PHD2 attenuates high-glucose-induced blood retinal barrier breakdown in human retinal microvascular endothelial cells by regulating the Hif-1α/VEGF pathway
PHD2 attenuates high-glucose-induced blood retinal barrier breakdown in human retinal microvascular endothelial cells by regulating the Hif-1α/VEGF pathway
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PHD2 通过调节 Hif-1α/VEGF 途径减弱人视网膜微血管内皮细胞中高葡萄糖诱导的血视网膜屏障破坏
DOI：
10.1007/s00011-021-01518-2
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发表时间：
2021
影响因子：
6.7
通讯作者：
Xiaoling Liang
中科院分区：
文献类型：
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作者：
Jia Li;Xi Lu;Liqing Wei;Dan Ye;Jianqiang Lin;Xiaoyu Tang;Kaixuan Cui;Shanshan Yu;Yue Xu;Xiaoling Liang
目的糖尿病黄斑水肿（DME）是严重视力丧失的最常见原因之一。 DME的发病机制尚不完全清楚；然而，据推测，这是由于高血糖条件下血管内皮生长因子（VEGF）分泌引起的视网膜炎症导致血视网膜屏障（BRB）破坏所致。在这项研究中，我们发现缺氧诱导因子 1 (HIF-1) 的上游调节因子 Prolyl-4-羟化酶 2 (PHD2) 可调节 VEGF 表达，从而保留小鼠视网膜中的 BRB 功能。 材料和方法原代人视网膜微血管内皮细胞 (hRMEC) 在人内皮无血清生长培养基中培养，并暴露于 高血糖。通过 MTT 测定研究细胞活力的变化。通过细胞旁通透性测定和跨内皮电阻（TEER）揭示各组的 BRB 功能。通过occludin 和 zonula occlusionns-1 (ZO-1) 的免疫荧光染色研究 BRB 的形态变化。通过逆转录定量PCR（RT-qPCR）、蛋白质印迹分析和ELISA检测紧密连接蛋白PHD2、HIF-1α和VEGF的mRNA和蛋白水平。结果在高血糖条件下，hRMECs活力下降，PHD2表达下调，伴随细胞旁通透性增加和降低。 跨内皮电阻。此外，HIF-1α和VEGF表达水平升高，而紧密连接蛋白（包括occludin和ZO-1）的表达水平降低，BRB功能受损。 PHD2激活剂R59949（二酰基甘油激酶抑制剂II）改变了这些病理变化，而PHD2抑制剂二甲基乙二酰甘氨酸（DMOG）则产生相反的效果。结论这些结果表明，在高血糖条件下，PHD2通过抑制hRMECs中HIF-1α的表达来抑制HIF-1活性，从而导致HIF-1α表达下调。 血管生成因子 VEGF 的表达，从而有助于维持 hRMEC 的功能。因此，有理由认为 PHD2 可能成为治疗 DME 或具有类似发病机制的其他疾病的潜在新靶点。
ObjectiveDiabetic macular edema (DME) is one of the most frequent causes of severe vision loss. The pathogenesis of DME is still not fully understood; however, it is hypothesized to result from breakdown of the blood–retinal barrier (BRB) due to retinal inflammation by vascular endothelial growth factor (VEGF) secretion under hyperglycemic conditions. In this investigation, we discovered that Prolyl-4-hydroxylase 2 (PHD2), an upstream regulator of hypoxia-inducible factor 1 (HIF-1) modulates VEGF expression and thus preserves BRB function in the mouse retina.Materials and methodsPrimary human retinal microvascular endothelial cells (hRMECs) were cultured in human endothelial serum-free growth medium and exposed to hyperglycemia. Changes in cell viability were investigated by an MTT assay. BRB function in each group was revealed by a paracellular permeability assay and trans-endothelial electrical resistance (TEER). Morphological changes in the BRB were investigated by immunofluorescence staining of occludin and zonula occludens-1 (ZO-1). The mRNA and protein levels of the tight junction proteins, PHD2, HIF-1α, and VEGF were measured by reverse transcription-quantitative PCR (RT-qPCR), western blot analysis and ELISA.ResultsUnder hyperglycemic conditions, the viability of hRMECs was decreased, and PHD2 expression was downregulated, accompanied by increased paracellular permeability and decreased trans-endothelial electrical resistance. Additionally, HIF-1α and VEGF expression levels were increased, whereas the expression levels of tight junction proteins, including occludin and ZO-1, were decreased and BRB function was compromised. The PHD2 activator R59949 (diacylglycerol kinase inhibitor II), altered these pathological changes, and the PHD2 inhibitor dimethyloxalylglycine (DMOG) resulted in the opposite effects.ConclusionThese results demonstrated that PHD2 inhibited HIF-1 activity by inhibiting HIF-1α expression in hRMECs under hyperglycemic conditions, which led to the downregulation of the expression of the angiogenic factor VEGF, and thus helped to maintain the functions of hRMECs. Therefore, it is reasonable to propose that PHD2 could be a potential novel target for the treatment of DME or other diseases with a similar pathogenesis.