Identification of fibroblast‐derived proteins important for endothelial cell sprouting and lumen formation

A role for fibroblasts in angiogenesis is now well recognized, however the precise mechanisms underlying their action have not been determined. Using a 3D fibrin gel angiogenesis model we find that, in contrast to fibroblasts, tumor cells that are highly angiogenic in vivo fail to support angiogenic sprouting in vitro. A combination of a candidate gene approach, 2D gel electrophoresis, column chromatography and mass spectrometry was then used to identify two classes of fibroblast‐derived factors – one that supports vessel sprouting but not lumen formation, and a second that promotes lumen formation. In the absence of fibroblasts a combination of ANG‐1, angiogenin, HGF, TGF‐α and TNF drives robust EC sprouting, however, only the subsequent addition of fibroblast‐conditioned medium supports lumenogenesis. Using siRNA‐mediated knockdown we show that five genes expressed in fibroblasts – Collagen 1, PCOLCE, SPARC, βig‐h3 and IGFBP7 – are necessary for lumen formation. Moreover, blocking of the collagen‐binding integrin α2β1 in fibrin gels disrupts sprouting and lumen formation, confirming the role of collagen and collagen cross‐linking proteins. These findings highlight the critical role that fibroblast‐derived extracellular matrix components play in EC lumen formation and provide insight into the role of fibroblasts in the tumor microenvironment. Supported by NIH RO1 H2 60067