Inhibition of vascular endothelial growth factor-associated tyrosine kinase activity with SU5416 blocks sprouting in the microvascular endothelial cell spheroid model of angiogenesis.

The angiogenic vascular endothelial growth factor (VEGF) is believed to play a critical role in endothelial cell proliferation, differentiation, and sprouting. Small molecules that selectively inhibit the VEGF receptor-associated tyrosine kinase activities of Flk-1 (KDR) and Flt-1 have been developed. These agents, a prototype being SU5416, have effects on the proliferation of cultured endothelial cells, constrain angiogenesis in vivo, and have been proposed as antitumor drugs. Although SU5416 inhibits in vivo angiogenesis, it is not clear which of the complex processes leading to angiogenesis are impacted by VEGF receptor-associated tyrosine kinase inhibition. We utilized SU5416 and a microvascular endothelial cell line derived from mouse heart (SMHEC4) to specifically examine the role of VEGF receptor-associated tyrosine kinase activity on in vitro models of angiogenesis. We characterized spheroid formation and sprouting, a new model of angiogenesis, in this stable cell line. SU5416 inhibits (approximately 50%) VEGF (50 ng/ml) stimulated and basal DNA synthesis of SMHEC4 cultured in monolayer. SU5416 does not prevent the aggregation and organization of SMHEC4 into tri-dimensional spheroids. CD31, a marker of differentiated endothelial cells, is negligibly expressed in monolayer cultures but highly expressed in SMHEC4 spheroids. The content and biochemical characteristics of spheroidal CD31 are unaltered by SU5416. SU5416 also does not prevent the spontaneous and rapid (approximately 3-h) alignment into cords by SMHEC4 on Matrigel. These two models suggest that the organization and differentiation of endothelial cells is independent of VEGF receptor-associated tyrosine kinase signaling. SMHEC4 spheroids embedded in collagen gels spontaneously and rapidly (approximately 6 h) sprout capillary-like projections and subsequently (1-2 days) form complex self-anastomosing networks. In addition, VEGF (50 ng/ml) markedly stimulates sprouting of capillary-like projections from SMHEC4 spheroids. Both the spontaneous and the VEGF-stimulated sprouting are nearly eliminated by SU5416. This demonstrates that VEGF receptor-associated tyrosine kinase activity is essential to the formation of capillary-like structures from SMHEC4 spheroids. Overall, these observations demonstrate that (a) the spheroid sprouting model is appropriate for the study of angiogenesis since it appears to recapitulate many of its steps and (b) SU5416 can inhibit endothelial cell proliferation and sprouting without impacting the organization and differentiation of endothelial cells.