PI3Kδ促进低氧诱导VEGF产生的分子机制及其在抗视网膜新生血管形成中的应用

视网膜新生血管形成是增殖性糖尿病视网膜病变(PDR)的主要特点之一，目前许多此类患者对血管内皮生长因子(VEGF)的靶向治疗仍效果欠佳。申请人前期研究发现磷脂酰肌醇3-激酶(PI3K)δ在PDR增殖膜血管内皮细胞中高表达，且PI3Kδ失活显著减少氧诱导视网膜病变小鼠玻璃体中VEGF的含量，但其具体分子机制尚不清楚。基于既往研究，我们提出PI3Kδ通过Akt/MDM2/HIF1α信号通路增强低氧诱导VEGF表达的科学假说。本项目以视网膜微血管内皮细胞、巨噬细胞和氧诱导视网膜病变小鼠模型为研究对象，采用先导编辑、CRISPR/Cas9、Western blot、ELISA、免疫荧光等方法验证此假说，并利用AAV-CRISPR/dCasX-KRAB阻断PI3Kδ的表达来抑制视网膜病理性新生血管形成。本研究将为开发以PI3Kδ为靶点治疗视网膜新生血管性眼病的新途径提供理论依据。

Retinal angiogenesis is one of the main characteristics of proliferative diabetic retinopathy (PDR), and at present there are still lots of patients with angiogenesis-associated retinal diseases who are not effectively treated by targeting vascular endothelial growth factor (VEGF). Our previous studies have demonstrated that phosphoinositide (PI3K)δ is highly expressed in vascular endothelial cells in fibrovascular membranes from patients with PDR, and PI3Kδ inactivation significantly reduces VEGF levels in the vitreous in a mouse model of oxygen-induced retinopathy; however, its exact molecular mechanism is unknown. Based on current publications, we hypothesize that PI3Kδ enhances hypoxia-induced VEGF expression through a signaling pathway of Akt/MDM2/HIF1α. To test this hypothesis we will employ retinal microvascular endothelial cells, macrophages and the mouse model of oxygen-induced retinopathy with methods including prime editing, CRISPR/Cas9, Western blot, ELISA, immunofluorescence; in addition, AAV-CRISPR/dCasX-KRAB will be utilized to block expression of p110δ, a catalytic subunit of PI3Kδ, in order to prevent retinal pathological angiogenesis. The resultant data will provide a strong foundation for developing novel therapeutic approaches to angiogenesis-associated retinal diseases by targeting PI3Kδ.