Therapy of ischemic lesions in systemic sclerosis

Systemic sclerosis (SSc), or scleroderma, is an autoimmune connective tissue disease cha-racterized by structural and functional vascular abnormalities, perivascular mononuclear cell infiltration, and increased deposition of extracellular matrix in skin and internal organs. An early hallmark is the microvascular damage causing a reduced blood flow and hypoxia. Under normal physiological conditions, hypoxia induces angiogenesis, but this mechanism is disturbed in SSc, which is a reason for the development of fingertip ulcers. A very potent mediator of angiogenesis is vascular endothelial growth factor (VEGF). In SSc-patients VEGF- expression seems to be uncontrolled and chronic. However, a very high VEGF-expression is associated with the lack of fingertip ulcers. Therefore, local therapy of ischemic lesions with VEGF seems to be a good option. We want to test this approach in UCD-200 and 206 chickens, which is the only animal model showing all hallmarks of human SSc and thus perfectly suited for testing new therapeutic concepts, and for the investigation of underlying mechanisms. In order to mimic the binding of VEGF to extracellular matrix and regulation of release by cell- associated proteolytic activities found in nature, we will covalently link modified VEGF121 to a fibrin matrix before applying it locally to dermal lesions of comb and neck. This makes a controlled and cell demanded release of VEGF possible, which is necessary for the formation of mature and stable blood vessels. Within this study we want to answer the following questions: 1. Is VEGF121 -fibrin effective in healing and/or preventing ischemic skin lesions? 2. What effects has VEGF121 -fibrin on endothelial cell apoptosis and angiogenesis? 3. Does therapy with VEGF121 -fibrin have an influence on the synthesis of pro-fibrotic cytokines and the development of fibrosis? Beyond the relevance for the treatment of fingertip ulcers in SSc, a positive outcome of this study would also have great impact on the therapy of other diseases of ischemia including chronic wounds (pressure ulcers, venous ulcers) and diabetic foot.

The main achievement of the present project was the development of an efficient therapy for the treatment of ischemic lesions in an animal model of systemic sclerosis (SSc). SSc is a devastating autoimmune connective tissue disease characterized by structural and functional vascular abnormalities, perivascular inflammation, and increased deposition of extracellular matrix in skin and internal organs. An early hallmark is the microvascular damage causing a reduced blood flow and hypoxia. Under normal physiological conditions, hypoxia induces angiogenesis, but this mechanism is disturbed in SSc. A very potent mediator of angiogenesis is vascular endothelial growth factor (VEGF), if temporally and spatially strictly controlled. In SSc patients, however, VEGF expression is uncontrolled and chronic. This results in chaotic vessels and intractable digital ulcers. We have addressed this therapeutic dilemma by a novel approach using a VEGF variant that binds covalently to fibrin and gets released enzymatically on cellular demand. We tested this approach in UCD-206 chickens, a spontaneous animal model showing all hallmarks of the human disease, by applying VEGF-fibrin gel onto comb and neck skin lesions. After one week the clinical outcome was assessed and underlying mechanisms analyzed. Our study in UCD-206 chickens convincingly showed the clinical efficacy of the topical VEGF-fibrin therapy. In most cases of early treatment, i.e. in combs with edema but no ulceration, VEGF-fibrin prevented the development of ischemic ulcers. On existing comb and neck skin ulcers, VEGF-fibrin had a healing effect. Overall, 79.3% of the VEGF-fibrin treated lesions showed clear clinical improvement, whereas 71.0% of fibrin treated controls and 93.1% of untreated lesions had deteriorated. This was accompanied by significantly increased growth of stable blood vessels, a normalization of VEGF receptor expression, and increased endogenous endothelial VEGF expression. These results suggest that cell-demanded release of modified VEGF from the fibrin matrix induces controlled angiogenesis, and thus a lasting revascularization. Since ischemic ulcers are not only a problem in SSc, but also in other diseases of ischemia including pressure ulcers, venous ulcers and diabetic foot, our findings might also be relevant for the therapeutic approach in these diseases.