Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
J Mol Cell Cardiol. 2010 Sep;49(3):490-8. doi: 10.1016/j.yjmcc.2010.05.003. Epub 2010 May 15.
The morphogen Sonic hedgehog (Shh) promotes neovascularization in adults by inducing pro-angiogenic cytokine expression in fibroblasts; however, the direct effects of Shh on endothelial cell (EC) function during angiogenesis are unknown. Our findings indicate that Shh promotes capillary morphogenesis (tube length on Matrigel increased to 271+/-50% of the length in untreated cells, p=0.00003), induces EC migration (modified Boyden chamber assay, 191+/-35% of migration in untreated cells, p=0.00009), and increases EC expression of matrix metalloproteinase 9 (MMP-9) and osteopontin (OPN) mRNA (real-time RT-PCR), which are essential for Shh-induced angiogenesis both in vitro and in vivo. Shh activity in ECs is mediated by Rho, rather than through the "classic" Shh signaling pathway, which involves the Gli transcription factors. The Rho dependence of Shh-induced EC angiogenic activity was documented both in vitro, with dominant-negative RhoA and Rho kinase (ROCK) constructs, and in vivo, with the ROCK inhibitor Y27632 in the mouse corneal angiogenesis model. Finally, experiments performed in MMP-9- and OPN-knockout mice confirmed the roles of the ROCK downstream targets MMP-9 and OPN in Shh-induced angiogenesis. Collectively, our results identify a "nonclassical" pathway by which Shh directly modulates EC phenotype and angiogenic activity.
形态发生素 Sonic hedgehog（Shh）通过诱导成纤维细胞中促血管生成细胞因子的表达来促进成年人的新生血管形成；然而，Shh 对血管生成过程中内皮细胞（EC）功能的直接影响尚不清楚。我们的研究结果表明，Shh 促进毛细血管形态发生（在未经处理的细胞中，Matrigel 上的管长度增加到未处理细胞长度的 271+/-50%，p=0.00003），诱导 EC 迁移（改良 Boyden 室测定法，未处理细胞的迁移率为 191+/-35%，p=0.00009），并增加 EC 表达基质金属蛋白酶 9（MMP-9）和骨桥蛋白（OPN）mRNA（实时 RT-PCR），这对于 Shh 在体外和体内诱导的血管生成都是必不可少的。Shh 在 EC 中的活性是通过 Rho 介导的，而不是通过涉及 Gli 转录因子的“经典”Shh 信号通路。在体外使用显性失活 RhoA 和 Rho 激酶（ROCK）构建体以及体内使用 ROCK 抑制剂 Y27632 在小鼠角膜血管生成模型中，都证明了 Shh 诱导的 EC 血管生成活性依赖 Rho。最后，在 MMP-9 和 OPN 基因敲除小鼠中进行的实验证实了 ROCK 下游靶标 MMP-9 和 OPN 在 Shh 诱导的血管生成中的作用。总之，我们的研究结果确定了 Shh 直接调节 EC 表型和血管生成活性的“非经典”途径。
