Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, United States.
Invest Ophthalmol Vis Sci. 2019 Feb 1;60(2):748-760. doi: 10.1167/iovs.18-25068.
To test the hypothesis that high glucose and matrix metalloproteinases (MMPs) contribute to the diabetes-induced loss of platelet endothelial cell adhesion molecule-1 (PECAM-1) in the retinal microvasculature.
PECAM-1 and MMP protein, activity, and interactions with PECAM-1 were assessed using western blotting, zymography, immunofluorescence, or coimmunoprecipitation assays. These assays were conducted using primary rat retinal microvascular endothelial cells (RRMECs) grown either in normal glucose (5 mM) or high glucose (25 mM) conditions and using retinas collected from streptozotocin-induced diabetic or control rats. The broad-spectrum MMP inhibitor GM6001 was administered in vivo and in vitro to ascertain the role of MMPs in the hyperglycemia-induced loss of PECAM-1.
A dramatic decrease in PECAM-1 (western blotting, immunofluorescence) was observed in both the diabetic retina and in hyperglycemic RRMECs. The decrease in PECAM-1 was accompanied by a significant increase in the presence and activity of matrix metalloproteinase-2 (MMP-2) (but not matrix metalloproteinase-9 [MMP-9]) in the diabetic plasma (P < 0.05) and in hyperglycemic RRMECs (P < 0.05). Moreover, RRMEC PECAM-1 significantly decreased when treated with plasma collected from diabetic rats. Several MMP-2 cleavage sites on PECAM-1 were identified using in silico analysis. Moreover, PECAM-1/MMP-2 interactions were confirmed using coimmunoprecipitation. PECAM-1 was significantly decreased in RRMECs treated with MMP-2 (P < 0.05), but became comparable to controls with the MMP inhibitor GM6001 in both the diabetic retina and hyperglycemic RRMECs.
These results indicate a possible role of MMP-2 in hyperglycemia-induced PECAM-1 loss in retinal endothelial cells.
检验高血糖和基质金属蛋白酶（MMPs）导致糖尿病状态下视网膜微血管中血小板内皮细胞黏附分子-1（PECAM-1）丢失的假说。
采用 Western blot、酶谱法、免疫荧光法或免疫共沉淀法检测 PECAM-1 和 MMP 蛋白、活性及其与 PECAM-1 的相互作用。这些检测方法采用正常葡萄糖（5 mM）或高葡萄糖（25 mM）培养条件下的原代大鼠视网膜微血管内皮细胞（RRMEC）以及链脲佐菌素诱导的糖尿病或对照大鼠的视网膜进行。体内和体外给予广谱 MMP 抑制剂 GM6001，以确定 MMP 在高血糖诱导的 PECAM-1 丢失中的作用。
在糖尿病视网膜和高糖培养的 RRMEC 中，PECAM-1 （Western blot、免疫荧光）明显减少。PECAM-1 的减少伴随着糖尿病血浆（P < 0.05）和高糖 RRMEC（P < 0.05）中基质金属蛋白酶-2（MMP-2）（而非基质金属蛋白酶-9 [MMP-9]）的存在和活性显著增加。此外，用来自糖尿病大鼠的血浆处理 RRMEC 时，PECAM-1 显著减少。使用计算机分析鉴定了 PECAM-1 上几个 MMP-2 切割位点。此外，通过免疫共沉淀证实了 PECAM-1/MMP-2 相互作用。用 MMP-2 处理的 RRMEC 中 PECAM-1 明显减少（P < 0.05），但在糖尿病视网膜和高糖 RRMEC 中用 MMP 抑制剂 GM6001 处理后，与对照组相当。
这些结果表明 MMP-2 在高血糖诱导的视网膜内皮细胞 PECAM-1 丢失中可能发挥作用。
