Department of Medicine, College of Medicine and Life Sciences,, University of Toledo,, Toledo, OH, USA.
Feinberg Cardiovascular Research Institute, Northwestern University, Chicago, IL, USA.
Lab Invest. 2018 May;98(5):682-691. doi: 10.1038/s41374-018-0028-5. Epub 2018 Feb 16.
Sonic Hedgehog (Shh) signaling induces neovascularization and angiogenesis. It is not known whether the hedgehog signaling pathway in endothelial cells is essential to angiogenesis. Smoothened (Smo) transduces hedgehog signaling across the cell membrane. This study assessed whether endothelial Smoothened-dependent Shh signaling is required for Shh-mediated angiogenesis and ischemic tissue repair. Endothelial-specific smoothened knockout mice, eSmo were created using Cre-lox recombination system. eSmo mice had no observable phenotype at baseline and showed normal cardiac function. Smoothened in CD31+ cells isolated from eSmo hearts was significantly reduced compared to CD31+ cells from eSmo littermate control hearts. Fluorescence immunostaining of eSmo heart sections showed Smo expression in endothelial cells was abolished. The hind-limb ischemia (HLI) model was used to assess the response to ischemic injury. Perfusion ratio, limb motor function, and limb necrosis were not significantly different after HLI between eSmo mice and eSmo. Capillary densities in the ischemic limb in eSmo mice were also similar to eSmo at 4 weeks after HLI. Next, response to exogenous Shh was assessed in the corneal angiogenesis model. There was no significant difference in corneal angiogenesis induced by administration of Shh pellets between eSmo and eSmo mice. Furthermore, in vitro experiments demonstrated that direct Shh had limited effects on endothelial cell proliferation and migration. However, conditioned media from Shh-treated fibroblasts had a more potent effect on endothelial cell proliferation and migration than non-treated conditioned media. Furthermore, Shh treatment of fibroblasts dramatically stimulated angiogenic growth factor expression, including PDGF-B, VEGF-A, HGF and IGF. PDGF-B was the most upregulated and may contribute to the large neo-vessels associated with Shh-induced angiogenesis. Taken together, these data demonstrate that Shh signaling via Smoothened in endothelial cells is not required for angiogenesis and ischemic tissue repair. Shh signaling via stromal cells likely mediates its angiogenic effects.
Sonic Hedgehog (Shh) 信号诱导新生血管形成和血管生成。目前尚不清楚内皮细胞中的 Hedgehog 信号通路是否对血管生成至关重要。Smoothened (Smo) 将 Hedgehog 信号跨细胞膜转导。本研究评估了内皮细胞 Smoothened 依赖性 Shh 信号是否需要 Shh 介导的血管生成和缺血组织修复。使用 Cre-lox 重组系统创建了内皮细胞特异性 Smoothened 敲除小鼠，eSmo。eSmo 小鼠在基线时没有观察到明显的表型，并且表现出正常的心脏功能。与 eSmo 同窝对照心脏的 CD31+细胞相比，eSmo 心脏分离的 CD31+细胞中的 Smoothened 显著减少。eSmo 心脏切片的荧光免疫染色显示内皮细胞中的 Smo 表达被消除。使用后肢缺血 (HLI) 模型评估对缺血损伤的反应。HLI 后，eSmo 小鼠和 eSmo 之间的灌注比、肢体运动功能和肢体坏死没有明显差异。HLI 后 4 周，eSmo 小鼠缺血肢体中的毛细血管密度也与 eSmo 相似。接下来，在角膜血管生成模型中评估对外源性 Shh 的反应。eSmo 和 eSmo 小鼠之间 Shh 丸给药诱导的角膜血管生成没有显着差异。此外，体外实验表明，直接 Shh 对内皮细胞增殖和迁移的影响有限。然而，来自 Shh 处理成纤维细胞的条件培养基对内皮细胞增殖和迁移的影响比未经处理的条件培养基更强。此外，Shh 处理成纤维细胞可显著刺激血管生成生长因子的表达，包括 PDGF-B、VEGF-A、HGF 和 IGF。PDGF-B 的上调最为明显，可能与 Shh 诱导的血管生成相关的大新生血管有关。总之，这些数据表明，内皮细胞中 Smoothened 介导的 Shh 信号传导对于血管生成和缺血组织修复不是必需的。基质细胞中的 Shh 信号传导可能介导其血管生成作用。
