Cells Derived from Regenerated Endothelium of the Porcine Coronary Artery Contain More Oxidized Forms of Apolipoprotein-B-100 without a Modification in the Uptake of Oxidized LDL

Abstract

Increased accumulation of lipoproteins and cholesterol within cells from regenerated endothelium may be responsible for their reported dysfunction. This study compared the presence and uptake of oxidized forms of low-density lipoprotein (LDL) in cells derived from native and regenerated endothelium. Four weeks after balloon denudation, primary cultures of native and regenerated endothelial cells were prepared from porcine coronary arteries. Regenerated endothelium stained more strongly using an antibody against oxidized lipoproteins. The increase in oxidized forms of apolipoprotein-B-100 exhibited by cells from regenerated endothelium was not due to an increase in extracellular-induced oxidation of native LDL, measured as the production of thiobarbituric-acid-reactive substances, being identical in both cell types. Intracellular cholesterol and cholesterol ester content were unchanged in regenerated cells. Using flow cytometry, accumulation of oxidized LDL was investigated further by quantifying the uptake of a mildly oxidized preparation of 1,1’-dioctadecyl-3,3,3’,3-tetramethyl-indocarbocyanine perchlorate-labelled LDL. The parameters of uptake, EC₅₀ and E_max, were not different between cells from native and regenerated endothelium suggesting that the number of LOX-1 receptors was identical in the two cell types. Moreover, a negative correlation between the increased uptake of acetylated LDL and decreased cGMP production in response to bradykinin was observed in cells from regenerated endothelium. Thus, the increased incorporation of modified LDL and their intracellular oxidation could be responsible for the alteration in NO production. The presence of oxidized forms of LDL may be a marker of endothelium regeneration and could be involved in the endothelial dysfunction of pig coronary arteries 4 weeks after balloon denudation.

Differential genomic changes caused by cholesterol- and PUFA-rich diets in regenerated porcine coronary endothelial cells

“…Hence, the present findings provide a genomic explanation for the similar blunting of endothelium-dependent relaxations in coronary arteries lined with regenerated endothelium and those harvested from hypercholesterolemic pigs and humans (10,35). They indirectly reinforce the earlier interpretation that abnormal handling of lipids is a key factor leading to endothelial dysfunction following regeneration (11,14,20,38). This abnormal handling of lipids in native cultures from cholesterol-fed pigs is exemplified by the appearance of FABP4.…”

“…11,14,19,20,26,31,[33][34][35]. Primary cultures were used for the gene expression studies to maintain the original genotype of both native and regenerated endothelial cells observed under similar experimental conditions (1,11,14,20). The microarray approach has been used successfully in earlier work on regenerated and senescent endothelial cell cultures (20,21).…”

Regeneration of the Endothelium in Vascular Injury

“…To attempt to unravel the molecular mechanisms underlying the dysfunction of the regenerated endothelium primary cell cultures were grown derived from the endothelium harvested from segments of coronary arteries of the same heart covered with either native or regenerated endothelium [30][31][32][33][34]. Compared to the former, those derived from regenerated endothelium exhibited the following phenotypic changes: a] the presence of multiple enlarged cells and cells containing several nuclei, an appearance characteristic of early senescence, as confirmed by the greater presence of β-galactosidase; b] a reduced expression and activity of eNOS; c] a greater production of oxygen-derived free radicals (ROS); d] a greater uptake of modified low-density lipoprotein cholesterol (LDL), a phenomenon confirmed in ex vivo measurements in intact coronary arteries lined with regenerated endothelium; e] a greater generation of oxidized LDL (oxLDL); f] a reduced activity of Gi-proteins despite the immunostaining demonstration of their unchanged presence, a phenomenon exacerbated by a hypercholesterolemic diet; and g] an indication of accelerated apoptosis.…”

Oxidized Low-Density Lipoprotein

“…The presence and amounts of MDA has always been used as a yard stick for minimally or terminally oxidized LDL (100-103), although numerous publications concluded a short-term oxidation always resulted in a “mildly” oxidized LDL as opposed to long-term oxidations (104–107). Yet, enzymatic (lipoxygenase, peroxidase) reactions and treatment with peroxynitrite that generated lower amounts of MDA were always considered fully oxidized LDL due the ability of macrophages to engulf such LDL.…”