CONTACT-DEPENDENT INHIBITION OF ANGIOGENESIS BY CARDIAC FIBROBLASTS IN 3-DIMENSIONAL FIBRIN GELS IN-VITRO - IMPLICATIONS FOR MICROVASCULAR NETWORK REMODELING AND CORONARY COLLATERAL FORMATION

Angiogenesis and coronary artery collateral formation can improve bloo d flow and thereby prevent myocardial ischemia. The role of perivascul ar fibroblasts in neovascularization remains incompletely understood. Here we investigated the effects of epicardial and myocardial fibrobla sts on angiogenesis in vitro by using a serum-free microcarrier-based fibrin gel angiogenesis system. To clearly distinguish between differe nt cell types, we either stained endothelial cells or fibroblasts in t he living with 3,3,3',3'-tetramethyl-indocarbocyanine-perchlorate (DiI ). In cocultures, low numbers of heart fibroblasts stimulated endothel ial sprouting, and capillary growth was also induced by fibroblast-con ditioned media, indicating a paracrine mechanism. Capillary formation was decreased by increasing the density of fibroblasts in the cocultur es, indicating contact-dependent inhibition. Using time-lapse studies, it turned out that close contacts between fibroblasts and endothelial cells resulted in rapid retraction of endothelial cells or, rarely, i n cell death. Depending on the local ratio of fibroblasts to endotheli al cell numbers, fibroblasts determined the location of capillary grow th and the size of developing capillaries and thereby contributed to c apillary network remodeling. In contrast to primary heart fibroblasts, NIH 3T3 fibroblasts did not display contact-dependent inhibition of e ndothelial sprouts. NM fibroblasts were frequently seen in close assoc iation with endothelial capillaries, resembling pericytes. Contact-dep endent inhibition of angiogenesis by epicardial fibroblasts could not be reversed by addition of neutralizing anti-TGF-pl antibodies, by add ition of serum, of medium conditioned by hypoxic turner cells or myoca rdium, by various cytokines or by growing cocultures under hypoxic con ditions. Our results implicate a pivotal role of periendothelial mesen chymal cells for the regulation of microvascular network remodeling an d collateral formation.