Toxicity of Mildly Modified Low-Density Lipoproteins to Cultured Retinal Capillary Endothelial Cells and Pericytes

Abstract

To investigate the role of modified low-density lipoproteins (LDL) in the pathogenesis of diabetic retinopathy, we studied the cytotoxicity of normal and mildly modified human LDL to bovine retinal capillary endothelial cells and pericytes in vitro. Pooled LDL was incubated (in phosphate-buffered saline-EDTA, 3 days, 37 degrees C) under 1) nitrogen with additional chelating agents and 2) air, to prepare normal and minimally oxidized LDL, respectively. Similar conditions, but with the addition of 50 mM D-glucose, were used to prepare glycated and glycoxidized LDL. None of the LDL preparations was recognized by the macrophage scavenger receptor, confirming limited modification. Retinal capillary endothelial cells and pericytes were grown to confluence and then exposed for 2 or 3 days to serum-free medium (1% albumin) supplemented with normal or modified LDL (100 mg/l) or to serum-free medium alone. Cytotoxicity was assessed by cell counting (live and total cells) and by cell protein determination. Compared with normal LDL, modified LDL were cytotoxic to both cell types at both time points, causing highly significant decreases in live and total cell counts (P < 0.001) (analysis of variance). Reductions in cell protein also were significant for pericytes at day 3 (P = 0.016) and of borderline significance for endothelial cells at day 2 (P = 0.05) and day 3 (P = 0.063). Cytotoxicity increased as follows: normal < glycated < or = minimally oxidized < glycoxidized LDL. We conclude that, in diabetes, mild modification of LDL resulting from separate or combined processes of glycation and oxidation may contribute to chronic retinal capillary injury and thus to the development of diabetic retinopathy.

Stable Expression of Angiopoietin-1 and Other Markers by Cultured Pericytes: Phenotypic Similarities to a Subpopulation of Cells in Maturing Vessels During Later Stages of Angiogenesis In Vivo

“…Our method differs from earlier reports (Challier et al, 1995;de la Rubia et al, 1992;D'Amore, 1992;He and Spiro, 1995;Helmbold et al, 2001;Hoying et al, 1996;Ivarsson et al, 1996;Lyons et al, 1994;Morse and Sidikaro, 1990;Sato et al, 1990;Schor et al, 1991;Schor and Schor, 1986;Shojaee et al, 1998;Wisse et al, 1996) in several additional respects. One is the use of a second purification step involving the use of 3G5 monoclonal antibodies linked to magnetic beads.…”

“…Studies of pericyte function have been hindered because, although they can be isolated, pericytes exhibit an unstable phenotype in culture (Challier et al, 1995;D'Amore, 1992;de la Rubia et al, 1992;He and Spiro, 1995;Helmbold et al, 2001;Hoying et al, 1996;Lyons et al, 1994;Morse and Sidikaro, 1990;Sato et al, 1990;Schor et al, 1991;Schor and Schor, 1986;Shojaee et al, 1998;Wisse et al, 1996). They quickly alter their expression of several different cell typespecific markers and ultimately differentiate into collagen Type I-producing cells that have the characteristics of fibroblasts .…”

Antioxidant treatment of experimental diabetic retinopathy in rats with nicanartine

“…The assumption that antioxidants may prevent retinopathy in diabetes was based on studies showing increased generation of ROS under hyperglycaemic conditions and the toxicity of these products for microvascular cells in vitro [7,10,14]. However, our study demonstrates that antioxidant treatment has only a limited beneficial effect on diabetes-induced The antioxidant potency of nicanartine on in vitro lipid peroxidation has been reported to be higher than vitamin E, especially in the VLDL fraction, and nicanartine inhibits endothelial-induced LDL oxidation more effectively than probucol, as measured by TBARS [20].…”

“…Based on these experimental observations, it has been postulated that diabetic retinopathy may be amenable to antioxidant treatment [10,14]. However, only preliminary experiments are available showing that probucol -a lipid lowering and antioxidant compound -ameliorates disturbed vascular permeability in the retinae of short-term diabetic rats [19].…”

Glycated LDL increases monocyte CC chemokine receptor 2 expression and monocyte chemoattractant protein-1-mediated chemotaxis

“…Indeed, LDL glycation and oxidation [11,12], alone or in combination, may contribute to the increased atherogenic risk in DM patients [13]. The toxicity of AGE-LDL [14] and its role in the pathogenesis of atherosclerosis may relate to its prolonged presence in the circulation [15], which results from impaired cellular uptake [16,17].…”