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. Specific oxysterols, however, have differential effects on endothelial stiffness with 7-ketocholesterol and 7α-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. oxLDL-induced stiffening is accompanied by incorporation of oxysterols into endothelial cells. We find significant accumulation of three oxysterols, 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol, in mouse aortas of dyslipidemic ApoE⁻/⁻ mice at the early stage of atherosclerosis. Remarkably, these are the same oxysterols we have identified to induce endothelial stiffening.
Highlights
Endothelial dysfunction is a key step in atherosclerosis development
We show that progressive LDL oxidation results in continuous increase in cell stiffness (TBARS values ranging between 3 ± 0.5 and 27 ± 1.2 MDA/mg protein) (Fig. 1)
An increase in endothelial stiffness is apparent from the right shift in the histograms of the elastic moduli measured in cells exposed to oxLDL compared with cells exposed to nonmodified LDL (Fig. 1A), as well as from the increase in the mean elastic moduli measured for cells exposed to progressively oxidized LDL (Fig. 1B, C)
Summary
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. 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. We demonstrate that oxysterols play a major role in the increase in endothelial stiffness and identify specific oxysterols responsible for this effect
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