[Vince001]

Misschien moeten we toch maar eens een aparte thread openen voor fibrose en TGF-b e.d...? Hier iig weer iets over fibrose (/tgf-b1), hoe het werkt en de ontwikkeling van een topical. Ik zal 't later nog wel ff wat mooier neerzetten.. En ook zorgen dat ik het zelf helemaal gelezen heb

Topical Application of a Peptide Inhibitor of Transforming Growth Factor-1 Ameliorates Bleomycin-Induced Skin Fibrosis
Begoña Santiago*,1, Irene Gutierrez-Cañas*,1, Javier Dotor†, Guillermo Palao*, Juan José Lasarte†, Juan Ruiz‡, Jesús Prieto†, Francisco Borrás-Cuesta† and José L Pablos*

*Unidad de Investigación, Hospital 12 de Octubre, Madrid, Spain
†Division of Hepatology and Gene Therapy, Centre for Applied Medical Research (CIMA), Universidad de Navarra, Pamplona, Spain
‡DIGNA Biotech, Madrid, Spain
Correspondence: José L. Pablos, Unidad de Investigación, Hospital 12 de Octubre, 28041 Madrid, Spain. Email: jlpablos@h12o.es

1B. Santiago and I. Gutierrez-Cañas contributed equally to this work.

Received 3 February 2005; Revised 15 April 2005; Accepted 13 May 2005.

Top of pageAbstract
Transforming growth factor- (TGF-) plays a crucial role in the pathogenesis of skin fibrotic diseases. Systemic TGF- inhibitors effectively inhibit fibrosis in different animal models; however, systemic inhibition of TGF- raises important safety issues because of the pleiotropic physiological effects of this factor. In this study, we have investigated whether topical application of P144 (a peptide inhibitor of TGF-1) ameliorates skin fibrosis in a well-characterized model of human scleroderma. C3H mice received daily subcutaneous injections of bleomycin for 4 wk, and were treated daily with either a lipogel containing P144 or control vehicle. Topical application of P144 significantly reduced skin fibrosis and soluble collagen content. Most importantly, in mice with established fibrosis, topical treatment with P144 lipogel for 2 wk significantly decreased skin fibrosis and soluble collagen content. Immunohistochemical studies in P144-treated mice revealed a remarkable suppression of connective tissue growth factor expression, fibroblast SMAD2/3 phosphorylation, and -smooth muscle actin positive myofibroblast development, whereas mast cell and mononuclear cell infiltration was not modified. These data suggest that topical application of P144, a peptide inhibitor of TGF-1, is a feasible strategy to treat pathological skin scarring and skin fibrotic diseases for which there is no specific therapy.

Keywords: antagonists, fibrosis, skin, topical administration, transforming growth factor

Abbreviations: -SMA, -smooth muscle-actin; CTGF, connective tissue growth factor; PBS, phosphate-buffered saline; TGF-, transforming growth factor-

Excessive accumulation of extracellular matrix (ECM) proteins is the hallmark of fibrotic skin conditions such as hypertrophic scarring, keloids, and localized or systemic sclerosis (scleroderma). This process is dependent on the activation of ECM synthesis in interstitial fibroblasts that often develop into -smooth muscle actin (-SMA)-positive myofibroblasts (Jimenez et al, 1996;Jelaska and Korn, 2000). One of the key molecular factors involved in both processes is transforming growth factor- (TGF-), which is consistently overexpressed in most fibrotic diseases and displays a variety of profibrotic effects in fibroblasts (Querfeld et al, 1999;Schiller et al, 2004). Activation of TGF- receptors leads to the activation of several kinase signaling cascades, leading to the phosphorylation of SMAD proteins as well as the activation of SMAD-independent kinases that collectively activate ECM synthesis and fibroblast growth and differentiation into myofibroblasts (Shi and Massague, 2003;Daniels et al, 2004). Connective tissue growth factor (CTGF) is a soluble mediator selectively and rapidly induced in fibroblasts by the action of TGF- (Leask et al, 2004). CTGF has also been specifically detected in skin fibrotic diseases (Igarashi et al, 1996), and in animal models, it enhances and perpetuates the profibrotic effects of TGF- (Frazier et al, 1996).

Although most fibrotic diseases are usually initiated by variable degrees of inflammation, anti-inflammatory therapies are ineffective in targeting chronic fibrotic diseases that represent an important group of disorders for which there is no specific therapy. TGF- appears as an attractive target for the therapy of fibrotic diseases, and several anti-TGF- strategies have been successfully assayed in animal models of fibrosis, including several murine models of scleroderma (McCormick et al, 1999;Yamamoto et al, 1999b;Zhang et al, 2003;Lakos et al, 2004). Systemic inhibition of TGF-, however, raises important safety concerns, because this factor displays pleiotropic and potent effects in immunomodulation, inflammation, and tumor development (Akhurst, 2002). Consistently, in TGF-1-deficient mice, skin scarring is reduced but they develop a severe wasting syndrome accompanied by a generalized inflammatory response and tissue necrosis, resulting in organ failure and death (Bottinger et al, 1997). Therefore, local rather than systemic TGF- inhibition, or targeting of downstream factors involved in TGF- profibrotic signaling represent alternative strategies for the development of anti-fibrotic therapies (Daniels et al, 2004;Lakos et al, 2004). Local inhibition of TGF- has previously been attempted by the direct application of neutralizing antibodies on skin or corneal open wounds, but the application of antibodies or large peptides through the epidermal barrier appears to be an unpractical approach (Jester et al, 1997;Brahmatewari et al, 2000).

We have previously reported that the peptide P144: TSLDASIIWAMMQN, encompassing aminoacids 730–743 (accession number Q03167, SwissProt) from human TGF-1 type III receptor (-glycan), was able to block the biological activity of TGF-1 (Ezquerro et al, 2003). This peptide is derived from the membrane-proximal ligand-binding domain of -glycan (Esparza-Lopez et al, 2001), and similar to soluble -glycan (Lopez-Casillas et al, 1994), was able to interfere with TGF-1 binding to its cellular receptors on Mv1Lu cells (Ezquerro et al, 2003). P144 prevented TGF-1-dependent inhibition of Mv1Lu cell proliferation and, in cultured fibroblasts, it induced a concentration-dependent decrease on TGF-1-dependent stimulation of a reporter gene under the control of human 2(I) collagen promoter (Ezquerro et al, 2003). Intraperitoneal administration of P144 also showed potent in vivo anti-fibrotic activity in the liver of rats receiving CCl4 (Ezquerro et al, 2003). Its small size and highly lipophilic character may allow its local use by topical application in skin fibrotic diseases, thereby reducing potential systemic effects. To examine the potential anti-fibrotic effects of the topical application of this peptide in vivo, we have tested P144 on a lipogel vehicle in an animal model of skin sclerosis induced by bleomycin. This model reproduces most of the features of human scleroderma such as skin-inflammatory cell infiltration, vascular damage, mast cell activation, and prolonged skin fibrosis (Yamamoto et al, 1999c). In this model, previous studies have demonstrated that either the administration of anti-TGF- antibodies or genetic SMAD3 deficiency ameliorates fibrosis development, strongly supporting a key role for TGF- (Yamamoto et al, 1999b;Lakos et al, 2004).

Top of pageResults
In order to study the anti-fibrotic effect of P144 (a peptide inhibitor of TGF-1) on bleomycin-induced skin fibrosis, we measured the changes induced in mice treated with bleomycin for 4 wk with and without P144 administration. It was found that bleomycin-treated mice showed a marked increase of the collagen matrix of the dermis. The dermis showed an increase of thickness that partially replaced the subcutaneous fat when compared with phosphate-buffered saline (PBS)-treated mice (Figure 1a). An increase in the collagen matrix around the upper fascia of the paniculus carnosus muscle was also observed, and it was particularly evident in Masson's trichrome-stained sections of bleomycin-treated mice skin (Figure 1b). An abundant inflammatory infiltrate, mainly composed of mononuclear cells as well as an increased number of mast cells, many of them showing degranulation features, was also observed in bleomycin-treated mice (data not shown). Mice treated with P144 anti-TGF-1 peptide showed a decrease of the dermal and hypodermal collagen area compared with vehicle-treated mice (Figure 1a, b). The thickness of the dermis was significantly decreased in P144-treated mice compared with vehicle-treated mice, which showed a thickness similar to that found in untreated mice (Figure 2). To confirm the histological observation of decreased fibrosis in P144-treated mice, we determined the pepsin-soluble collagen content of 4 mm punch skin biopsies by a colorimetric Sircol-based assay. This analysis showed a significant decrease of the soluble collagen content in P144-treated mice (Figure 2). Changes in the density of inflammatory cell infiltration, mast cell infiltration, or morphological changes of the epidermis were not observed in the P144 or vehicle-treated mice compared with those receiving only bleomycin injections (data not shown).

Discussion
The effectiveness of systemic strategies targeting TGF- during the development of experimental skin fibrosis has been previously demonstrated. The natural human latency-associated peptide, and neutralizing anti-TGF-1 antibodies have shown to prevent the development of skin fibrotic lesions effectively in different experimental models (McCormick et al, 1999;Yamamoto et al, 1999b;Zhang et al, 2003). These molecules are large enough to prevent its diffusion through the epidermal barrier. We have tested the feasibility of using a smaller lipophilic peptide, based on a conserved region of human type III TGF-1 receptor, as a topical therapy for skin fibrosis.

Our data consistently show that daily application of this peptide for 4 wk in parallel to fibrogenic bleomycin subcutaneous injections prevents fibrosis. Furthermore, and more importantly, regarding human skin fibrotic diseases, established fibrosis was also significantly reduced following topical application of peptide P144 for 2 wk. Improvement of established skin fibrosis in this model by post-onset therapy has been previously demonstrated with systemic interferon-, or superoxide dismutase therapy but not with systemic TGF- inhibitors (Yamamoto et al, 1999a,b,2000). We decided to test topical application of P144 because it was thought that in the case of bleomycin-induced scleroderma, this would be more efficacious than systemic administration of this peptide inhibitor. Also, in the event of P144 being toxic, topical application might reduce toxic side-effects that might be encountered following systemic administration of P144.

In previous studies in the bleomycin-induced scleroderma model, treatment with systemic anti-TGF- antibodies reduced fibrosis in parallel to a reduction in mast cell and inflammatory cell infiltration (Yamamoto et al, 1999b). The relevance of mast cells in skin fibrosis models is uncertain, because previous studies in mast cell-deficient mice have shown their dispensable contribution to fibrosis development (Everett et al, 1995;Yamamoto et al, 2001). Inflammatory cell infiltration plays an important role in the early stages of fibrosis development but its role is less clear at later stages, where it can either resolve or persist independent of the progression of fibrosis. Indeed, at later stages, fibrosis usually progresses in the absence of significant inflammatory cell infiltration. Our data and similar data using latency-associated peptide in a model of graft versus host scleroderma, or in SMAD3-deficient mice challenged with bleomycin, suggest that fibrosis can be decreased by antagonizing TGF- independent of inflammatory cell infiltration (Zhang et al, 2003;Lakos et al, 2004).

The mainstay of therapy for dermatological diseases remains topical therapy because it can readily target lesional skin decreasing systemic effects of the active principles; however, delivery of large peptides is limited by their size and physicochemical properties. We took advantage of the small size of P144 peptide and its lipophilic properties, which allowed for its application as a lipogel. Although dermal absorption of the peptide is yet to be demonstrated, our data suggest that topical application of this peptide efficiently interferes with TGF- action on dermal fibroblasts as critical players of TGF- profibrotic responses. Alternatively, its local accumulation in the epidermis could have potentially contributed to its anti-fibrotic effects. In this regard, cross-talk between the epidermis and the dermis during fibrosis development may occur, as profibrotic factors such as TGF- and monocyte chemoattractant protein 1 (MCP-1) have been detected in the epidermal layer of fibrotic skin (Galindo et al, 2001;Flanders et al, 2002). Indeed, keratinocyte overexpression of TGF-1 in transgenic mice induces dermal fibrosis (Ito et al, 2001;Yang et al, 2001;Chan et al, 2002). Interestingly, our study points to CTGF induction in skin keratinocytes of bleomycin-treated mice, which was reduced by topical anti-TGF- therapy to a higher extent than in dermal fibroblasts. Although the role of epidermal CTGF has not been established in fibrosis, previous studies demonstrate that it is expressed by normal keratinocytes in vivo (Quan et al, 2002). Also, its downregulation by ultraviolet (UV) radiation has been linked to the reduction in procollagen synthesis induced by UV radiation (Quan et al, 2002).

The demonstration of the effectiveness of topical application of a peptide inhibitor of TGF-1 provides a potentially fruitful strategy for the therapy of pathological scarring and skin fibrotic diseases. Experiments are being carried out to determine as to what extent P144 might be systemically absorbed through the skin. These experiments, together with a study of the potential toxicity of P144, will determine whether this peptide is suitable for human therapy.

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