Wang H, Keiser J A
Department of Vascular and Cardiac Disease, Therapeutics, Parke-Davis Pharmaceutical Research, Ann Arbor, MI, USA.
Circ Res. 1998 Oct 19;83(8):832-40. doi: 10.1161/01.res.83.8.832.
Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis that stimulates proliferation, migration, and proteolytic activity of endothelial cells. Although the mitogenic activity of VEGF is endothelial cell specific, recent reports indicate VEGF is able to stimulate chemotaxis and tissue factor production in monocytes. VEGF-stimulated activity in monocytes is mediated by the VEGF receptor flt-1. The purpose of the present study was to investigate the effects of VEGF on another major cell type in the vascular wall, namely, the vascular smooth muscle cell (SMC). Using cultured cells, we showed that VEGF has a minimal mitogenic effect on SMCs, which is in accordance with published data. However, VEGF treatment significantly enhanced production of matrix metalloproteinase (MMP)-1, -3, and -9 by human SMCs. The upregulation of MMP-1 and MMP-9 was pronounced, and the stimulation for MMP-3 was less prominent. Stimulation could be demonstrated at both protein and mRNA levels, as reflected by ELISA, zymography, and Northern blot analysis. To explore the signal transduction pathway for the effect of VEGF on SMCs, we studied the expression of 2 high-affinity VEGF receptors, the kinase insert domain-containing receptor (KDR) and flt-1, in human SMCs. Both reverse transcriptase-polymerase chain reaction and immunoblotting revealed the expression of flt-1. Immunoprecipitation followed by immunoblotting illustrated phosphorylation of the flt-1 receptor after VEGF treatment. Similar methodology failed to detect expression of KDR in human SMCs. These data suggest the role of flt-1 in mediating VEGF-stimulated MMP expression of SMCs. The physiological relevance of MMP upregulation was studied by examining VEGF-stimulated SMC migration through 2 synthetic extracellular matrix barriers, Matrigel and Vitrogen. Our results indicate that VEGF treatment accelerated SMC migration through both barriers, and that this response was blocked by MMP inhibition in Matrigel, which supports a permissive role of MMP in SMC migration. These data are the first to show a direct effect of VEGF on SMCs. SMC-derived MMPs may be an additional source of proteases to digest vascular basement membrane, which is a crucial step in the initial stage of angiogenesis. The MMPs may also contribute to SMC migration in angiogenesis and atherogenesis.
血管内皮生长因子（VEGF）是血管生成的关键调节因子，可刺激内皮细胞的增殖、迁移和蛋白水解活性。尽管VEGF的促有丝分裂活性具有内皮细胞特异性，但最近的报告表明VEGF能够刺激单核细胞的趋化性和组织因子生成。单核细胞中VEGF刺激的活性由VEGF受体flt-1介导。本研究的目的是探讨VEGF对血管壁中另一种主要细胞类型，即血管平滑肌细胞（SMC）的影响。使用培养细胞，我们发现VEGF对SMC的促有丝分裂作用极小，这与已发表的数据一致。然而，VEGF处理显著增强了人SMC中基质金属蛋白酶（MMP）-1、-3和-9的产生。MMP-1和MMP-9的上调明显，对MMP-3的刺激则不太显著。通过ELISA、酶谱分析和Northern印迹分析反映出，在蛋白质和mRNA水平均能证明这种刺激作用。为了探索VEGF对SMC作用的信号转导途径，我们研究了人SMC中两种高亲和力VEGF受体，即含激酶插入结构域受体（KDR）和flt-1的表达。逆转录聚合酶链反应和免疫印迹均显示了flt-1的表达。免疫沉淀后进行免疫印迹表明，VEGF处理后flt-1受体发生了磷酸化。类似的方法未能检测到人SMC中KDR的表达。这些数据表明flt-1在介导VEGF刺激的SMC的MMP表达中起作用。通过检测VEGF刺激的SMC通过两种合成细胞外基质屏障（基质胶和维特根）的迁移，研究了MMP上调的生理相关性。我们的结果表明，VEGF处理加速了SMC通过两种屏障的迁移，并且在基质胶中这种反应被MMP抑制所阻断，这支持了MMP在SMC迁移中的促进作用。这些数据首次表明VEGF对SMC有直接作用。SMC衍生的MMPs可能是消化血管基底膜的蛋白酶的另一个来源，这是血管生成初始阶段的关键步骤。MMPs也可能在血管生成和动脉粥样硬化中促进SMC迁移。
