Oxidised, glycated LDL selectively influences tissue inhibitor of metalloproteinase-3 gene expression and protein production in human retinal capillary pericytes

Abstract

Aims/hypothesis Matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitor of metalloproteinases (TIMPs), regulate important biological processes including the homeostasis of the extracellular matrix, proteolysis of cell surface proteins, proteinase zymogen activation, angiogenesis and inflammation. Studies have shown that their balance is altered in retinal microvascular tissues in diabetes. Since LDLs modified by oxidation/ glycation are implicated in the pathogenesis of diabetic vascular complications, we examined the effects of modified LDL on the gene expression and protein production of MMPs and TIMPs in retinal pericytes.Methods Quiescent human retinal pericytes were exposed to native LDL (N-LDL), glycated LDL (G-LDL) and heavily oxidised and glycated LDL (HOG-LDL) for 24 h. We studied the expression of the genes encoding MMPs and TIMPs mRNAs by analysis of microarray data and quantitative PCR, and protein levels by immunoblotting and ELISA. Results Microarray analysis showed that MMP1, MMP2, MMP11, MMP14 and MMP25 and TIMP1, TIMP2, TIMP3 and TIMP4 were expressed in pericytes. Of these, only TIMP3 mRNA showed altered regulation, being expressed at significantly lower levels in response to HOG-vs N-LDL. Quantitative PCR and immunoblotting of cell/matrix proteins confirmed the reduction in TIMP3 mRNA and protein in response to HOG-LDL. In contrast to cellular TIMP3 protein, analysis of secreted TIMP1, TIMP2, MMP1 and collagenase activity indicated no changes in their production in response to modified LDL. Combined treatment with N-and HOG-LDL restored TIMP3 mRNA expression to a level comparable with that after N-LDL alone. Conclusions/interpretation Among the genes encoding for MMPs and TIMPs expressed in retinal pericytes, TIMP3 is uniquely regulated by HOG-LDL. Reduced TIMP3 expression might contribute to microvascular abnormalities in diabetic retinopathy.

Intraretinal Leakage and Oxidation of LDL in Diabetic Retinopathy

“…More recently, we demonstrated that HOG-LDL has unique effects on one of the inhibitors of matrix metalloproteinases, TIMP-3. 20 It is noteworthy that although glycated LDL has less drastic effects than HOG-LDL on pericytes, as witnessed by our gene array study, 17 modification by glycation is important not only for its own effects but also because it may predispose LDL to oxidative damage and enhance its tendency to become sequestered after extravasation, again increasing the likelihood of oxidation. The sublethal effects of oxidized LDL in the retina merit further study, but our present study addresses its lethal effects on pericytes.…”

“…[17][18][19][20] Briefly, HRCPs (Cambrex, Walkersville, MD) were cultured at 37°C with 5% CO 2 in medium containing 5% fetal bovine serum, 0.1% human epithelial growth factor (hEGF), 0.04% hydrocortisone, 0.1% vascular epithelial growth factor (VEGF), 0.4% human fibroblast growth factor (hFGF)-B, 0.1% R 3 -insulinlike growth factor (IGF)-1, 0.1% ascorbic acid, and 0.1% GA-1000 (Clonetics, Walkersville, MD). HRCP from passages 4 to 13 were used.…”

“…Supporting this, we have shown adverse effects of in vitro-modified LDL (glycated and oxidized) on retinal capillary cells, specifically in mediating cytotoxicity and altered gene expression. [17][18][19][20] However, to date there is little direct evidence of the presence of modified LDL in the diabetic retina. In the present study, using immunohistochemistry, we demonstrate the presence of extravasated ApoB100 and oxidized LDL in retinas from diabetic patients, correlating with the severity of DR. Further, in PDR, the presence of macrophages and their colocalization with ApoB was observed.…”

Diabetic retinopathy in type 1 diabetes—a contemporary analysis of 8,784 patients

“…There is long-standing experimental evidence that elevated and biochemically altered lipids may confer cytotoxicity to retinal capillary cells [42]. Moreover, some clinical studies have suggested a link between triacylglycerol and retinopathy [43,44].…”

Biomarkers in Diabetic Retinopathy

“…Lyons' team has shown that extravasated and modified LDL and immune-complexed oxLDL are present in the retina of diabetic patients. They have also demonstrated that glycated LDL and/or oxLDL exert cytotoxicity to cultured human retinal capillary endothelial cells [192], pericytes [166,[192][193][194][195], retinal pigment epithelium [165], and Muller glial cells [196]. Finally, these glycated LDL could be shown to cause changes in cell signaling, gene expression, cell apoptosis, and autophagy [165,192,193,195,196].…”