SIRT1 缺失通过下调 VEGF-A/VEGFR-2 和 MMP14 损害视网膜内皮细胞迁移。
SIRT1 Deletion Impairs Retinal Endothelial Cell Migration Through Downregulation of VEGF-A/VEGFR-2 and MMP14.
机构信息
School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China.
State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, China.
出版信息
Invest Ophthalmol Vis Sci. 2018 Nov 1;59(13):5431-5440. doi: 10.1167/iovs.17-23558.
PURPOSE
Silent information regulator protein 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase that is abundantly expressed in vascular endothelial cells (VECs), and it has an essential role in angiogenesis. However, its contribution to retinal vascular development remains unclear. Here we characterize its involvement in regulating this process under both physiological and pathologic conditions.
METHODS
Endothelium-specific Sirt1 knockout mice were established using the Cre-lox system. VECs were isolated using magnetic beads and identified by immunostaining. Retinal whole-mount staining analyzed the retinal vascular patterns. SIRT1 was knocked down or overexpressed in human retinal microvascular endothelial cells (HRMECs) using small interfering RNA (siRNA) or lentivirus infection, respectively. Scratch assay, Transwell, and Matrigel angiogenesis assay evaluated cell migration and tube formation, respectively. Quantitative RT-PCR analyzed genes regulating VEC migration. Western blotting determined protein expression. Coimmunoprecipitation detected the interaction of hypoxia-inducible factor 1α (HIF-1α) and SIRT1 as well as acetylation status of HIF-1α.
RESULTS
Specific deletion of Sirt1 in VECs dramatically delayed retinal vessel expansion and reduced vessel density. In the oxygen-induced retinopathy (OIR) mouse model, Sirt1 ablation markedly suppressed retinal revascularization and consequently increased retinal avascularity. SIRT1 downregulation in HRMECs inhibited cell migration and tube formation, while overexpression of SIRT1 had the opposite effects. Vascular endothelial growth factor-A (VEGF-A)/VEGF receptor-2 (VEGFR-2), and matrix metalloproteinases 14 (MMP14) expression significantly declined in Sirt1-null VECs, as well as SIRT1 siRNA-transfected HRMECs. SIRT1 downregulation upregulated the HIF-1α acetylation status. Conversely, SIRT1 overexpression decreased this response.
CONCLUSIONS
SIRT1 contributes to both physiological and pathologic retinal angiogenesis through promoting retinal VEC migration. Its underlying molecular mechanism involves SIRT1-mediated deacetylation of HIF-1α and subsequent upregulation of VEGF-A/VEGFR-2 and MMP14 expression.
目的
沉默信息调节蛋白 1（SIRT1）是一种烟酰胺腺嘌呤二核苷酸依赖性去乙酰化酶，在血管内皮细胞（VECs）中大量表达，在血管生成中具有重要作用。然而，它在视网膜血管发育中的作用尚不清楚。在这里，我们描述了它在生理和病理条件下调节这一过程的作用。
方法
使用 Cre-lox 系统建立内皮细胞特异性 Sirt1 敲除小鼠。使用磁珠分离 VECs，并通过免疫染色进行鉴定。视网膜全铺片染色分析视网膜血管模式。使用小干扰 RNA（siRNA）或慢病毒感染分别在人视网膜微血管内皮细胞（HRMECs）中敲低或过表达 SIRT1。划痕实验、Transwell 和 Matrigel 血管生成实验分别评估细胞迁移和管形成。定量 RT-PCR 分析调节 VEC 迁移的基因。Western blot 确定蛋白表达。免疫共沉淀检测缺氧诱导因子 1α（HIF-1α）与 SIRT1 的相互作用以及 HIF-1α的乙酰化状态。
结果
VEC 中 Sirt1 的特异性缺失显著延迟了视网膜血管扩张并降低了血管密度。在氧诱导的视网膜病变（OIR）小鼠模型中，Sirt1 缺失显著抑制了视网膜再血管化，从而增加了视网膜无血管区。HRMECs 中 SIRT1 的下调抑制了细胞迁移和管形成，而过表达 SIRT1 则产生相反的效果。Sirt1 缺失的 VECs 以及 SIRT1 siRNA 转染的 HRMECs 中血管内皮生长因子-A（VEGF-A）/血管内皮生长因子受体-2（VEGFR-2）和基质金属蛋白酶 14（MMP14）的表达显著下降。SIRT1 下调上调了 HIF-1α的乙酰化状态。相反，SIRT1 的过表达降低了这种反应。
结论
SIRT1 通过促进视网膜 VEC 迁移促进生理和病理视网膜血管生成。其潜在的分子机制涉及 SIRT1 介导的 HIF-1α去乙酰化以及随后上调 VEGF-A/VEGFR-2 和 MMP14 的表达。
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