oxLDL facilitates flow-induced realignment of aortic endothelial cells

Abstract

(ECs) in the direction of the flow is considered a key factor in maintaining endothelial integrity in an active hemodynamic environment. Our recent studies showed that exposure to oxidized LDL (oxLDL), one of the major proatherogenic lipoproteins, significantly increases the stiffness of human aortic ECs, suggesting that oxLDL may have a significant impact on the sensitivity of ECs to mechanical stimuli. In this study, we show that oxLDL strongly enhances the ability of ECs to realign in the direction of the flow and facilitates the formation of F-actin stress fibers under static and flow conditions. The impact of oxLDL on the flow-induced realignment is observed on whole cell and single-fiber levels. We also show that, similar to the effect of oxLDL on endothelial stiffness, the impact of oxLDL on flow-induced realignment can be simulated by methyl-␤-cyclodextrininduced cholesterol depletion, supporting the hypothesis that oxLDL acts as cholesterol acceptor, rather than cholesterol donor, for ECs. Finally, we propose that oxLDL/cholesterol depletion-induced sensitization of ECs to flow may be a result of an increase in cellular stiffness and a respective increase in membrane-cytoskeleton tension.cholesterol; lipid rafts HEMODYNAMIC FORCES GENERATED by blood flow are known to play prominent roles in the acute control of vascular tone, regulation of arterial structure, and localization of atherosclerotic lesions (12,19,31). The primary tissue that is affected by the hemodynamic environment is vascular endothelium, a single-cell layer that constitutes the inner lining of the blood vessels and exists on the blood-vascular wall interface. Multiple studies have shown that endothelial cells (ECs) respond to flow by a variety of mechanisms, including cytoskeleton rearrangement, activation of multiple signaling pathways, and changes in gene expression (for review see 24,25,34). Although the exact mechanisms responsible for conversion of mechanical stimuli to the biological responses are not fully understood, it is generally accepted that the cytoskeleton provides the structural framework for transmission of mechanical signals throughout the cell. Flow-induced changes of endothelial morphology, such as realignment in the direction of flow and cell elongation, are suggested to be important for maintaining endothelial integrity in active flow environments (9,20,25).Our recent studies showed that endothelial mechanical properties are strongly affected by exposure of cells to the oxidized form of low-density lipoproteins (oxLDL) (6). More specifically, exposure to oxLDL, but not the unoxidized form of LDL, resulted in a significant decrease in the deformability/increase in stiffness of aortic ECs, as measured by micropipette aspiration (microaspiration), as well as an increase in the ability of ECs to generate force on the cell-substrate interface (6). Depletion of cellular cholesterol had the same effect (6) and also increased membrane-cytoskeleton adhesion (37). Multiple studies have shown that deformability of the cellu...

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

“…Our earlier studies showed that effects of oxLDL on endothelial cell biomechanical properties, such as cell stiffness and reorientation in the direction of flow, can be simulated by cholesterol depletion (10,80), whereas cholesterol enrichment has a rescuing effect (11). However, we did not find a significant effect of oxLDL on endothelial membrane cholesterol.…”

“…We have also shown that cholesterol depletion paradoxically strengthens membrane-cytoskeleton adhesion, as estimated by pulling membrane tethers (7). Moreover, our studies further demonstrate that exposure to oxLDL induces an increase in F-actin stress fibers (80) and that endothelial stiffening induced by cholesterol depletion critically depends on the integrity of F-actin (6). In terms of the mechanism that links insertion of oxidized lipids to endothelial cell stiffening, our recent study demonstrates that for oxLDL and oxysterols, stiffening is mediated by the activation of the RhoA/Rock/PLCP/MLC2 pathway (56).…”

Oxidized LDL signals through Rho-GTPase to induce endothelial cell stiffening and promote capillary formation

“…Because it is known that RhoA is a key factor in stress-fiber formation, these data suggest that the stiffening is primarily a result of stress fiber formation. Indeed, our previous study demonstrated that exposure to oxLDL induces formation of stress fibers (35). In contrast, activation of Rac1, which was previously shown to be responsible for oxLDL-induced dephosphorylation of MLC in macrophages (27), does not contribute significantly to oxLDL-induced regulation of endothelial stiffness.…”

“…We also found that oxLDL and cholesterol depletion have similar effects on endothelial contractility, network formation (3,4), and flow-induced endothelial alignment (35). In this study, we focus on the downstream signaling mechanism that underlies oxLDL-induced endothelial stiffening.…”

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

“…Changes in cell stiffness are expected to have a major impact on cell mechanosensitivity and mechanotransduction. Specifically, we have shown recently that an increase in endothelial stiffness facilitates their sensitivity to flow 25 . We have also shown that endothelial stiffening is associated with an increase in their angiogenic potential 22 .…”