Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, China.
Diabetes. 2010 Sep;59(9):2297-305. doi: 10.2337/db09-1420. Epub 2010 Jun 8.
Vascular endothelial growth factor (VEGF-A or VEGF) is a major pathogenic factor and therapeutic target for diabetic retinopathy (DR). Since VEGF has been proposed as a survival factor for retinal neurons, defining the cellular origin of pathogenic VEGF is necessary for the effectiveness and safety of long-term anti-VEGF therapies for DR. To determine the significance of Müller cell-derived VEGF in DR, we disrupted VEGF in Müller cells with an inducible Cre/lox system and examined diabetes-induced retinal inflammation and vascular leakage in these conditional VEGF knockout (KO) mice.
Leukostasis was determined by counting the number of fluorescently labeled leukocytes inside retinal vasculature. Expression of biomarkers for retinal inflammation was assessed by immunoblotting of TNF-alpha, ICAM-1, and NF-kappaB. Vascular leakage was measured by immunoblotting of retinal albumin and fluorescent microscopic analysis of extravascular albumin. Diabetes-induced vascular alterations were examined by immunoblotting and immunohistochemistry for tight junctions, and by trypsin digestion assays for acellular capillaries. Retinal integrity was analyzed with morphologic and morphometric analyses.
Diabetic conditional VEGF KO mice exhibited significantly reduced leukostasis, expression of inflammatory biomarkers, depletion of tight junction proteins, numbers of acellular capillaries, and vascular leakage compared to diabetic control mice.
Müller cell-derived VEGF plays an essential and causative role in retinal inflammation, vascular lesions, and vascular leakage in DR. Therefore, Müller cells are a primary cellular target for proinflammatory signals that mediates retinal inflammation and vascular leakage in DR.
血管内皮生长因子（VEGF-A 或 VEGF）是糖尿病性视网膜病变（DR）的主要致病因子和治疗靶点。由于 VEGF 被认为是视网膜神经元的存活因子，因此确定致病 VEGF 的细胞来源对于 DR 的长期抗 VEGF 治疗的有效性和安全性至关重要。为了确定 Müller 细胞来源的 VEGF 在 DR 中的意义，我们使用诱导型 Cre/lox 系统破坏 Müller 细胞中的 VEGF，并在这些条件性 VEGF 敲除（KO）小鼠中检查糖尿病诱导的视网膜炎症和血管渗漏。
通过计数视网膜血管内荧光标记白细胞的数量来确定白细胞淤滞。通过免疫印迹 TNF-α、ICAM-1 和 NF-κB 评估视网膜炎症的生物标志物表达。通过免疫印迹视网膜白蛋白和血管外白蛋白的荧光显微镜分析测量血管渗漏。通过免疫印迹和紧密连接的免疫组织化学以及无细胞毛细血管的胰蛋白酶消化测定检查糖尿病诱导的血管改变。用形态学和形态计量学分析分析视网膜完整性。
与糖尿病对照小鼠相比，糖尿病条件性 VEGF KO 小鼠的白细胞淤滞、炎症生物标志物表达、紧密连接蛋白耗竭、无细胞毛细血管数量和血管渗漏均显著减少。
Müller 细胞来源的 VEGF 在 DR 中的视网膜炎症、血管病变和血管渗漏中发挥重要的因果作用。因此，Müller 细胞是介导 DR 中视网膜炎症和血管渗漏的促炎信号的主要细胞靶标。
