Angiogenesis, i.e. the formation of blood vessels by endothelial cells, plays a critical role in wound healing and in pathologic processes such as neoplastic growth, rheumatoid arthritis, diabetic retinopathy, and atherosclerosis. The long term goal of these studies is to elucidate the molecular mechanisms of angiogenesis and to facilitate the treatment of angiogenesis-dependent disorders. We have developed a serum-free in vitro model of angiogenesis which is based on the capacity of rat aorta rings to generate microvessels in collagen gels. Angiogenesis in this model is regulated by endogenous basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF). We also postulate a role for vascular endothelial growth factor (VEGF) based on its potent stimulatory effect on the system. Our hypothesis is that aortic fibroblasts stimulated by bFGF and PDGF promote the formation of microvessels by producing heparin-binding growth factors including VEGF. We also hypothesize that fibroblasts stabilize microvessels by promoting the perivascular deposition of basement membrane. Using a serum-free co-culture model recently developed in our laboratory we will evaluate the effect of fibroblasts on the proliferative and vasoformative response of endothelial cells to bFGF and PDGF. We will also investigate the inhibitory effects on rat aortic angiogenesis of neutralizing antibodies against these growth factors. Immunoprecipitation studies together with light and electron immunohistochemical analysis will evaluate how fibroblasts influence the perivascular production, deposition, and distribution of fibronectin, laminin, and type IV collagen, which are components of the basement membrane. The stabilizing effects of these molecules on the microvessels will be also evaluated. The production of VEGF by fibroblasts and by aortic cultures at different stages of angiogenesis will be studied by Western blotting, Northern blotting, reverse transcriptase-polymerase chain reaction (RT-PCR), and RNAase protection assay. The expression of the endothelial VEGF receptor during angiogenesis will be investigated by affinity labeling studies, RT-PCR, and RNAase protection assay. The function of endogenous VEGF in the rat aorta model will be inhibited with anti-VEGF antibodies and antisense oligonucleotides. By studying how fibroblasts regulate angiogenesis we will gain insight into the mechanisms of angiogenesis-dependent disorders which are often characterized by a combination of fibroblastic and vascular proliferation.
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