Regulation of Fibronectin and Laminin Synthesis by Retinal Capillary Endothelial Cells and Pericytes In Vitro

Abstract

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 μg -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 μg -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.