The overall objective of our research is to characterize the molecular mechanisms that regulate the generation of new blood vessels (angiogenesis) in the skin and in skin tumors. Our previous studies have identified vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), as a cytokine of central importance for normal and neoplastic skin angiogenesis. Recently, it has been suggested that VEGF-C, a new member of the VEGF family, may play a major role in skin lymphangiogenesis. Lymphatic vessels are the predominant path for the metastatic spread of skin cancers, and we found that VEGF-C, although barely detectable in normal epidermis, was upregulated in malignant squamous cell carcinomas of the skin. We now propose experiments to test our specific hypotheses: (1) that VEGF and VEGF-C exert important, yet distinct effects on blood vascular endothelium versus lymphatic endothelium in vivo and in vitro, (2) that VEGF-C, possibly in synergy with VEGF, promotes skin carcinogenesis, malignant tumor growth and lymphatic metastasis, and (3) that VEGF gene transcription and VEGF-induced downstream effects may serve as novel targets for the treatment of cutaneous neoplasias. Therefore, we propose to: 1. Investigate the effects of VEGF and VEGF-C on skin angiogenesis, lymphangiogenesis, vascular permeability and leukocyte recruitment in vivo, studying transgenic mice with skin specific overexpression of both VEGF and VEGF-C; 2. Investigate the biological effects of VEGF and VEGF-C on cultured human dermal lymphatic endothelial cells versus human dermal microvascular endothelial cells, selectively isolated by our novel immunomagnetic protocol; 3. Investigate the importance of VEGF-C for the malignant growth and metastatic spread of experimental squamous cell carcinomas and malignant melanomas of the skin, using cell lines transfected to overexpress VEGF- C, VEGF or both VEGF and VEGF-C; 4. Investigate chemically induced skin carcinogenesis in transgenic mice which overexpress both VEGF and VEGF-C in the skin, and determine the induction and cell-type specificity of VEGF gene transcription during skin carcinogenesis in a novel transgenic mouse model for expression of green fluorescent protein under control of the VEGF promoter. This experimental model could then be used as a novel in vivo reporter assay system to investigate the effects of drugs with potential benefit for the treatment of (pre)cancerous skin lesions on VEGF gene transcription.
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