Haspel Howard C, Scicli Gloria M, McMahon Gerald, Scicli A Guillermo
Department of Anesthesiology, Henry Ford Health System, Detroit, Michigan 48202, USA.
Microvasc Res. 2002 May;63(3):304-15. doi: 10.1006/mvre.2001.2383.
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.
血管生成性血管内皮生长因子（VEGF）被认为在内皮细胞增殖、分化和出芽过程中发挥关键作用。已经开发出了选择性抑制Flk-1（KDR）和Flt-1的VEGF受体相关酪氨酸激酶活性的小分子。这些药物，以SU5416为原型，对培养的内皮细胞增殖有影响，在体内抑制血管生成，并已被提议作为抗肿瘤药物。尽管SU5416在体内抑制血管生成，但尚不清楚导致血管生成的复杂过程中哪些受到VEGF受体相关酪氨酸激酶抑制的影响。我们利用SU5416和源自小鼠心脏的微血管内皮细胞系（SMHEC4）来专门研究VEGF受体相关酪氨酸激酶活性在体外血管生成模型中的作用。我们在这个稳定的细胞系中对球体形成和出芽这一血管生成的新模型进行了表征。SU5416抑制（约50%）VEGF（50 ng/ml）刺激的以及单层培养的SMHEC4的基础DNA合成。SU5416并不阻止SMHEC4聚集并组织成三维球体。CD31，一种分化内皮细胞的标志物，在单层培养中表达可忽略不计，但在SMHEC4球体中高度表达。球体CD31的含量和生化特性不受SU5416影响。SU5416也不阻止SMHEC4在基质胶上自发且快速（约3小时）排列成条索状。这两个模型表明内皮细胞的组织和分化独立于VEGF受体相关酪氨酸激酶信号传导。嵌入胶原凝胶中的SMHEC4球体自发且快速（约6小时）长出毛细血管样突起，随后（1 - 2天）形成复杂的自我吻合网络。此外，VEGF（50 ng/ml）显著刺激SMHEC4球体长出毛细血管样突起。SU5416几乎消除了自发的和VEGF刺激的出芽。这表明VEGF受体相关酪氨酸激酶活性对于从SMHEC4球体形成毛细血管样结构至关重要。总体而言，这些观察结果表明：（a）球体出芽模型适用于血管生成研究，因为它似乎概括了血管生成的许多步骤；（b）SU5416可以抑制内皮细胞增殖和出芽，而不影响内皮细胞的组织和分化。
