State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, No.7 Jinsui Rd, Tianhe District, Guangzhou, Guangdong, People's Republic of China, 510030.
Eye Hospital of Wenzhou Medical University, Hangzhou Xihu Zhijiang Eye Hospital, No.7 Jinsui Rd, Hangzhou, Zhejiang, People's Republic of China, 310024.
Inflamm Res. 2022 Jan;71(1):69-79. doi: 10.1007/s00011-021-01518-2. Epub 2021 Nov 13.
Diabetic 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.
Primary 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.
Under 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.
These 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.
糖尿病性黄斑水肿（DME）是导致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，它是由于在高血糖条件下血管内皮生长因子（VEGF）分泌引起视网膜炎症导致血视网膜屏障（BRB）破裂而发生的。在本研究中，我们发现脯氨酰-4-羟化酶 2（PHD2），缺氧诱导因子 1（HIF-1）的上游调节剂，调节 VEGF 的表达，从而在小鼠视网膜中维持 BRB 功能。
原代人视网膜微血管内皮细胞（hRMECs）在人内皮无血清生长培养基中培养并暴露于高血糖中。通过 MTT 测定法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）揭示每组的 BRB 功能。通过 occludin 和 zonula occludens-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）、western blot 分析和 ELISA 测量紧密连接蛋白、PHD2、HIF-1α 和 VEGF 的 mRNA 和蛋白水平。
在高血糖条件下，hRMECs 的活力降低，PHD2 表达下调，同时细胞旁通透性增加，跨内皮电阻降低。此外，HIF-1α 和 VEGF 的表达水平增加，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 抑制剂二甲氧酰基甘氨酸（DMOG）则产生相反的效果。
这些结果表明，PHD2 通过抑制高血糖条件下 hRMECs 中的 HIF-1α 表达抑制 HIF-1 活性，从而导致血管生成因子 VEGF 的表达下调，从而有助于维持 hRMECs 的功能。因此，有理由提出 PHD2 可能是治疗 DME 或其他具有相似发病机制的疾病的潜在新靶点。
