion

In conclusion, results support our hypothesis that systemic administration of IGF-I improves healing in collagenous extracellular matrices. Growth hormone alone did not result in any significant improvement contrary to our hypothesis, while GH + IGF-I produced remarkable improvement in hindlimb unloaded animals. Interestingly, addition of IGF-I or GH + IGF-I in HU animals resulted in recovery of strength measures to a level equal to ambulatory controls, indicating that in fact IGF-I may be a plausible therapy for overcoming reduced tissue healing due to disuse from bed rest, immobilization, or microgravity. Additionally, although supplementation with IGF-I in ambulatory animals did not result in full recovery of the mechanical properties at 3 weeks, treatment resulted in an ~60% increase in tissue strength demonstrating the potential for IGF-I to improve tissue healing. These changes in tissue healing with tissue loading or IGF-I supplementation raise important questions regarding the essential role of mechanical stress for collagen matrix organization in connective tissues and the mechanisms by which systemic IGF-I or GH+IGF-I lead to improved tissue healing.

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Methods

Animal model and study design

This study was approved by the institutional animal use and care committee and meets N.I.H. guidelines for animal welfare. Seventy-two male Sprague-Dawley rats (248 ± 6 grams) were used as an animal model. These animals were randomly divided into nine groups each containing eight rats: 1) sham control surgery – ambulatory + saline (Sham), 2) healing – ambulatory + saline (Amb + Sal), 3) healing – hindlimb unloaded + saline (HU + Sal), 4) healing – ambulatory + GH (Amb + GH), 5) healing – hindlimb unloaded + GH (HU + GH), 6) healing – Ambulatory + IGF-I (Amb + IGF), 7) healing – hindlimb unloaded + IGF-I (HU + IGF), 8) healing – ambulatory + GH + IGF-I (Amb + GH + IGF), 9) healing – hindlimb unloaded + GH + IGF-I (HU + GH + IGF). Each rat in the ambulatory healing and hindlimb suspension (unloaded) groups underwent aseptic surgery to disrupt both knee MCLs. Rats were anesthetized with an isofluorane anesthetic administered by facemask using a non-rebreathing delivery system. The incision area was clipped and prepared for surgery. A skin incision approximately 8 mm long was made on the medial side of the stifle and fascia was incised to expose the MCL. The MCL was exposed and transected at the joint line, after which the muscles and skin were closed with suture. Sham control animals were subjected to identical surgical procedures without MCL disruption. All animals were given an analgesic (Tylenol^®/codeine) in their water for 72 hours post-surgery. Animals in the sham control and ambulatory healing groups were allowed unrestricted cage movement. Hindlimb unloaded healing rats were subjected to hindlimb unloading (24 hours after surgery), which induced substantial stress reduction by eliminating ground reaction forces, using the noninvasive tail suspension protocol of the NASA-Ames center [72]. Only the hindlimbs were suspended (unloaded) and the animals were allowed to move freely within the cages using the forelimbs while being restricted from kicking the sides of the cages. For GH and IGF-I delivery, recombinant human GH or IGF-I (Genentech, South San Francisco, CA) was dissolved in sterile saline at a concentration of 0.25 mg/mL. Rats received two 0.5 mL injections daily (0830 and 1530 hours) of GH, IGF-I, or GH+IGF-I subcutaneously in the nape of the neck. The dosage of GH and IGF-I were based upon previous studies in rats [73–75] based on initial body weight and was not adjusted during the course of the study. Combined GH and IGF-I were administered since previous reports have shown that increased levels of serum IGF-I and IGF-I binding proteins are obtained after co-injection of GH and IGF-I when compared to either GH or IGF-I alone [61,62]. All animals were housed at 24°C under a 12 hr light and 12 hr dark cycle, fed Purina rat chow, and watered ad libitum. Rats were checked twice daily for overall health, skin incision healing, food and water consumption, and the condition of their tails (the harness should prevent slippage without restricting circulation). After 3 weeks the animals were euthanized. Immediately after death, animal hindlimbs were flash frozen and stored at -80°C until testing. Of the sixteen ligaments per group, six ligaments were used for mechanical testing, six for histology and immunohistochemistry, and four for Western blotting.

Biomechanical testing

On the day of testing, hindlimbs were thawed at room temperature from -80°C. It has been shown that postmortem storage by freezing does not change the biomechanical properties of ligament [76]. Tissue harvest and testing were performed using methods similar to those previously described [48,77]. Extraneous tissue was carefully dissected away to expose each MCL and the femur-MCL-tibia complex was removed. Ligaments were kept moist in PBS at 25°C to prevent dehydration (pH = 7.4), and cross sectional area measured optically. While remaining hydrated the femur-MCL-tibia complex was placed into a custom designed tissue bath system with special structures to hold the femoral and tibial bone sections of the sample along the longitudinal axis of the MCL where all fibers appear to load simultaneously (~70° flexion). For tissue strain measurement, optical markers (impregnated carbon grease) were placed onto the ligament tissue near the insertion sites and the tissue bath containing the MCL samples were inserted into our custom testing machine. A small preload of 0.1 N was applied in order to obtain a uniform start (zero) point. The ligaments were preconditioned (~1% strain for 10 cycles) to allow the tissues to settle into the grips of the testing bath and then they were pulled to failure in displacement control at 10 %/s. Tissue deformation was examined and recorded until post-failure. Digital images of each test were analyzed to assess the structural failure location. For the case of a potential tibial avulsion the distal end of the ruptured tissue was examined for bone.

Tissue displacement was obtained using video dimensional analysis (resolution = 10 μm). The change in distance between optical markers was calculated by analyzing digital frames from the test with N.I.H. Image using a custom macro to calculate the change in x-y coordinate center (centroid coordinates) of each marker. Force (resolution 0.005 N) was obtained, displayed on the video screen and synchronized with displacement. From displacement data, engineering strain was calculated where L₀ is the initial length of the tissue at preload (0.1 N) and L is the current distance between strain markers:

The stretch ratio is represented by λ and is the current length divided by the original length. Lagrangian stress was calculated as the current force (F) divided by the initial undeformed area (A₀):

Stress versus stretch-ratio curves were created and fit with the microstructural model presented by Hurschler and co-workers [78] to obtain the elastic modulus. Biomechanical parameters for comparison are ultimate force, ultimate stress, strain at failure, and elastic modulus (determined from the microstructural model).

Histology and immunohistochemistry

Immediately following tissue harvest, samples for histology were fixed in 10% formalin. After standard histology procedures, sections underwent hematoxylin and eosin (H&E) staining. Slides were coverslipped and viewed with light microscopy. For immunohistochemistry, fixed ligaments were flash frozen in Optimal Cutting Temperature Media (OCT) at -70°C. Cryosections (6 μm) of each MCL were mounted onto lysine coated slides for staining. Mounted specimens were washed in a solution of PBS and 0.1% Tween-20 (PBST) between all incubation steps. Endogenous peroxidase activity was blocked by incubating slides in 3% hydrogen peroxide for 10 minutes, followed by blocking with 5% goat serum for 30 minutes at room temperature in a moist chamber. Tissue sections were then incubated for one hour at room temperature in a 1:1 dilution of SP1.D8 (University of Iowa Developmental Studies Hybridoma Bank), a primary antibody against the amino-terminal of type I procollagen. Sections were then incubated in rat adsorbed secondary antibody (Innovex Biosciences, Parsippany, New Jersey) for 20 minutes at room temperature, followed by incubation in horseradish peroxidase (HRP) labeled strepavidin (Innovex Biosciences, Parsippany, New Jersey) for 20 minutes at room temperature. Slides were stained with DAB (3,3′-diaminobenzidine; Innovex Biosciences, Parsippany, New Jersey) for 5 minutes at room temperature, and then washed in distilled water (twice) for 5 minutes. No counterstaining was performed and no primary antibody and a species appropriate IgG antibody served as negative controls. All slides were dehydrated in graded ethanol solutions, and then cleared in xylenes prior to cover-slipping. Light microscopy was used to examine each specimen and digital images of each MCL specimen were taken at 100× magnification. The area of staining was measured using ImageJ software after establishing a constant threshold across images.

Multiphoton laser scanning microscopy

Multiphoton Laser Scanning Microscopy (MPLSM) was performed on hematoxylin and eosin stained slides using an optical workstation [79,80] to better examine collagen organization in tissue sections. Since, MPLSM of formalin fixed paraffin embedded sections produces images of collagen structure and organization that far exceed the detail provided with standard histology under brightfield illumination, MPLSM was employed to detect potential differences in collagen matrix organization that have been previously reported in healing ligaments from ambulatory and hindlimb unloaded animals using scanning electron microscopy [48]. The excitation source was a Ti:sapphire laser (Spectra-Physics-Millennium/Tsunami, Mountain View, CA) producing around 100fs pulse widths and tuned to 890 nm. The beam was focused onto the sample with a Nikon 40X Plan Fluor oil-immersion lens (N.A. = 1.4).

Western blot analysis

Ligaments were finely minced and boiled in 3X Laemmli buffer for 10 minutes. Following centrifugation to remove tissue debri, protein was separated by SDS-PAGE on 7.5% gel, and electro-transferred to PVDF membrane (Millipore, Billerica, MA). Membranes were blocked with TBST (10 mM Tris, 150 mM NaCL, 0.3% Tween-20) plus 5% Blotto (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with either rabbit anti-collagen type-I (Biotrend, Germany) or mouse anti-collagen type-III (Abcam; FH-7A) for 30 minutes at 37°C or anti-IGF-I receptor antibody (Cell Signaling Technology, Danvers, MA) overnight at 4°C. The membrane was then incubated in secondary HRP-conjugated donkey anti-rabbit or anti-mouse antibody (Jackson ImmunoResearch Lab; 0.8 μg/mL) and visualized using the ECL Plus detection reagent (Amersham, Piscataway, NJ). For analysis of the ratio of collagen I to III the membranes were probed for type III collagen then stripped and re-developed to confirm the absence of residual signal, and then reprobed for type I collagen. IGF-I receptor levels were normalized to levels of GAPDH (anti-GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA). Following automated development of a number of exposures to confirm linearity, densitometry analysis was performed with ImageJ software. The values for each group were normalized to the Sham group to allow comparison of data across multiple Western blot experiments. Specificity of the collagen antibodies was confirmed by blotting purified type I and III collagen (BD Biosciences) and a panel tissues known to be composed of types I and III collagen. Cross-reactivity of the antibodies for other collagens was negligible (data not shown). The anti-collagen type I antibody did not cross-react with type III collagen consistent with the manufacturers report that the antibody has minimal cross reactivity to types II, III, IV, V, and VI collagen. The anti-collagen type III antibody did not substantially cross-react with type I collagen consistent with the manufacturers data indicating that the antibody does recognize collagen types I, II, IV, V, VI and X.

Statistical analysis

Due to the non-factorial nature of our experimental design, statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by pair wise comparisons. The level of significance was set to 0.05 and analysis was performed with SAS PROC MIXED (SAS Institute, Inc., Cary, NC). Fisher’s protected least significant difference test was used for post-hoc multicomparisons. For analyzing failure location data, the non-parametric Kruskal-Wallis test was performed.

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Abbreviations

Extracellular matrix (ECM), Growth hormone (GH), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hindlimb Unloaded (HU), Insulin-like growth factor I (IGF-I), Insulin-like growth factor receptor (IGFR), Medial collateral ligament (MCL), Multiphoton Laser Scanning Microscopy (MPLSM), Platelet derived growth factor (PDGF); Group Abbreviations: sham control surgery – ambulatory + saline (Sham), healing – ambulatory + saline (Amb + Sal), healing – hindlimb unloaded + saline (HU + Sal), healing – ambulatory + GH (Amb + GH), healing – hindlimb unloaded + GH (HU + GH), healing – Ambulatory + IGF-I (Amb + IGF), healing – hindlimb unloaded + IGF-I (HU + IGF), healing – ambulatory + GH + IGF-I (Amb + GH + IGF), healing – hindlimb unloaded + GH + IGF-I (HU + GH + IGF).

L-Ascorbic acid 2-phosphate promotes IGF-1 expression and stimulates hair follicle growth

Abstract

BACKGROUND:

l-Ascorbic acid 2-phosphate (Asc 2-P), a derivative of l-ascorbic acid, promotes elongation of hair shafts in cultured human hair follicles and induces hair growth in mice.

OBJECTIVES:

To investigate whether the promotion of hair growth by Asc 2-P is mediated by insulin-like growth factor-1 (IGF-1) and, if so, to investigate the mechanism of the Asc 2-P-induced IGF-1 expression.

METHODS:

Dermal papilla (DP) cells were cultured and IGF-1 level was measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay after Asc 2-P treatment in the absence or presence of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Also, hair shaft elongation in cultured human scalp hair follicles and proliferation of cocultured keratinocytes were examined after Asc 2-P treatment in the absence or presence of neutralizing antibody against IGF-1. In addition, keratinocyte proliferation in cultured hair follicles after Asc 2-P treatment in the absence or presence of LY294002 was examined by Ki-67 immunostaining.

RESULTS:

IGF-1 mRNA in DP cells was upregulated and IGF-1 protein in the conditioned medium of DP cells was significantly increased after treatment with Asc 2-P. Immunohistochemical staining showed that IGF-1 staining is increased in the DP of cultured human hair follicles by Asc 2-P. The neutralizing antibody against IGF-1 significantly suppressed the Asc 2-P-mediated elongation of hair shafts in hair follicle organ culture and significantly attenuated Asc 2-P-induced growth of cocultured keratinocytes. LY294002 significantly attenuated Asc 2-P-inducible IGF-1 expression and proliferation of follicular keratinocytes in cultured hair follicles.

CONCLUSIONS:

These data show that Asc 2-P-inducible IGF-1 from DP cells promotes proliferation of follicular keratinocytes and stimulates hair follicle growth in vitro via PI3K.

PMID:

19416266

[PubMed – indexed for MEDLINE]

A Role for Insulin-Like Growth Factor-I (IGF-1) in the Regulation of Schwann Cell Survival

Daniel E. Syroid,1 Todd S. Zorick,1,3 Christophe Arbet-Engels,2 Trevor J. Kilpatrick,1 Walter Eckhart,2 and Greg Lemke1
1Molecular Neurobiology Laboratory and 2Molecular Biology and Virology Laboratory, The Salk Institute for BiologicalStudies, La Jolla, California 92037, and 3Department of Neurosciences, University of California San Diego, La Jolla, California 92093

During postnatal development in the peripheral nerve, differentiating Schwann cells are susceptible to apoptotic death. Schwann cell apoptosis is regulated by axons and serves as one mechanism through which axon and  Schwann cell numbers are correctly matched. This regulation is mediated in part by the provision of limiting axon-derived trophic molecules, although neuregulin-1 (NRG-1) is the only trophic factor shownto date to support  Schwann cell survival. In this report, we identify insulin-like growth factor-I (IGF-I) as an additional trophin that can promote Schwann cell survival in vitro. We find that IGF-I, like NRG-1, can prevent the apoptotic death of postnatal rat Schwann cells cultured under conditions of serum withdrawal. Moreover, we show that differentiating Schwann cells in the rat sciatic nerve express both the IGF-I receptor (IGF-I R) and IGF-I throughout  postnatal development. These results indicate that IGF-I is likely to control Schwann cell viability in the developing peripheral nerve and, together with other findings, raise the interesting possibility that such survival regulation may switch during postnatal development from an axon-dependent mechanism to an autocrine and/or paracrine one.
Key words: peripheral nervous system; glia; Schwann cell; myelination; apoptosis; insulin-like growth factor-I

Programmed death is a cell fate adopted by multicellular organisms to control cell number in development, homeostasis, and defense. Such physiological cell death, which most often proceeds through a series of well-defined  alterations in cellular morphology termed apoptosis, is genetically programmed and results in cell suicide. Importantly, apoptosis must be tightly regulated such that only certain cells are specified to die, and it is now clear that both cell-extrinsic and cell-intrinsic signals can render cells susceptible to apoptosis (Steller, 1995; Fraser et al., 1996; White, 1996). During development, programmed cell death is often controlled by the positive  selection of cells via specific ligand–receptor tyrosine kinase interactions. Approximately 50% of immature postmitotic neurons normally undergo apoptotic death. In this instance, extrinsic signals delimit neuronal number and establish appropriate innervation patterns through competition for limiting target-derived trophins (Barde, 1989; Oppenheim, 1991). Death occurs when neurons fail to secure access to these trophins and consequently are unable to suppress a constitutive cell death program (Raff, 1992; Raff et al., 1993). Apoptosis also occurs in differentiating oligodendrocytes and Schwann cells, the myelinating glial cells of the CNS and peripheral nervous  system, respectively. Approximately 50% of newly generated oligodendrocytes die during development of the rodent optic nerve (Raff et al., 1993; Barres and Raff, 1994), and differentiating Schwann cells undergo apoptotic death during both embryonic (Ciutat et al., 1996) and early postnatal (Grinspan et al., 1996; Syroid et al., 1996; Nakao et al., 1997) development.  Cell death in these glial lineages is thought to be in part regulated by the limited availability of axon-derived trophic factors and represents one mechanism whereby the appropriate stoichiometry between glia and the axons they myelinate is achieved (Raff et al., 1993; Barres and Raff, 1994; Zorick and emke, 1996). Understanding the regulation of Schwann cell apoptosis requires an identification of the trophins that control Schwann cell viability. Apoptosis in Schwann cell precursors in vitro (Dong et al., 1995) and in  postnatal Schwann cells in vivo (Grinspan et al., 1996; Trachtenberg and Thompson, 1996; Kopp et al., 1997) and in vitro (Syroid et al., 1996) can be prevented by neuregulin-1 (NRG-1), which is normally supplied by axons  (Carraway and Burden, 1995; Lemke, 1996). Another set of factors that may mediate Schwann cell survival is the insulin-like growth factors (IGFs)-I and -II. As autocrine and/or paracrine factors, the IGFs are thought to play  an important role in the development an regeneration of the nervous system (Hansson, 1993; Ishii et al., 1993; Lewis et al., 1993a; de Pablo and de la Rosa, 1995). IGF-I, for example, regulates oligodendrocyte development  and myelination in vitro and in vivo (McMorris et al., 1986, 1993; McMorris and Dubois-Dalcq, 1988; Saneto et al., 1988; Mozell and McMorris, 1991; Barres and Raff, 1994; Yao et al., 1995). Reduced Received Oct. 23, 1998; revised Dec. 22, 1998; accepted Dec. 28, 1998. This work was supported by postdoctoral fellowships from the National Multiple Sclerosis Society to D.E.S. and from the Howard Hughes Medical Institute and the Bushell  Fellowship of the Royal Australasian College of Physicians to T.J.K., by predoctoral fellowships from the Department of Defense and the National Institutes of Health to T.S.Z., and by grants from the National Institutes of   Health to W.E. and G.L. We thank Danny Ortun˜o, Patrick Burrola, and Darcie Baynes for excellent technical assistance, Stefano Bertuzzi for help with RNase protection assays, Dan Peterson of the Gage laboratory for use of  their confocal microscope, Jill Meisenhelder  or peptide synthesis, and Bob Hyman for kindly providing anti-Thy-1antibodies.

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Effect of locally delivered IGF-1 on nerve regeneration during aging: an experimental study in rats

Abstract

Age is an important predictor of neuromuscular recovery after peripheral nerve injury. Insulin-like growth factor 1 (IGF-1) is a potent neurotrophic factor that is known to decline with increasing age. The purpose of this study was to determine if locally delivered IGF-1 would improve nerve regeneration and neuromuscular recovery in aged animals. Young and aged rats underwent nerve transection and repair with either saline or IGF-1 continuously delivered to the site of the nerve repair. After 3 months, nerve regeneration and neuromuscular junction morphology were assessed. In both young and aged animals, IGF-1 significantly improved axon number, diameter, and density. IGF-1 also significantly increased myelination and Schwann cell activity and preserved the morphology of the postsynaptic neuromuscular junction (NMJ). These results show that aged regenerating nerve is sensitive to IGF-1 treatment.

Keywords: aging, IGF-1, nerve injury, nerve regeneration, neuromuscular junction

Age is thought to be the most important predictor of neuromuscular recovery after peripheral nerve injury.¹ Of the 18 million extremity injuries recorded annually in the USA, >50% occur in patients 45 years of age or older.² Children often regain near-normal function after nerve injury,^3,4 but adults or aged patients have slow or absent neuromuscular recovery. Over half of patients >50 years of age do not achieve any functional recovery following nerve repair,¹ resulting in impaired activities of daily living. Aging is known to affect the structure and function of the peripheral nerve,^5,6 but the exact etiology of age-related impairment in regeneration is unknown, although delayed axonal regeneration^7,8 and instability of the neuromuscular junction (NMJ) have been implicated.⁹

Insulin-like growth factor 1 (IGF-1) is a single-chain, 70-amino-acid polypeptide, with a molecular mass of 7.6 kDa, similar in structure to pro-insulin, and is highly conserved across species.¹⁰ IGF-1 plays a significant role in neuronal development, ^11,12 recovery from neuronal injury,^13,14 neuronal survival,¹⁵ and neurite outgrowth following crush injury.¹⁶ In vitro studies have suggested that IGF-1 is produced locally by non-neuronal cells following injury and stimulates regeneration.¹⁷ Hansson et al. first showed that IGF-1 is secreted from Schwann cells in an autocrine fashion after peripheral nerve injury.¹³ IGF-1 has been shown to affect multiple facets of Schwann cell function in vitro, which likely contributes to improved nerve regeneration, ¹⁸ including proliferation,¹⁹ mobilization, myelination,²⁰ and Schwann cell–axon interaction. IGF-1 knockout mice have defects in neurologic development²¹ in addition to impaired recovery from neuronal injuries.²² Thus, there is substantial evidence that IGF-1 plays a key role in neuromuscular recovery following injury.

Age and developmental stage are the major determinants of IGF-1 levels. IGF-1 levels peak during puberty, initiating and maintaining the pubertal growth spurt.²³ Following the pubertal growth spurt, IGF-1 levels decline²⁴ at a rate of approximately 14% per decade.^25,26 With continued aging, IGF-1 concentrations fall to levels of 20–80% of values for young adults,²⁷ concordant with the decline in functional recovery following nerve transection and repair in aged adults.¹

Given the wide-ranging effects of IGF-1 on nerve regeneration and the known decline in IGF-1 with age, we sought to determine whether IGF-1 replacement would improve nerve regeneration in aged animals. Previously, we found that increasing systemic IGF-1 via pulsatile growth hormone injection did not improve nerve regeneration (unpublished data). Thus, the purpose of this study was to examine the effects of IGF-1 locally delivered to a nerve transection site in aged rats, and to evaluate neuromuscular recovery, axonal regeneration, myelination, Schwann cell activity, and the resulting changes at the NMJ. The hypothesis was that age-related decline in neuromuscular recovery following transection would be ameliorated by IGF-1 administered to the regenerating nerve.

METHODS

Animal Model

Thirty-two Fischer 344 × Brown Norway rats were obtained from the National Institute of Aging colony at the Harlan Sprague-Dawley facility (Indianapolis, Indiana). The animals were divided into two groups: young adult (8 months) and aged (24 months). Each age group was subdivided into saline and IGF-1 treatment groups. There were 6 young adult saline, 6 young adult IGF, 10 aged saline, and 10 aged IGF-1 animals. Animals underwent nerve transection and repair, as detailed in what follows, and were then killed after 12 weeks.

Surgical Procedure

Each animal was anesthetized with isoflurane, and an incision was made over the posterior aspect of the left thigh. Under the operating microscope, the sciatic nerve was exposed, and the tibial nerve was isolated and transected 1 cm from its insertion into the gastrocnemius. The nerve stumps were placed at opposing ends of a custom-made T-tube and the middle arm was attached to a minipump (Fig. 1). The minipump was buried subcutaneously under the skin of the back. The nerve conduit portion of the T-tube was made from 1016-µm internal diameter, semiporous tubing (Micro-Renathane; Braintree Scientific, Braintree, Massachusetts), and the T-arm was constructed of Silastic tubing (Dow Corning, Midland, Michigan). An Alzet 2004 mini-osmotic pump (Durect Corp., Cupertino, California) delivered either normal saline or IGF-1 at a rate of 0.25 µl/h. Half of the animals received recombinant human IGF-1 (Bachem AG, Torrance, California) at a 0.10-µg/µl (100 µg/ml) concentration. Thus, final IGF-1 delivery was 0.025 µg/h, modeled after previous studies of IGF-1 infusion.²⁸ The other animals received normal saline only. The subcutaneous pumps were replaced under anesthesia after 6 weeks. At 12 weeks after nerve transection and repair, tissue was harvested, and the animals were overdosed with isoflurane and perfused with fixative.

FIGURE 1 Diagram of experimental setup. The tibial nerve was transected 1 cm from the insertion into the gastrocnemius and allowed to relax to create a 7-mm gap. A custom-made T-tube device was interposed between the nerve ends and secured with a 9-0 nylon suture. (more …)

Animals were group-housed and allowed food and water ad libitum. All experiments were conducted under a protocol approved by the institutional animal care and use committee. The animals were housed in an appropriate facility in accordance with standards established by the Association for Assessment and Accreditation of Laboratory Animal Care.

Muscle and Tissue Harvest

At study termination, the animals were anesthetized with isoflurane, and the gastrocnemius was harvested. The muscle was cleaned, rinsed, flash frozen in liquid nitrogen, and stored at −80°C. Following removal of the gastrocnemius, the animals were transcardially perfused with warm saline followed by ice-cold 4% paraformaldehyde. The regenerated nerve was then removed and immersion-fixed in 2.5% glutaraldehyde.

Nerve Histomorphometry

The midpoint of the regenerated nerve was secondarily fixed in 1% osmium tetroxide, embedded in resin, sectioned to 1 µm, and stained with toluidine blue. All images were acquired microscopically (AxioImager M1; Carl Zeiss, Jena, Germany) using AxioVision version 4.5 software. Images were imported into ImageJ (National Institutes of Health, Bethesda, Maryland) for analysis. For the peripheral nerve, the following measurements were taken: nerve diameter; nerve cross-sectional area; axon number; axon density (axons per mm²); average axon diameter; and the average g-ratio (ratio of axon diameter to fiber diameter). Nerve diameter and nerve area were measured on 100× or 200× images. For axon number and axon density, two or three non-overlapping 400× fields were obtained from each of the four quadrants of the nerve. For average axon diameter and the average g-ratio, three or four non-overlapping 1000× fields were obtained from each of the four quadrants of the nerve. In each 400× high-power field, the myelinated axons were counted, and the area sampled was calculated, yielding axon density. For axon diameter and g-ratio, a minimum of 100 nerve fibers were analyzed. In order to focus analysis on motor neurons, axons <1 µm were not measured. For axon diameter, the measurement was from made from the inner table of the myelin sheath and did not include the myelin layer. The g-ratio was calculated by measuring axon diameter and total fiber diameter.

mRNA Expression of GAP43

The lateral gastrocnemius was pulverized, and total RNA was extracted with Trizol (Sigma-Aldrich, St. Louis, Missouri). Quantity and purity of the RNA was assessed using a spectrophotometer (ND-1000; NanoDrop Technologies, Wilmington, Delaware), and quality of RNA was assessed by microcapillary gel electrophoresis (Model 2100 Bioanalyzer; Agilent Technologies, Santa Clara, California) prior to downstream applications. The mRNA was transcribed to cDNA using oligo-dT primers (Invitrogen, Carlsbad, California) and Superscript II (Invitrogen, Carlsbad, California). Primers were obtained from Applied Biosystems for GAP43 (NM_017195.3). Relative quantitative reverse transcript–polymerase chain reaction (qRT-PCR) was then performed (Model 7900HT; Applied Biosystems) with a 384-well block. Muscle creatine kinase (MCK) was used as an endogenous control, as it has been shown to be stably expressed in skeletal muscle with age.²⁹

Histology of Postsynaptic NMJs

The medial gastrocnemius was pinned to resting length and immersed in isopentane cooled with dry ice. The muscle was sectioned on a cryotome longitudinally in 25-µm sections. The sections were permeabilized with 0.1% Triton X-100 for 20 minutes, and then treated with AlexaFluor 488–conjugated α-bungarotoxin (1:200; Invitrogen, Carlsbad, California) for 1 hour. Sections were rinsed and fixed in 1% paraformaldehyde for 1 hour. Confocal images were obtained on a laser scanning confocal microscope (Zeiss 510) with argon and helium–neon lasers. Slice thickness was set at ≤1.2 µm. Slices were then merged to create the final image. The NMJs were then qualitatively evaluated.

Statistical Analysis

All data were imported into SigmaStat version 3.11 (Systat Software, Inc., San Jose, California) for analysis. For each outcome measure, a two-way analysis of variance (ANOVA) model was established with age and treatment as the independent variables. Post hoc pairwise comparisons were made with the Student–Newman–Keuls test. Transformations to ranked sums were made when the data were nonparametric. Type 1 error was set at 0.05.

RESULTS

IGF-1 Increases Axon Number, Diameter, and Density in Regenerated Nerve of both Young and Aged Animals

Examination of the peripheral nerve revealed that, in aged saline-treated nerves, there were fewer total axons, fewer axons per mm², and less myelination than in young saline-treated nerves. IGF-1 had a marked effect on both young and aged animals, increasing axon number, diameter, and density, in addition to increasing myelination (Fig. 2). Quantitative data are presented in Figure 3. For the number of axons per nerve, IGF-1 increased total axon number in both young and aged animals; however, this was only statistically significant for aged animals. For axon density, IGF-1 increased axon density in both young and aged animals; however, this was only statistically significant for aged animals. For average axonal diameter, there was a significant increase in average axonal diameter in both young and aged animals when they were treated with IGF-1. There was no difference between the young IGF-1 group and aged IGF-1 group for number of axons per nerve, axon density, and average axonal diameter.

FIGURE 2 Cross-section of regenerated nerve segment. After 3 months, the regenerated nerve segment was removed, sectioned to 1 µm, stained with toluidine blue, and viewed at 1000×. (A) Young saline, (B) aged saline, (C) young IGF-1, and (D) aged (more …)

FIGURE 3 Effect of age and IGF-1 on axon number, density, and diameter. Quantification of histologic images reveals a significant effect of IGF-1 on (A) axons per nerve, (B) axon density (axons per mm2), and (C) average axonal diameter. Although there were differences (more …)

IGF-1 Increases Myelination and Schwann Cell Activity in Regenerated Nerve of both Young and Aged Animals

Three parameters of Schwann cell activity and myelination were examined: (1) myelin thickness; (2) axon/fiber ratio (g-ratio); and (3) GAP43 mRNA expression, a marker of Schwann cell activity. ^30,31 We found that IGF-1 significantly affected all measured aspects of Schwann cell activity and myelination (Fig. 4). For myelin thickness and g-ratio, this effect was not age-dependent. However, for GAP43 expression, aged animals had a greater response to IGF-1 than young animals (Fig. 4C).

FIGURE 4 Effect of age and IGF-1 on measures of myelination. Quantification of histologic images revealed a significant effect of IGF-1 on multiple facets of myelination and Schwann cell function. (A) Myelin thickness was significantly increased in IGF-1–treated (more …)

IGF-1 Preserves Morphology of Postsynaptic NMJs in Aged Animals

The postsynaptic NMJs were stained with fluorescent-labeled α-bungarotoxin and qualitatively examined using laser scanning confocal microscopy (Fig. 5). In saline-treated young animals, the postsynaptic NMJs were highly contiguous, complex, and had deep gutters, similar to uninjured control NMJs (images not shown). In young IGF-1 animals, the NMJs were very similar to those in the young saline group. In the aged saline group, numerous NMJs displayed a loss of complexity, with markedly more shallow gutters, and a loss of perimeter and endplate area. This is consistent with the morphology of denervated NMJs.^32,33 In contrast, in the aged IGF-1 animals, the complexity, gutter depth, and overall morphology more closely resembled that of young animals.

FIGURE 5 Response of the neuromuscular junction to saline or IGF-1 treatment. (A) Young saline, (B) aged saline, (C) young IGF-1, and (D) aged IGF-1. The postsynaptic NMJ was stained with fluorescent-labeled α-bungarotoxin and imaged under a confocal microscope. (more …)

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DISCUSSION

In this study we sought to determine whether IGF-1 would improve neuromuscular recovery after nerve injury during aging. We found that IGF-1 improved nerve regeneration by acting on the axons and Schwann cells, and secondarily on the NMJs. These results suggest that there is no loss of sensitivity to IGF-1 with age and that IGF-1 can improve regeneration after nerve injury during aging.

Previous studies have examined the effect of systemically administered IGF-1 during neuromuscular recovery in neonatal rats. Vergani et al. found that systemic IGF-1 promoted neuronal survival following crush injury and improved muscle reinnervation.³⁴ Kanje et al. found that locally delivered IGF-1 following crush injury in juveniles led to improved sensory function.³⁵ Tiancgo et al. found that IGF-1 locally delivered to an end-to-side repair improved muscle function in young rats.³⁶ To the authors’ knowledge, the current study is the first to examine the effect of IGF-1 during nerve regeneration in aged animals.

Axonal outgrowth is central to nerve regeneration, and IGF-1 is a well-documented promoter of motor neuron survival,³⁷ axonal growth,³⁸ and axonal branching.¹⁸ In the regenerating nerve, IGF-1 is a key promoter of initial sprouting and subsequent elongation of axons³⁹ (for review, see Rabinovsky²). Although there has been extensive investigation of the effect of IGF-1 on neuron outgrowth and survival, nearly all of the studies were performed in vitro or in embryonic animals. Thus, it is not known how increased age affects the neuronal response to IGF-1. In this study we found that, for metrics of neuronal trophism, aged animals had a robust response to IGF-1 treatment, increasing axonal number, diameter, and density in the regenerated nerve segment. Thus, the neuronal response to IGF-1 of the regenerating axon does not appear to be limited due to age.

The Schwann cell is a key factor in promotion of nerve regeneration. The effect of IGF-1 on Schwann cells has also been studied. IGF-1 is known to promote Schwann cell survival,⁴⁰ motility,⁴¹ proliferation, ⁴² and myelination.⁴³ However, the physiology of Schwann cells from aged animals is largely unknown. This study has addressed some of the parameters of Schwann cell function in vivo, particularly myelination and GAP43 expression. GAP43 is upregulated in nearly all myelinating Schwann cells of the distal stump following axotomy.³¹ Expression of GAP43 is postulated to play a role in the interaction between the regenerating axon and Schwann cell.³⁰ In this study, we found that GAP43 expression was significantly upregulated in both young and aged animals with IGF-1 treatment. In addition, GAP43 expression was higher in aged animals treated with IGF-1 than in similarly treated young animals, suggesting that age increases sensitivity to IGF-1 for this aspect of Schwann cell function. However, due to pooling of the whole muscle mRNA, in this study we could differentiate between an increase in Schwann cell number and an increase in activity. Furthermore, because the sample was from muscle, the increase in GAP43 expression was due to intramuscular neurons, terminal Schwann cells, or both. GAP43 expression in the regenerated segment itself was not assayed. In addition to GAP43 activity, IGF-1 increased myelination in the regenerated nerve, independent of the increase in axon diameter. Thus, at the Schwann cell level, age does not impair the response to IGF-1 and results in an increased regenerative response.

Failure to achieve reinnervation of the muscle is thought to contribute to impaired neuromuscular recovery after nerve injury.⁴⁴ In this study we found by histologic examination that the motor endplate regained an innervated, pre-injury morphology in all but the aged saline group. This failure to reinnervate may have been due to delayed nerve regeneration or instability of the postsynaptic NMJ, both of which have been shown to occur with aging.⁹ In this study, treatment with IGF-1 may have accelerated axonal regeneration, leading to earlier reinnervation of the muscle, and thus preservational of the postsynaptic NMJ.

One limitation of our study is the lack of functional and electrical assessments. Functional recovery is the gold standard for clinical outcomes in human subjects, but, historically, functional recovery in rodent models has been difficult to assess.⁴⁵ In rats, there is a poor correlation between functional, electrophysiologic, and histologic outcomes. ⁴⁶ In this study, electrical measures of function were performed and showed a trend toward improvement with IGF-1 treatment, but this was not statistically significant (data not shown). Nonetheless, we were able to demonstrate that IGF-1 improved nerve regeneration in aged animals.

An important strength of our study is the in vivo comparison between young adult and aged rats after a complete nerve transection. Previous studies of IGF-1 on nerve regeneration utilized in vitro, neonatal, or juvenile animal models. In addition, many previous studies utilized crush injury models. Crush injuries in rodents typically have a near 100% rate of recovery and do not accurately model clinical scenarios wherein neuromuscular recovery is often very poor. Although previous studies have helped to answer many questions about the effects of IGF-1, this study is the first to examine in vivo the effects of age and IGF-1 on neuromuscular recovery following nerve transection.

Overall, our study has provided substantial evidence that locally delivered IGF-1 improves neuromuscular recovery after nerve injury in aged animals. We showed that, when local IGF-1 levels were equally supplemented in young and aged animals, nerve regeneration and NMJ preservation were similar, suggesting that IGF-1 may be a contributing factor in age-related impairment of neuromuscular recovery. However, our study has not conclusively demonstrated that IGF-1 deficiency is the immediate cause of age-related impairments in neuromuscular recovery after injury.

Insulin and IGF-1 are important hair growth promoters – new candidates in the fight against hair loss?

Effects of insulin and insulin-like growth factors on cultured human hair follicles: IGF-I at physiologic concentrations is an important regulator of hair follicle growth in vitro.

Philpott MP, Sanders DA, Kealey T.

Source

Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, U.K.

Abstract

Insulin stimulated hair follicle growth in a dose-dependent manner over the range of 0.01 to 100 micrograms/ml. Maximum rates of hair follicle growth were observed when follicles were maintained in medium containing 10 micrograms/ml insulin, which is supraphysiologic. Hair follicles maintained in the absence of insulin or at physiologic levels showed premature entry into a catagen-like state. Insulin-like growth factor (IGF)-I and -II had no significant effect on hair follicle growth when maintained in the presence of 10 micrograms/ml insulin. However, in the absence of insulin, both IGF-I (0.01-100 ng/ml) and IGF-II (0.01-100 ng/ml) stimulated hair follicle growth in a dose-dependent manner. IGF-I was more potent than either insulin or IGF-II, stimulating maximum rates of hair follicle growth at 10 ng/ml, whereas IGF-II gave maximum stimulation at 100 ng/ml. The rates of hair follicle growth stimulated by 10 ng/ml IGF-I were identical to those stimulated by 10 micrograms/ml insulin. IGF-II (100 ng/ml), however, was unable to stimulate hair follicle growth to the same extent as insulin. Both IGF-I (10 ng/ml) and IGF-II (100 ng/ml) were more potent than insulin at preventing hair follicles from entering into a catagen-like state. Growth hormone had no effect on hair follicle growth or morphology in the absence of insulin. These data suggest that in vitro IGF-I may be an important physiologic regulator of hair growth and possibly the hair growth cycle. Moreover, the removal of insulin from tissue culture medium may be a useful method of generating large numbers of catagen hair follicles for further in vitro studies.

PMID:

8006448

[PubMed – indexed for MEDLINE]

Raspberry extract on your skin and head – combat baldness and rejuvenate your skin, as it raises IGF-1 levels

BUY IGF-1 LR3 or DES-IGF FROM US! superhumangear@gmail.com

Effect of topical application of raspberry ketone on dermal production of insulin-like growth factor-I in mice and on hair growth and skin elasticity in humans.

Harada N, Okajima K, Narimatsu N, Kurihara H, Nakagata N.

Source

Department of Translational Medical Science Research, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.

Abstract

Sensory neurons release calcitonin gene-related peptide (CGRP) on activation. We recently reported that topical application of capsaicin increases facial skin elasticity and promotes hair growth by increasing dermal insulin-like growth factor-I (IGF-I) production through activation of sensory neurons in mice and humans. Raspberry ketone (RK), a major aromatic compound contained in red raspberries (Rubus idaeus), has a structure similar to that of capsaicin. Thus, it is possible that RK activates sensory neurons, thereby increasing skin elasticity and promoting hair growth by increasing dermal IGF-I production. In the present study, we examined this possibility in mice and humans. RK, at concentrations higher than 1 microM, significantly increased CGRP release from dorsal root ganglion neurons (DRG) isolated from wild-type (WT) mice and this increase was completely reversed by capsazepine, an inhibitor of vanilloid receptor-1 activation. Topical application of 0.01% RK increased dermal IGF-I levels at 30 min after application in WT mice, but not in CGRP-knockout mice. Topical application of 0.01% RK increased immunohistochemical expression of IGF-I at dermal papillae in hair follicles and promoted hair re-growth in WT mice at 4 weeks after the application. When applied topically to the scalp and facial skin, 0.01% RK promoted hair growth in 50.0% of humans with alopecia (n=10) at 5 months after application and increased cheek skin elasticity at 2 weeks after application in 5 females (p<0.04). These observations strongly suggest that RK might increase dermal IGF-I production through sensory neuron activation, thereby promoting hair growth and increasing skin elasticity.

PMID:

18321745

[PubMed – indexed for MEDLINE]