Mandarino L J, Sundarraj N, Finlayson J, Hassell H R
Diabetes Division, University of Texas Health Science Center at San Antonio 78284-7886.
Exp Eye Res. 1993 Nov;57(5):609-21. doi: 10.1006/exer.1993.1166.
Retinal capillaries are composed of endothelial cells resting on a basement membrane, in which are embedded pericytes. In diabetes mellitus, the basement membrane becomes thickened, and there is a loss of pericytes. The relative contributions of endothelial cells and pericytes to the synthesis of extracellular matrix proteins which are components of the basement membrane are not well-characterized. To determine how a selective loss of pericytes might affect the composition of retinal capillary basement membranes, we used primary cultures of bovine retinal capillary endothelial cells and pericytes to determine the forms and quantify the amounts of laminin and fibronectin synthesized and secreted by these cell types as well as to determine how high glucose concentrations alter these parameters. Results of ELISAs showed that pericyte cell/matrix layers contained nearly ten times more fibronectin than endothelial cells (288 +/- 24 vs. 34 +/- 5 ng micrograms-1 DNA, P < 0.001), but the amounts of laminin were similar. D-glucose (40 mM) tripled the amount of fibronectin incorporated into the endothelial cell/matrix layer (102 +/- 4 vs. 34 +/- 5 ng micrograms-1 DNA, P < 0.05), but had a lesser effect on pericytes. The non-metabolizable analogue L-glucose, also increased the amount of fibronectin incorporated in both pericyte and endothelial cell/matrix layers. The effects of D- and L-glucose on fibronectin secreted into the medium by both cell types were similar to the effects on incorporation of fibronectin into cell/matrix layers. Glucose had no effect on laminin synthesis. [35S]methionine radiolabeling and immunoprecipitation showed that pericytes and endothelial cells synthesize different forms of fibronectin. Both pericytes and endothelial cells synthesized an A and two B chains of laminin which were of similar apparent size, but the two cell types post-translationally modified the subunits differently. We conclude that pericytes and endothelial cells may contribute different forms and amounts of fibronectin and laminin to the retinal capillary basement membrane, so the preferential loss of pericytes in diabetes could result in basement membrane abnormalities which might lead to endothelial cell dysfunction.
视网膜毛细血管由位于基底膜上的内皮细胞组成，周细胞镶嵌于基底膜中。在糖尿病中，基底膜增厚，周细胞丢失。内皮细胞和周细胞对作为基底膜成分的细胞外基质蛋白合成的相对贡献尚未完全明确。为了确定周细胞的选择性丢失如何影响视网膜毛细血管基底膜的组成，我们使用牛视网膜毛细血管内皮细胞和周细胞的原代培养物来确定这些细胞类型合成和分泌的层粘连蛋白和纤连蛋白的形式并对其含量进行定量，以及确定高糖浓度如何改变这些参数。酶联免疫吸附测定（ELISA）结果显示，周细胞细胞/基质层中的纤连蛋白含量比内皮细胞多近十倍（288±24对34±5 ng微克-1 DNA，P<0.001），但层粘连蛋白的含量相似。D-葡萄糖（40 mM）使掺入内皮细胞/基质层的纤连蛋白量增加两倍（102±4对34±5 ng微克-1 DNA，P<0.05），但对周细胞的影响较小。不可代谢的类似物L-葡萄糖也增加了掺入周细胞和内皮细胞/基质层的纤连蛋白量。D-葡萄糖和L-葡萄糖对两种细胞类型分泌到培养基中的纤连蛋白的影响与对纤连蛋白掺入细胞/基质层的影响相似。葡萄糖对层粘连蛋白合成无影响。[35S]甲硫氨酸放射性标记和免疫沉淀显示，周细胞和内皮细胞合成不同形式的纤连蛋白。周细胞和内皮细胞均合成了一条A链和两条B链的层粘连蛋白，其表观大小相似，但两种细胞类型对亚基的翻译后修饰不同。我们得出结论，周细胞和内皮细胞可能对视网膜毛细血管基底膜贡献不同形式和数量的纤连蛋白和层粘连蛋白，因此糖尿病中周细胞的优先丢失可能导致基底膜异常，进而可能导致内皮细胞功能障碍。
