The evidence for the role of transforming growth factor‐beta in the formation of abnormal scarring

Abstract

The complex biological and physiological mechanisms that result in poor quality scarring are still not fully understood. This review looks at current evidence of the role of transforming growth factor‐beta (TGFβ) in this pathological process.

INTRODUCTION

Wound healing is a complex dynamic process that involves the coordination of numerous physiological, immunological and cellular pathways (1). Transforming growth factor‐beta (TGFβ) is a 25‐kDa homodimeric protein with three distinct genes (TGFβ1, TGFβ2 and TGFβ3) identified in mammalian cells (2). It is produced by numerous cells within the body including activated T‐cells, macrophages and neutrophils (3), platelets (4) and bone (5). Since its identification and purification in 1983 4, 6, it has been shown to be an important cytokine in regulation of various biological processes including angiogenesis (7), bone formation and chondrogenesis 8, 9, haematopoiesis (10), carcinogenesis (11) and the production of granulation tissue 7, 12. With TGFβ contributing to various aspects of normal wound healing physiology, it is therefore implicated in pathological processes leading to abnormal scarring 13, 14. This review looks at the literature related to the role TGFβ has in wound healing leading to the formation of abnormal scar tissue. This review was performed as part of the Master of Science/Postgraduate Diploma in Wound Healing and Tissue Repair, Cardiff University.

ABNORMAL SCARRING

Hypertrophic scarring (HTS) and keloid scarring (KS) represent abnormal healing responses to a cutaneous injury. A keloid scar clinically extends beyond the margins of the original injury whilst a hypertrophic scar does not (15). The process is complex, multifactorial and the true pathogenesis is not yet established (16). They have similar pathological mechanisms that result in the deposition of excess extracellular matrix and collagen within the healing wound and as such the terminologies have often been used interchangeably (17). Whilst the normal healing cycle of inflammation, proliferation and maturation is prolonged in the hypertrophic scar, the keloid scar does not follow this process and it is now regarded that they are two separate processes (16).

Investigations into normal scar formation and fibrotic disease such as pulmonary fibrosis (18) and scleroderma (19) have identified TGFβ and its isoforms as important mediators in the development of abnormal scarring. In addition, the study on foetal scarring has strongly linked TGFβ with scar promotion. In a study by Krummel et al. (20), a rabbit foetal model showed no sign of scar by day 7 whilst addition of exogenous TGFβ to the foetus induced a prompt inflammatory reaction, fibroblast proliferation and fibrosis. Interestingly at higher doses, TGFβ appeared to be inhibitory which adds to the diversity of the cytokine.

The main body of work in this area has focused on the influence TGFβ has on fibroblast function. The fibroblast has an extremely important role in wound healing and subsequently in the abnormal scarring process. Early in the proliferative phase of healing, stimulated by activated platelets and macrophages, fibroblasts stimulate keratinocyte migration and epithelialisation (21). Growth factors from platelets and macrophages such as platelet‐derived growth factor (PDGF) and epidermal growth factor (EGF) promote fibroblast proliferation (22) and formation of extracellular matrix products such as collagen, fibronectin, proteoglycans, hyaluronic acid, proteases and granulation tissue (23). Production of TGFβ1 by macrophages promotes differentiation into contractile myofibroblasts to aid wound contraction and closure (12). Montesano and Orchi demonstrated that TGFβ was able to induce normal human fibroblasts to strongly contract in an in vitro collagen‐matrix system suggesting TGFβ promotes myofibroblast function in the contracting wound (24).

Bettinger et al. investigated the effect of TGFβ on fibroblasts derived from a number of black patients (14). The samples were from keloid excisions, adjacent normal skin or from normal skin. The fact that we are not told about the demographics of the subjects, the sites of the biopsies and whether ethical approval was gained for this study is not mentioned. Excising normal tissue adjacent to the keloid excision risks recurrence rates of 40–100% and is not currently recommended (25). The authors found that exposure to TGFβ at a 5·0 ng/ml concentration significantly increased absolute collagen synthesis as defined by DNA content, in the keloid fibroblasts and did not alter collagen synthesis in the normal fibroblasts. This supports the hypothesis that keloid fibroblasts have an augmented response to TGFβ. Another finding of note was that out of two normal and four keloid fibroblast samples, type I procollagen transcript levels increased in all the keloid lines tested and one of the normal lines, whilst type III procollagen was increased in all cell lines tested. This is not remarked upon in the discussion and does not support other research that found an increase in type I procollagen expression in keloid fibroblasts but none in type III procollagen expression (26). This disparity may represent the short comings of in vitro studies where the numerous cellular interactions and autocrine and paracrine effects of cytokines cannot be easily replicated. The literature does not appear to be consistent on the relative concentrations of type I versus type III collagen in keloid scars. This may represent the dynamic nature of these scars. Abergel et al. quantified relative concentrations of collagen in keloid scars in a series of nine black patients (27). In the normal skin, the ratio of type I to type III was 3:1. In the keloid tissue, the ratio was approaching 15:1. This represent keloid scars only and should not be directly compared to hypertrophic scars.

It is important to point out that the process of wound healing and subsequently abnormal scarring is complex and relies on multiple regulatory mechanisms involving various cytokines other than TGFβ (17). Polo et al. looked at the composition of peripheral blood samples from patients with burn injuries with and without hypertrophic scarring and showed that patients with hypertrophic burn scars demonstrated higher systemic levels of inflammatory cytokines such as Interleukin (IL)‐1), IL‐6, tumour necrosis factor‐alpha and TGFβ compared to controls (28). Zhang et al. investigated whether decorin, a proteoglycan could exert any effects on wound healing when added to a fibroblast‐populated collagen lattice (FPCL) (29). Addition of TGFβ to the hypertrophic scar fibroblasts resulted in a greater contractile ability in the hypertrophic scar fibroblasts whilst addition of decorin inhibited both basal and TGFβ contraction of the matrix in normal and hypertrophic scar fibroblast. It also inhibits TGFβ1‐induced alpha‐smooth muscle actin (αSMA) and plasminogen activator inhibitor. There may be a therapeutic role for decorin in the inhibition of TGFβ wound contraction.

Schmid et al. investigated the expression patterns of the TGFβ type 1 and 2 receptors (RI and RII) in normal skin, granulating tissue, healed scar and post‐burn hypertrophic scar (30). The authors found that in quiescent fibroblasts in the non wounded skin, the expression of TGFβ receptor expression was low. However, in the granulating wound there was a strong expression of both RI and RII receptors. This then resolved over time corresponding to the healing wound. In contrast, the hypertrophic scar fibroblasts showed an over‐expression of RI and RII receptors up to 2 years post injury. With TGFβ known to have the ability to locally upregulate TGFβ1 mRNA transcription via autocrine mechanisms which propagate the TGFβ effects (31), the authors propose that abnormal scar formation is due to an inability of the wound to downregulate fibroblastic cells sensitive to TGFβ. The study was also able to show that there was a failure to detect TGFβ receptors in a HTS after 8 years which is consistent with a slowly regressing scar.

The role of TGFβ in scar modulation is further supported by Shah et al. (32). The authors used an adult rat incisional model to show that addition of neutralising antibody to reduced levels of TGFβ1 and TGFβ2 resulted in reduced collagen content and improved orientation of the extracellular matrix in the wound and less scarring compared with controls. Later, Shah et al. showed that addition of TGFβ3 reduced levels of scarring in the rat model, suggesting an antagonistic role for TGFβ3 (33). This is now thought to be controlled by TGFβ3 downregulation of fibroblasts by TGFβ‐dependent and independent pathways (34). Further studies using TGFβ1 over‐expressed transgenic mice by Shah et al. showed that, whilst predicting that mice with high levels of circulating TGFβ1 will develop exaggerated scars that form more rapidly, what they actually discovered was that in the incisional wounded mice TGFβ1 was reduced whilst TGFβ3 was increased as was the TGFβ receptor II (35). In addition, the transgenic mice that had subcutaneous PVA sponges inserted had upregulation of TGFβ1, TGFβ2 and TGFβ3 as well as TGFβ receptors I and II, resulting in an increase in matrix production and deposition. The authors concluded that increased circulating levels of systemic TGFβ1 does not necessarily lead to an increased collagen response. The location of the incisional wounds and the PVA sponges on the dorsum and ventral aspect of the mice, respectively, may have contributed to an altered local cytokine response that was not controlled for during the study. Whilst the wounds were examined up to 80 days post wounding, the fact that human keloid scars may take longer to develop means that application of this model to pathological human scarring is limited. What it does add is that early imbalances of the three TGFβ subtypes in a healing wound can direct a healing wound towards an abnormal scar phenotype (34).

Smith et al. examined whether exogenous TGFβ2 would have an effect on an in vitro FPCL derived from keloid scars, burn hypertrophic scars and normal skin fibroblasts (36). On addition of TGFβ2, both the abnormal scar derived fibroblasts showed a significantly greater contraction than normal skin fibroblasts. Of interest this occurred at day 1 for the keloid group and days 4 and 5 in the hypertrophic group. The delay in response between cell lines is not discussed but may represent as yet undefined differences in the fibroblast populations in keloid versus hypertrophic scars. In addition, the authors added an anti‐TGFβ2 antibody to the FPCL and found that contraction was reduced in all fibroblast cell lines including those fibroblasts pretreated with TGFβ2. Whilst this study duration is only 5 days and hence application to a prolonged dynamic scarring process is limited, it does suggest that systemic treatment with monoclonal antibody to TGFβ may have a role in a future therapeutic management.

Further evidence of the link between keloid fibroblasts and TGFβ comes from studies that have shown higher levels of TGFβ1 and TGFβ2 proteins in keloid fibroblasts than in normal dermal fibroblasts (37). Additionally, Chin et al. demonstrated that TGFβ signalling pathways were upregulated in keloid fibroblasts with an increased expression of TGFβ receptors I and II (38). They go onto propose that elevated levels of receptors will lead to excessive signalling and production of TGFβ as seen in keloid pathogenesis. Fujiwara et al. identified that TGFβ1 reduces the production of metalloproteinase‐1 (MMP‐1) in the keloid fibroblasts cell lines whilst MMP‐2 was increased after treatment (39). As MMP‐1 can degrade the collagen triple helix, they play a central role in fibroblast migration meaning that over‐expression of TGFβ in keloid scarring may affect cellular migration, propagation and thus scarring.

There are a number of areas that do not comprehensively provide evidence to support the central role TGFβ has in excessive scarring. Clearly there is a genetic predisposition to keloid scarring primarily by racial predisposition (16). However, for a cytokine so closely related to these wound healing processes there is yet to be any genetic associations found in the population between the TGFβ gene and the TGFβ receptor gene 40, 41. There are multifactoral causes for the development of abnormal scarring and as keloid scars only occur in humans the use of animal models will always be an issue. However, the need for animal studies is obvious when considering the ethical implications of inducing or propagating a keloid scar in a patient. One of the questions remaining is whether the presence of TGFβ is as a result of the keloid or hypertrophic scar or the cause of it? Campaner et al. looked into this very question (42). TGFβ1 is produced in a latent inactive form bound to the latent‐TGFβ1‐binding protein. By genetically modifying normal human dermal fibroblasts to express a latent or an active form of TGFβ1, they were placed intradermally into the back of a mouse model. Only the active form of TGFβ1 in combination with smooth muscle cell (SMC) α‐actin was able to mount a fibroproliferative response and form keloid‐like lesion in situ. This showed that it was the activity of TGFβ1 rather than the presence of the protein (inactive TGFβ1) that was driving the response.

Therapies for keloid and hypertrophic scars are numerous and include pressure therapy, silicon hydration, steroids, surgery and radiotherapy (1). Therapies that focus directly on modulating TGFβ are less numerous. The use of interferons such as IFN‐α2b has been shown to be successful at reducing the recurrence rate after excisional surgery when compared to steroid injection or excision alone (43) with antagonistic interaction with TGFβ, resulting in reduced collagen production (44). More recently, tamoxifen (anti‐oestrogen) has been shown to reduce the expression of TGFβ in keloid cells (45), whilst the steroid, triamcinolone has been used in treatment of hypertrophic and keloid scars effectively for many years. In an in vitro study by Carroll et al., triamcinolone was shown to downregulate TGFβ in the keloid fibroblasts (46).

SUMMARY

TGFβ has been shown to be an important regulator of the wound healing process. In particular, it is the interaction with dermal fibroblasts and the subsequent production of extracellular products including collagen that highlights its central role in abnormal scarring. It is clear, however, that there are still areas that need further research. The fibroblasts that inhabit the hypertrophic and keloid wound are more responsive to TGFβ, but as yet there is still no definitive reason why. Whilst it is almost certainly the cytokine milieu, autocrine function and cell‐mediated control of TGFβ that is regulated in normal scar formation, the precise details of the physiology and biochemistry in abnormal scarring is still not available. Finally as more is known about the role of cytokines such as TGFβ in both keloid and hypertrophic scar pathologies, more targeted treatments can be developed.