Activation of integrin α5 mediated by flow requires its translocation to membrane lipid rafts in vascular endothelial cells

Abstract

Local flow patterns determine the uneven distribution of atherosclerotic lesions. Membrane lipid rafts and integrins are crucial for shear stress-regulated endothelial function. In this study, we investigate the role of lipid rafts and integrin α5 in regulating the inflammatory response in endothelial cells (ECs) under atheroprone versus atheroprotective flow. Lipid raft proteins were isolated from ECs exposed to oscillatory shear stress (OS) or pulsatile shear stress, and then analyzed by quantitative proteomics. Among 396 proteins redistributed in lipid rafts, integrin α5 was the most significantly elevated in lipid rafts under OS. In addition, OS increased the level of activated integrin α5 in lipid rafts through the regulation of membrane cholesterol and fluidity. Disruption of F-actin-based cytoskeleton and knockdown of caveolin-1 prevented the OS-induced integrin α5 translocation and activation. In vivo, integrin α5 activation and EC dysfunction were observed in the atheroprone areas of low-density lipoprotein receptor-deficient (Ldlr −/− ) mice, and knockdown of integrin α5 markedly attenuated EC dysfunction in partially ligated carotid arteries. Consistent with these findings, mice with haploinsufficency of integrin α5 exhibited a reduction of atherosclerotic lesions in the regions under atheroprone flow. The present study has revealed an integrin-and membrane lipid raft-dependent mechanotransduction mechanism by which atheroprone flow causes endothelial dysfunction.integrin | lipid rafts | shear stress | proteomics | endothelial dysfunction S hear stress imposed on vascular endothelial cells (ECs) influences vascular phenotype and function. Atherosclerosis preferentially develops at branches and curvatures in the arterial tree where flow is disturbed. In contrast, pulsatile shear stress (PS) in the straight parts of the arteries is atheroprotective (1). At the cellular and molecular levels, disturbed flow pattern increases, while PS inhibits, the inflammatory response in ECs, including the expression of intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), and interleukin 1β (IL-1β) (2). We found that integrins in ECs are shear stress-sensitive (3). Many subsequent studies demonstrated that integrin activation is essential for transmitting mechanical stimuli to intracellular biochemical pathways (4-6). Additionally, mounting evidence indicates that lipid raft microdomains, membrane receptors, cytoskeletal proteins, and extracellular matrices are linked to integrin activation in the context of mechanotransduction (7-11). However, the key mechanism underlying the differential effects of atheroprone versus atheroprotective flows in activating integrins that in turn induces inflammatory response in ECs remains to be elucidated.Lipid rafts are membrane microdomains that are enriched in cholesterol, sphingolipids, and a variety of signaling molecules, which function as cellular signaling platforms. These microdomains are more ordered and tightly packed than the surrounding membran...

Lipid rafts sense and direct electric field-induced migration

“…Inhibition of raft and integrin polarization from Cav-1 knockdown cells suggested reciprocal interactions among raft, integrin, and Cav-1, and the integrity of all three components was necessary for EF-directed migration. Activation of integrin by extracellular matrix proteins has been shown to change integrin partitioning (35)(36)(37), stabilize lipid rafts (38), and modulate EFinduced motility and directionality (4,39). Integrin activation may also induce src signaling that phosphorylates Cav (40).…”

“…Lipid rafts have been shown to mediate mechanotransduction through spatial or allosteric regulation of protein functions (37,49). However, it is not clear what initially leads to the changes in raft organization.…”

Lipid raft-mediated miR-3908 inhibition of migration of breast cancer cell line MCF-7 by regulating the interactions between AdipoR1 and Flotillin-1

“…Lipid raft fractions were purified from MCF-7 cells by a modified sucrose density gradient ultracentrifugation procedure [25, 26]. Briefly, MCF-7 cells were lysed with ice-cold MBS buffer.…”

Quantitative proteomics reveals key proteins regulated by eicosapentaenoic acid in endothelial activation

“…HUVECs were cultured for at most five passages with previously described methods [11]. One day before the experiments, HUVECs were plated in 10 cm dishes at 50% confluency to minimize cell overgrowth during the next three days of experiments.…”