Key Laboratory of Vision Loss and Restoration, Ministry of Education, Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases, Department of Ophthalmology, Peking University People's Hospital, Beijing, China.
Parkway Health, Inc., Shanghai, China.
Invest Ophthalmol Vis Sci. 2015 Jan 8;56(2):816-26. doi: 10.1167/iovs.14-14734.
The present study aimed to investigate the effects of αA-crystallin on pathologic ocular neovascularization.
Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study, and αA-crystallin-knockout (CRYAA[-/-]) mice were used for the in vivo study. αA-crystallin was knocked down in HUVECs by using a specific small interfering RNA (siRNA), and the effects of αA-crystallin knockdown on proliferation, migration, tube formation, and apoptosis were evaluated. Enzyme-linked immunosorbent assays were performed to investigate extracellular concentrations of vascular endothelial growth factor (VEGF). In vivo mouse models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) were generated by using CRYAA(-/-) mice. The nonperfused area in the OIR model was measured in flat-mounted retinas, and angiogenesis resulting in CNV was evaluated in retinal sections. Western blot analysis was performed to investigate the phosphorylation status of vascular endothelial growth factor receptor 2 (VEGFR2, high affinity receptor for VEGF), AKT, PLCγ1, FAK, Src, p42/p44MAPK, p38MAPK, caspase-3, and caspase-9 in cultured HUVECs, as well as in the OIR and CNV models.
The CRYAA siRNA not only induced HUVEC apoptosis but also inhibited exogenous and endogenous VEGF-induced cell activities, including proliferation, migration, and tube formation. Pathologic neovascularization was attenuated in the CRYAA(-/-) murine OIR and CNV models. Both in vitro and in vivo, the inhibition of angiogenesis was mediated by the suppression of VEGF secretion and the inhibition of the VEGFR2 signaling pathway; VEGFR2, AKT, PLCγ1, FAK, Src, p42/p44 MAPK, and p38 MAPK all showed reduced phosphorylation levels. In addition, CRYAA knockout led to increased levels of cleaved caspase-9 and caspase-3.
Knockout of αA-crystallin inhibited pathologic neovascularization through the VEGF and VEGFR2 signaling pathways both in vitro and in vivo. These results suggest that αA-crystallin could be a novel pharmaceutical target for the prevention of ocular neovascularization.
本研究旨在探讨αA-晶体蛋白对病理性眼部新生血管形成的影响。
本研究采用人脐静脉内皮细胞（HUVEC）进行体外研究，并用αA-晶体蛋白敲除（CRYAA[-/-]）小鼠进行体内研究。采用特异性小干扰 RNA（siRNA）敲低 HUVEC 中的αA-晶体蛋白，评估αA-晶体蛋白敲低对增殖、迁移、管形成和凋亡的影响。通过酶联免疫吸附试验（ELISA）检测血管内皮生长因子（VEGF）的细胞外浓度。采用 CRYAA（-/-）小鼠建立氧诱导视网膜病变（OIR）和激光诱导脉络膜新生血管（CNV）的体内小鼠模型。在 flat-mounted 视网膜上测量 OIR 模型中的无灌注区，在视网膜切片上评估导致 CNV 的血管生成。采用 Western blot 分析培养的 HUVEC 中血管内皮生长因子受体 2（VEGFR2，VEGF 的高亲和力受体）、AKT、PLCγ1、FAK、Src、p42/p44MAPK、p38MAPK、caspase-3 和 caspase-9 的磷酸化状态，以及在 OIR 和 CNV 模型中。
CRYAA siRNA 不仅诱导 HUVEC 凋亡，还抑制外源性和内源性 VEGF 诱导的细胞活性，包括增殖、迁移和管形成。CRYAA（-/-）小鼠的 OIR 和 CNV 模型中的病理性新生血管形成减弱。体外和体内均抑制血管生成，这是通过抑制 VEGF 分泌和 VEGFR2 信号通路介导的；VEGFR2、AKT、PLCγ1、FAK、Src、p42/p44MAPK 和 p38MAPK 的磷酸化水平均降低。此外，CRYAA 敲除导致 cleaved caspase-9 和 caspase-3 的水平增加。
体外和体内敲除αA-晶体蛋白均通过 VEGF 和 VEGFR2 信号通路抑制病理性新生血管形成。这些结果表明，αA-晶体蛋白可能成为预防眼部新生血管形成的新型药物靶点。
