CCN1/Cyr61-PI3K/AKT signaling promotes retinal neovascularization in oxygen-induced retinopathy.
CCN1/Cyr61-PI3K/AKT signaling promotes retinal neovascularization in oxygen-induced retinopathy.
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DOI：
10.3892/ijmm.2015.2371
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发表时间：
2015-12
影响因子：
5.4
通讯作者：
Chen X
中科院分区：
文献类型：
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作者：
Di Y;Zhang Y;Nie Q;Chen X
视网膜新生血管（RNV）是早产儿视网膜病变（ROP）的特征性病理改变。据报道，富含半胱氨酸的61 [Cyr 61，也称为CCN家族成员1（CCN 1）]介导血管生成。本研究旨在探讨CCN 1/Cyr 61-磷酸肌醇3-激酶（PI 3 K）/AKT信号通路在ROP中的作用机制。分别使用细胞计数试剂盒-8（CCK-8）和膜联蛋白V/碘化丙啶（PI）染色以及使用靶向CCN 1的siRNA（CCN 1 siRNA）来确定CCN 1对缺氧条件下人脐静脉内皮细胞（HUVEC）增殖和凋亡的贡献。暴露于缺氧的细胞也用PI 3 K/AKT抑制剂LY 294002处理。此外，对患有氧诱导视网膜病变（OIR）的小鼠幼仔给予CCN 1 siRNA的玻璃体内注射。RNV通过镁激活的腺苷二磷酸酶（ADP 13）染色来评估。采用RT-qPCR、western blot、免疫荧光和免疫组化等方法检测CCN 1、PI 3 K和AKT的分布和表达。缺氧组视网膜新生血管时数评分从1.23±0.49增加到5.60±0.73（P<0.05），视网膜前新生血管细胞数增加，CCN 1、PI 3 K和AKT的mRNA和蛋白表达水平增加（均P<0.05）。CCN 1 siRNA的注射降低了新生血管时钟小时分数和视网膜前新生血管细胞的数量（1.53±0.72 vs. 4.76±1.04; 12.0±2.8 vs. 31.4±2.6，均P<0.05），以及CCN 1、PI 3 K和AKT的mRNA和蛋白表达水平（蛋白质，分别为-45.3%、-22.5%和-28.4%; mRNA，分别为-43.7%、-58.7%和-42.9%，均P<0.05）。LY 294002可降低缺氧细胞中CCN 1的mRNA和蛋白表达水平（均P<0.05）。CCN 1 siRNA的应用可减轻OIR小鼠眼内新生血管的严重程度，提示CCN 1在ROP的RNV中起重要作用，可能成为ROP预防和治疗的潜在靶点。
Retinal neovascularization (RNV) is a characteristic pathological finding of retinopathy of prematurity (ROP). Cysteine-rich 61 [Cyr61, also known as CCN family member 1 (CCN1)] has been reported to mediate angiogenesis. The aim of the present study was to investigate the mechanisms of CCN1/Cyr61-phosphoinositide 3-kinase (PI3K)/AKT signaling in ROP. The contribution of CCN1 to human umbilical vein endothelial cell (HUVEC) proliferation and apoptosis under hypoxic conditions was determined using a cell counting kit-8 (CCK-8) and Annexin V/propidium iodide (PI) staining, respectively, as well as using siRNA targeting CCN1 (CCN1 siRNA). The cells exposed to hypoxia were also treated with the PI3K/AKT inhibitor, LY294002. In addition, mouse pups with oxygen-induced retinopathy (OIR) were administered an intravitreal injection of CCN1 siRNA. RNV was assessed by magnesium-activated adenosine diphosphatease (ADPase) staining. RT-qPCR, western blot analysis, immunofluorescence staining and immunohistochemistry were used to detect the distribution and expression of CCN1, PI3K and AKT. Exposure to hypoxia increased the neovascularization clock hour scores (from 1.23±0.49 to 5.60±0.73, P<0.05) and the number of preretinal neovascular cells, as well as the mRNA and protein expression levels of CCN1, PI3K and AKT (all P<0.05). The injection of CCN1 siRNA decreased the neovascularization clock hour scores and the number of preretinal neovascular cells (1.53±0.72 vs. 4.76±1.04; 12.0±2.8 vs. 31.4±2.6, respectively, both P<0.05), as well as the mRNA and protein expression levels of CCN1, PI3K and AKT (protein, −45.3, −22.5 and −28.4%; mRNA, −43.7, −58.7 and −42.9%, respectively, all P<0.05) compared to the administration of scrambled siRNA under hypoxic conditions. Treatment with LY294002 decreased the mRNA and protein expression levels of CCN1 in the cells exposed to hypoxia (both P<0.05). The administration of CCN1 siRNA resulted in less severe neovascularization in the eyes of the the mouse pups with OIR. Thus, out data suggest that CCN1 plays an important role in RNV in ROP, and may thus be a potential target for the prevention and treatment of ROP.