Mandriota S J, Pepper M S
Department of Morphology, University Medical Center, Geneva, Switzerland.
J Cell Sci. 1997 Sep;110 ( Pt 18):2293-302. doi: 10.1242/jcs.110.18.2293.
Induction of in vitro angiogenesis and upregulation of urokinase- and tissue type-plasminogen activator (uPA, tPA) expression are two hallmarks of vascular endothelial growth factor (VEGF) activity on cultured endothelial cells. We report here that neutralizing antibodies to basic fibroblast growth factor (bFGF) inhibit VEGF-induced in vitro angiogenesis in bovine microvascular endothelial (BME) cells. Analysis of VEGF receptor-2 (VEGFR-2) expression revealed no alteration in VEGFR-2 mRNA or total protein in anti-bFGF antibody-treated BME or bovine aortic endothelial (BAE) cells. Ethidium bromide/agarose gel electrophoresis on the cytosolic fraction of BME cells revealed a basal level of fragmented DNA that was increased by anti-bFGF antibodies to an extent not exceeding that observed in parallel cultures incubated with concentrations of transforming growth factor-ss1 that increase VEGF-induced in vitro angiogenesis. In both BME and BAE cells, antibodies to bFGF also decreased basal levels of cell-associated uPA activity, and completely blocked the VEGF-mediated increase in uPA and tPA expression observed in parallel cultures incubated with VEGF alone. In contrast, PA inhibitor-1 expression was strongly upregulated in BME and BAE cells incubated with antibodies to bFGF, either alone or in combination with VEGF. These findings demonstrate that: (1) VEGF-induced in vitro angiogenesis and PA expression are dependent on endogenous bFGF, (2) that this phenomenon is not mediated by a decrease in VEGFR-2 expression and that apoptosis does not necessarily correlate with inhibition of invasion, and (3) that inhibition of endogenous bFGF in VEGF-treated cells results in a net antiproteolytic (and possibly also anti-adherent) effect, which could account in part for the inhibitory effect of the anti-bFGF antibodies. These findings point to a novel and unsuspected role for endogenous bFGF in regulating VEGF-induced in vitro angiogenesis.
体外血管生成的诱导以及尿激酶型和组织型纤溶酶原激活剂（uPA、tPA）表达的上调是血管内皮生长因子（VEGF）对培养的内皮细胞作用的两个标志。我们在此报告，针对碱性成纤维细胞生长因子（bFGF）的中和抗体可抑制VEGF诱导的牛微血管内皮（BME）细胞的体外血管生成。对VEGF受体-2（VEGFR-2）表达的分析显示，抗bFGF抗体处理的BME或牛主动脉内皮（BAE）细胞中VEGFR-2 mRNA或总蛋白没有改变。对BME细胞胞质部分进行溴化乙锭/琼脂糖凝胶电泳显示，存在基础水平的片段化DNA，抗bFGF抗体使其增加，但增加程度不超过与能增强VEGF诱导的体外血管生成的转化生长因子-β1浓度孵育的平行培养物中观察到的程度。在BME和BAE细胞中，抗bFGF抗体也降低了细胞相关uPA活性的基础水平，并完全阻断了在仅与VEGF孵育的平行培养物中观察到的VEGF介导的uPA和tPA表达增加。相反，在单独或与VEGF联合使用抗bFGF抗体孵育的BME和BAE细胞中，PA抑制剂-1表达强烈上调。这些发现表明：（1）VEGF诱导的体外血管生成和PA表达依赖于内源性bFGF；（2）这种现象不是由VEGFR-2表达降低介导的，且细胞凋亡不一定与侵袭抑制相关；（3）VEGF处理的细胞中内源性bFGF的抑制导致净抗蛋白水解（可能还有抗黏附）作用，这可能部分解释了抗bFGF抗体的抑制作用。这些发现指出了内源性bFGF在调节VEGF诱导的体外血管生成中一种新的、意想不到的作用。
