Oxidative modifications of LDL are a major risk factor in the development of vascular disease and are known to induce endothelial dysfunction, one of the earliest manifestations of atherosclerosis (1, 2). Our studies focus on the oxidized LDL (oxLDL)-induced impact on endothelial biomechanics and its role in vascular dysfunction.Our recent studies showed that the stiffness of aortic endothelial cells (ECs) is significantly increased by exposing the cells to oxLDL in vitro or by dyslipidemia in the dietinduced porcine atherosclerosis model in vivo (3, 4). An increase in endothelial stiffness was accompanied by an increase in endothelial contractile forces generated on the cell-substrate interface and an enhanced ability of ECs to form branching networks in 3D cultures (3, 4), which is considered a prerequisite of angiogenesis (5). Moreover, earlier studies demonstrated a correlation between increased endothelial force and network formation across several endothelial subtypes (6). We proposed, therefore, that oxLDL-induced endothelial stiffening may lead to increased angiogenic activity of ECs during the development of atherosclerotic plaques. This process is expected to be of major clinical importance because neovascularization of the plaques is increasingly recognized as a critical process and a major risk factor for plaque vulnerability (7). The goal of this study is to elucidate the mechanism of oxLDL-induced endothelial stiffening and evaluate a link between this effect and the ability of ECs to form functional capillaries. Abstract Endothelial biomechanics is

The influence of two bioactive oxidized phospholipids on model bilayer properties, membrane packing, and endothelial cell biomechanics was investigated computationally and experimentally. The truncated tail phospholipids, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), are two major oxidation products of the unsaturated phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine. A combination of coarse-grained molecular dynamics simulations, Laurdan multiphoton imaging, and atomic force microscopy microindentation experiments was used to determine the impact of POVPC and PGPC on the structure of a multicomponent phospholipid bilayer and to assess the consequences of their incorporation on membrane packing and endothelial cell stiffness. Molecular simulations predicted differential bilayer perturbation effects of the two oxidized phospholipids based on the chemical identities of their truncated tails, including decreased bilayer packing, decreased bilayer bending modulus, and increased water penetration. Disruption of lipid order was consistent with Laurdan imaging results indicating that POVPC and PGPC decrease the lipid packing of both ordered and disordered membrane domains. Computational predictions of a larger membrane perturbation effect by PGPC correspond to greater stiffness of PGPC-treated endothelial cells observed by measuring cellular elastic moduli using atomic force microscopy. Our results suggest that disruptions in membrane structure by oxidized phospholipids play a role in the regulation of overall endothelial cell stiffness.

Regulation of cell volume is a fundamental property of all mammalian cells. Multiple signaling pathways are known to be activated by cell swelling and to contribute to cell volume homeostasis. Although cell mechanics and membrane tension have been proposed to couple cell swelling to signaling pathways, the impact of swelling on cellular biomechanics and membrane tension have yet to be fully elucidated. In this study, we use atomic force microscopy under isotonic and hypotonic conditions to measure mechanical properties of endothelial membranes including membrane stiffness, which reflects the stiffness of the submembrane cytoskeleton complex, and the force required for membrane tether formation, reflecting membrane tension and membrane-cytoskeleton attachment. We find that hypotonic swelling results in significant stiffening of the endothelial membrane without a change in membrane tension/membrane-cytoskeleton attachment. Furthermore, depolymerization of F-actin, which, as expected, results in a dramatic decrease in the cellular elastic modulus of both the membrane and the deeper cytoskeleton, indicating a collapse of the cytoskeleton scaffold, does not abrogate swelling-induced stiffening of the membrane. Instead, this swelling-induced stiffening of the membrane is enhanced. We propose that the membrane stiffening should be attributed to an increase in hydrostatic pressure that results from an influx of solutes and water into the cells. Most importantly, our results suggest that increased hydrostatic pressure, rather than changes in membrane tension, could be responsible for activating volume-sensitive mechanisms in hypotonically swollen cells.

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The impact of a Western-style high fat diet (HFD) on cardiovascular health is a chronic concern. Our previous studies showed that HFD results in significant endothelial cell (EC) stiffening, which was abrogated by the global deletion of scavenger receptor Cd36. Here we present studies into the role of endothelial Cd36 on EC stiffening and EC permeability in mice fed a short-term HFD of 6-8 weeks. Inducible EC-specific knockdown of Cd36 (EC-Cd36-KD) mice were generated by crossing EC-promoter Cre mice with Cd36fl/fl mice. Knockdowns were induced via tamoxifen injections at 8 weeks of age, after which mice were placed on HFD for 6-8 weeks (Cre-only and sham injected mice served as control groups). The efficiency and specificity of EC-Cd36-KD was verified through RT-qPCR and immunostaining. At the end of the study window, control and EC-Cd36-KD mice exhibited hallmarks of metabolic disease like significant increases in body weight and blood cholesterol (total and LDL-Chol). EC-Cd36-KD did not have an effect on these parameters. As described before, HFD results in significant endothelial stiffening in freshly-isolated intact aortas, assessed using atomic force microscopy. We show here that EC-Cd36-KD prevented HFD-induced EC stiffening. No effect was observed in EC-Cd36-KD mice fed low fat diet. In contrast no change was observed in vascular wall stiffness, assessed via pulse wave velocity analysis following Doppler echocardiography in the ventral short-axis. The EC stiffening effect was observed only in male mice, with female mice showing no EC stiffening and exhibiting lower levels of weight gain and blood cholesterol in comparison to males. EC permeability of the aorta was assessed by injecting Evans blue dye (EBD) into circulation and measuring its penetration into the vascular wall by fluorescent imaging. While HFD results in increased EC permeability, EC-Cd36-KD significantly reduced the penetration of EBD into the aortic wall indicating a protective barrier effect. Cre-only HFD-fed controls were unaffected. These results suggest that Cd36-depedent EC stiffening mediates EC permeability of the aorta. Mechanistically, we show that Cd36 is required for oxLDL-induced stiffening but not for the EC stiffening induced by 7-ketocholesterol and oxidized phospholipid PGPC, suggesting that these oxidized lipids induce endothelial stiffening downstream of Cd36. Finally, the knockdown of RhoGDI-1, an inactivator of RhoA, using siRNA prevents oxysterol-induced EC stiffening. We propose that oxidized lipids induce EC stiffening by uncoupling of RhoA from RhoGDI-1 leading to its activation. This work was supported by NHLBI grants HL141120, HL073965, HL083298 (IL), NHLBI trainee award T32HL144459 and an internal fellowship (VA). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.