The role of oxysterols in control of endothelial stiffness

Abstract

Multiple studies have shown that oxidative modifi cations of LDL (oxLDL) are a major factor in the development of atherosclerosis ( 1-6 ). The level of oxLDL increases dramatically with hypercholesterolemia in animal models of atherosclerosis ( 7,8 ), and in humans ( 9, 10 ), it is found in atherosclerotic lesions ( 11 ). It is also well established that dyslipidemia-induced dysfunction of vascular endothelial cells is a critical step in the early stage of atherosclerosis (e.g., Refs. 12-14 ) and a strong predictor of cardiovascular disease (CVD) development ( 15-17 ). The mechanisms, however, of dyslipidemia-induced endothelial dysfunction are still poorly understood. Our recent studies have shown that exposure to oxLDL in vitro or to plasma dyslipidemia in vivo signifi cantly increases the stiffness of aortic endothelial cells, which in turn is associated with an increase in endothelial contractility, enhanced angiogenic potential, and sensitivity to shear stress (18)(19)(20). Furthermore, unexpectedly, our studies showed that dyslipidemia-induced endothelial stiffening is caused not by cholesterol loading but by disruption of lipid packing of cholesterol-rich membrane domains in endothelial cells ( 18,19,21 ). Consistent with these observations, oxLDLinduced endothelial stiffness could be fully reversed by enriching the cells with cholesterol, even though oxLDL had no effect on the cholesterol content of endothelial membranes ( 19 ). These studies led us to the hypothesis that oxLDL induces endothelial dysfunction by inserting oxysterols into the plasma membrane, resulting in the disruption of cholesterol-rich membrane domains and endothelial stiffening. The goal of this study, therefore, was to determine the impact of oxysterols on endothelial stiffness.Abstract Endothelial dysfunction is a key step in atherosclerosis development. Our recent studies suggested that oxLDL-induced increase in endothelial stiffness plays a major role in dyslipidemia-induced endothelial dysfunction. In this study, we identify oxysterols, as the major component of oxLDL, responsible for the increase in endothelial stiffness. Using Atomic Force Microscopy to measure endothelial elastic modulus, we show that endothelial stiffness increases with progressive oxidation of LDL and that the two lipid fractions that contribute to endothelial stiffening are oxysterols and oxidized phosphatidylcholines, with oxysterols having the dominant effect. Furthermore, endothelial elastic modulus increases as a linear function of oxysterol content of oxLDL. Specifi c oxysterols, however, have differential effects on endothelial stiffness with 7-ketocholesterol and 7 ␣ -hydroxycholesterol, the two major oxysterols in oxLDL, having the strongest effects. 27-hydroxycholesterol, found in atherosclerotic lesions, also induces endothelial stiffening. For all oxysterols, endothelial stiffening is reversible by enriching the cells with cholesterol. ox-LDL-induced stiffening is accompanied by incorporation of oxysterols into endothelial cells. We fi nd signif...

Molecular-Scale Biophysical Modulation of an Endothelial Membrane by Oxidized Phospholipids

“…Our previous studies showed that endothelial cell stiffening induced by the incorporation of oxysterols could be reversed by loading the cells with cholesterol (14). Here, we showed that cholesterol loading had the same effect on oxPAPC-induced endothelial cell stiffening (Fig.…”

“…In contrast to the previous belief, exposing endothelial cells to oxLDL does not result in cholesterol loading of endothelial cells but, instead, results in a decrease in lipid order of the cell membrane (11). A similar effect is observed in cells exposed to specific oxysterols (14). Thus, consistent with our findings on the effects of cholesterol depletion, exposure to oxLDL results in fluidization of the endothelial membrane accompanied by an increase in endothelial cell stiffness.…”

“…This inverse relationship may be considered paradoxical, because disruption of lipid order is known to decrease the lipid bilayer elastic modulus (5) and is expected to disrupt the integrity of cholesterol-rich membrane domains-signaling platforms that serve as focal points for membrane-cytoskeleton interactions (9,71). Our previous findings indicate that cholesterol depletion and incorporation of specific oxysterols also induce endothelial cell stiffening (10,14). Moreover, this study extends our previous observations to demonstrate that cellular stiffening is not a specific effect of the loss of cholesterol or its substitution with a structurally different oxysterol, which is a possible explanation of our previous results.…”

“…Next we tested whether oxPC-induced disruption of lipid packing is accompanied by an increase in endothelial cell stiffness, measured using AFM, with a 10-mm-diameter bead attached to the cantilever, as we have previously shown for oxysterols (14). Three oxidized species were used to test this hypothesis.…”

“…To determine the impact of POVPC and PGPC on the properties of cell membranes, aortic endothelial cells were exposed to 10 mg/mL of each oxPC separately for 1 h, as described previously for oxysterols (14). The efficiency of incorporation of the oxPCs and their potential impact on the levels of LPC and oxysterols was determined using LC-MS/MS.…”