Harhaj Nicole S, Felinski Edward A, Wolpert Ellen B, Sundstrom Jeffrey M, Gardner Thomas W, Antonetti David A
Departments of Cellular and Molecular Physiology, Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA
Invest Ophthalmol Vis Sci. 2006 Nov;47(11):5106-15. doi: 10.1167/iovs.06-0322.
VEGF is a potent permeabilizing factor that contributes to the pathogenesis of diabetic retinopathy and brain tumors. VEGF-induced vascular permeability in vivo and in cell culture requires PKC activity, but the mechanism by which PKC regulates barrier properties remains unknown. This study was conducted to examine how VEGF and diabetes alter occludin phosphorylation and endothelial cell permeability.
Chemical PKC inhibitors and activators were used to treat primary retinal endothelial cells in culture. In vitro kinase assays and Western blot analysis of two-dimensional (2D) and one-dimensional (1D) gel retardation assays were used to analyze occludin phosphorylation. Endothelial cell permeability was determined by measuring the flux of 70-kDa dextran through a cell monolayer in culture. Exogenous expression of a dominant negative PKCbetaII mutant (S217A) was used to assess the PKC dependence of VEGF-induced occludin phosphorylation and endothelial permeability. Occludin phosphorylation was also determined in retinas of streptozotocin-induced diabetic rats.
VEGF stimulated the phosphorylation of occludin in primary retinal endothelial cells. Chemical inhibitors of PKC activity blocked the VEGF-induced increase in occludin phosphorylation, as assessed by 2D gel and gel retardation in Western blot analysis, and blocked part of the VEGF-induced monolayer permeability to 70-kDa dextran. Expression of a dominant negative PKCbetaII mutant blocked VEGF-induced occludin phosphorylation and endothelial permeability. Finally, elevated occludin phosphorylation was observed in the retina of diabetic animals.
These results strongly suggest that VEGF-induced endothelial permeability requires PKC-dependent phosphorylation of occludin. Regulation of PKC activity and tight junction protein modifications may have therapeutic implications for treatment of diabetic retinopathy and brain tumors.
血管内皮生长因子（VEGF）是一种强效的通透因子，在糖尿病视网膜病变和脑肿瘤的发病机制中起作用。VEGF在体内和细胞培养中诱导的血管通透性需要蛋白激酶C（PKC）的活性，但PKC调节屏障特性的机制尚不清楚。本研究旨在探讨VEGF和糖尿病如何改变闭合蛋白的磷酸化及内皮细胞通透性。
使用化学PKC抑制剂和激活剂处理培养的原代视网膜内皮细胞。采用体外激酶测定以及二维（2D）和一维（1D）凝胶阻滞分析的蛋白质印迹法分析闭合蛋白的磷酸化。通过测量70 kDa葡聚糖通过培养的细胞单层的通量来测定内皮细胞通透性。使用显性负性PKCβII突变体（S217A）的外源性表达来评估VEGF诱导的闭合蛋白磷酸化和内皮通透性对PKC的依赖性。还测定了链脲佐菌素诱导的糖尿病大鼠视网膜中闭合蛋白的磷酸化。
VEGF刺激原代视网膜内皮细胞中闭合蛋白的磷酸化。PKC活性的化学抑制剂阻断了VEGF诱导的闭合蛋白磷酸化增加，这通过蛋白质印迹分析中的2D凝胶和凝胶阻滞评估，并阻断了VEGF诱导的单层对70 kDa葡聚糖的部分通透性。显性负性PKCβII突变体的表达阻断了VEGF诱导的闭合蛋白磷酸化和内皮通透性。最后，在糖尿病动物的视网膜中观察到闭合蛋白磷酸化升高。
这些结果强烈表明，VEGF诱导的内皮通透性需要PKC依赖的闭合蛋白磷酸化。PKC活性的调节和紧密连接蛋白修饰可能对糖尿病视网膜病变和脑肿瘤的治疗具有治疗意义。
