State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China.
Graefes Arch Clin Exp Ophthalmol. 2012 Nov;250(11):1615-25. doi: 10.1007/s00417-012-2094-5. Epub 2012 Jul 10.
This paper aimed to determine the involvement of Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) in experimental murine corneal neovascularization (NV), and to study the effect of NHERF1 on the biological properties of HUVEC and related mechanisms.
Using loss- and gain-function, we investigated the biological effects of NHERF1 on HUVEC. Western blotting was used to detect the expression of NHERF1 in cells. A carboxyfluorescein succinimidyl ester (CFSE) labeling assay and scarification test were used to measure the proliferation and migration activity, respectively, of HUVEC. The cell cycle distribution of the cells was assessed by flow cytometry analysis. The effect of NHERF1 on the phosphorylation levels of Akt and the changes of matrix metalloproteinase (MMP)-2 and MMP-9 levels were detected by western blotting analysis. Change in the NHERF1 expression in the alkali burn-induced corneal NV model was detected by microarray, real-time PCR, and immunofluorescence.
Overexpression of exogenous NHERF1 in HUVEC slightly inhibited the proliferation and significantly reduced the migration of the cells. NHERF1 also significantly downregulated Akt1 phosphorylation induced by platelet-derived growth factor BB (PDGF-BB) and the secretion of MMP-2 and MMP-9 compared with control cells. NHERF1 was upregulated in corneas challenged with alkali burns.
Our results indicated that NHERF1 might serve as a potential target for manipulating neovascularization-related diseases. This discovery contributes to a better understanding of the bioactivity of NHERF1 in angiogenesis.
本研究旨在探讨 Na(+)/H(+) 交换调节因子 1（NHERF1）在实验性小鼠角膜新生血管（NV）中的作用，并研究 NHERF1 对 HUVEC 生物学特性的影响及其相关机制。
采用缺失和获得功能的方法，研究 NHERF1 对 HUVEC 的生物学效应。Western blot 检测细胞中 NHERF1 的表达。采用羧基荧光素琥珀酰亚胺酯（CFSE）标记实验和划痕实验分别检测 HUVEC 的增殖和迁移活性。采用流式细胞术分析细胞周期分布。Western blot 分析检测 NHERF1 对 Akt 磷酸化水平以及基质金属蛋白酶（MMP）-2 和 MMP-9 水平变化的影响。通过微阵列、实时 PCR 和免疫荧光检测碱烧伤诱导的角膜 NV 模型中 NHERF1 的表达变化。
外源性 NHERF1 在 HUVEC 中的过表达略微抑制了细胞的增殖，显著降低了细胞的迁移。与对照组细胞相比，NHERF1 还显著下调了血小板衍生生长因子 BB（PDGF-BB）诱导的 Akt1 磷酸化和 MMP-2、MMP-9 的分泌。碱烧伤后角膜中 NHERF1 的表达上调。
本研究结果表明，NHERF1 可能成为调控血管新生相关疾病的潜在靶点。这一发现有助于更好地理解 NHERF1 在血管生成中的生物活性。
