Yamagishi S, Yonekura H, Yamamoto Y, Fujimori H, Sakurai S, Tanaka N, Yamamoto H
Department of Biochemistry, Kanazawa University School of Medicine, Japan.
Lab Invest. 1999 Apr;79(4):501-9.
Angiogenesis is the process by which new vascular networks are formed from preexisting capillaries. The small vessels are composed of two types of cells, namely endothelial cells (EC) and pericytes, with the former being encircled by the latter. We previously showed that hypoxia, the principal cause of angiogenesis, can induce the proliferation of pericytes as well as EC. In this report we present evidence that the hypoxic induction of pericyte growth can be ascribed at least in part to vascular endothelial growth factor (VEGF) produced by this very cell type. First, the finding that hypoxia can stimulate the proliferation of pericytes was confirmed by cultivating bovine retinal pericytes in a controlled-atmosphere culture chamber containing various concentrations of oxygen and then assaying pericyte synthesis of DNA. Second, Northern blot analysis revealed that pericyte levels of mRNA encoding VEGF increased as the atmospheric oxygen tension was decreased; this was accompanied by an increase in de novo synthesis of VEGF proteins. Third, pericytes were able to respond to exogenously added VEGF, resulting in a dose-dependent increase in viable cell numbers. Fourth, polyclonal antibodies against VEGF efficiently blocked the hypoxic induction of pericyte growth. Fifth, pericytes expressed the gene for fms-like tyrosine kinase 1 (flt1) as the predominant form of VEGF receptor, and tyrosine phosphorylation of this receptor protein was enhanced when pericytes were exposed to hypoxia, as it was when cells were exposed to VEGF. Sixth, the antisense DNA complement of flt1 mRNA abolished the hypoxia-induced stimulation of pericyte growth. Finally, exogenous VEGF stimulated the migration of pericytes in a dose-dependent manner. The results thus suggest that VEGF, which has been thought to be a specific mitogen for EC, also acts on neighboring pericytes, probably in both autocrine and paracrine manners, and that the hypoxia-induced overproduction of VEGF could promote not only EC sprouting but also the recruitment of pericytes, thereby contributing to the maturation of newly formed microvessels.
血管生成是指从已有的毛细血管形成新的血管网络的过程。这些小血管由两种类型的细胞组成，即内皮细胞（EC）和周细胞，前者被后者环绕。我们之前表明，缺氧作为血管生成的主要原因，可诱导周细胞以及内皮细胞的增殖。在本报告中，我们提供证据表明，周细胞生长的缺氧诱导至少部分可归因于这种细胞类型产生的血管内皮生长因子（VEGF）。首先，通过在含有不同浓度氧气的可控气氛培养箱中培养牛视网膜周细胞，然后检测周细胞的DNA合成，证实了缺氧可刺激周细胞增殖这一发现。其次，Northern印迹分析显示，随着大气氧张力降低，编码VEGF的周细胞mRNA水平升高；这伴随着VEGF蛋白从头合成的增加。第三，周细胞能够对外源添加的VEGF作出反应，导致活细胞数量呈剂量依赖性增加。第四，抗VEGF多克隆抗体有效地阻断了周细胞生长的缺氧诱导。第五，周细胞表达fms样酪氨酸激酶1（flt1）基因作为VEGF受体的主要形式，当周细胞暴露于缺氧时，该受体蛋白的酪氨酸磷酸化增强，细胞暴露于VEGF时也是如此。第六，flt1 mRNA的反义DNA互补物消除了缺氧诱导的周细胞生长刺激。最后，外源性VEGF以剂量依赖性方式刺激周细胞迁移。因此，结果表明，一直被认为是内皮细胞特异性促有丝分裂原的VEGF，也可能以自分泌和旁分泌方式作用于邻近的周细胞，并且缺氧诱导的VEGF过量产生不仅可促进内皮细胞芽生，还可促进周细胞募集，从而有助于新形成微血管的成熟。
