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BACKGROUNDVascular endothelial cells (ECs) experience two different blood flow patterns: laminar and disturbed flows. Their responses to laminar flow contribute to vascular homeostasis, whereas their responses to disturbed flow result in EC dysfunction and vascular diseases. However, it remains unclear how ECs differentially sense laminar and disturbed flows and trigger signalings that elicit different EC responses. We aimed to investigate EC flow-sensing and signaling mechanisms, focusing on the role of the plasma membrane and mitochondria.METHODSWe exposed cultured human aortic ECs to laminar flow and disturbed flow in flow-loading devices and used real-time imaging with optical probes to examine changes in the lipid order of the plasma and mitochondria membranes and the mitochondrial adenosine triphosphate (ATP) production and hydrogen peroxide (H2O2) release.RESULTSThe lipid order of EC plasma membranes immediately decreased in response to laminar flow, while it increased in response to disturbed flow. Laminar flow also decreased the lipid order of mitochondrial membranes and increased mitochondrial ATP production. In contrast, disturbed flow increased the lipid order of mitochondrial membranes and increased the release of H2O2from mitochondria. Addition of cholesterol to the cells increased the lipid order of both membranes and abrogated the laminar flow-induced ATP production, while treatment of the cells with a cholesterol-depleting reagent, methyl-β cyclodextrin, decreased the lipid order of both membranes and abolished the disturbed flow-induced H2O2release, indicating that the changes in the membrane lipid order are closely linked to the flow-induced changes in the mitochondrial functions.CONCLUSIONSECs differentially sense laminar and disturbed flows by altering the lipid order of their plasma and mitochondrial membranes in opposite directions, which result in distinct changes in the mitochondrial functions, namely, increased ATP production for laminar flow and increased H2O2release for disturbed flow, leading to ATP- and H2O2-mediated signalings, respectively.

Cells in the tissues and organs of a living body are subjected to mechanical forces, such as pressure, friction, and tension from their surrounding environment. Cells are equipped with a mechanotransduction mechanism by which they perceive mechanical forces and transmit information into the cell interior, thereby causing physiological or pathogenetic mechano-responses. Endothelial cells (ECs) lining the inner surface of blood vessels are constantly exposed to shear stress caused by blood flow and a cyclic strain caused by intravascular pressure. A number of studies have shown that ECs are sensitive to changes in these hemodynamic forces and alter their morphology and function, sometimes by modifying gene expression. The mechanism of endothelial mechanotransduction has been elucidated, and the plasma membrane has recently been shown to act as a mechanosensor. The lipid order and cholesterol content of plasma membranes change immediately upon the exposure of ECs to hemodynamic forces, resulting in a change in membrane fluidity. These changes in a plasma membrane's physical properties affect the conformation and function of various ion channels, receptors, and microdomains (such as caveolae and primary cilia), thereby activating a wide variety of downstream signaling pathways. Such endothelial mechanotransduction works to maintain circulatory homeostasis; however, errors in endothelial mechanotransduction can cause abnormalities in vascular physiological function, leading to the initiation and progression of various vascular diseases, such as hypertension, thrombosis, aneurysms, and atherosclerosis. Recent advances in detailed imaging technology and computational fluid dynamics analysis have enabled us to evaluate the hemodynamic forces acting on vascular tissue accurately, contributing greatly to our understanding of vascular mechanotransduction and the pathogenesis of vascular diseases, as well as the development of new therapies for vascular diseases.

OBJECTIVEThere are no effective therapeutic drugs for cerebral aneurysms, partly because the pathogenesis remains unresolved. Chronic inflammation of the cerebral arterial wall plays an important role in aneurysm formation, but it is not clear what triggers the inflammation. The authors have observed that vascular endothelial P2X4 purinoceptor is involved in flow-sensitive mechanisms that regulate vascular remodeling. They have thus hypothesized that shear stress–associated hemodynamic stress on the endothelium causes the inflammatory process in the cerebral aneurysm development.METHODSTo test their hypothesis, the authors examined the role of P2X4 in cerebral aneurysm development by using P2X4−/− mice and rats that were treated with a P2X4 inhibitor, paroxetine, and subjected to aneurysm-inducing surgery. Cerebral aneurysms were induced by unilateral carotid artery ligation and renovascular hypertension.RESULTSThe frequency of aneurysm induction evaluated by light microscopy was significantly lower in the P2X4−/− mice (p = 0.0488) and in the paroxetine-treated male (p = 0.0253) and female (p = 0.0204) rats compared to control mice and rats, respectively. In addition, application of paroxetine from 2 weeks after surgery led to a significant reduction in aneurysm size in the rats euthanized 3 weeks after aneurysm-inducing surgery (p = 0.0145), indicating that paroxetine suppressed enlargement of formed aneurysms. The mRNA and protein expression levels of known inflammatory contributors to aneurysm formation (monocyte chemoattractant protein–1 [MCP-1], interleukin-1β [IL-1β], tumor necrosis factor–α [TNFα], inducible nitric oxide synthase [iNOS], and cyclooxygenase-2 [COX-2]) were all significantly elevated in the rats that underwent the aneurysm-inducing surgery compared to the nonsurgical group, and the values in the surgical group were all significantly decreased by paroxetine administration according to quantitative polymerase chain reaction techniques and Western blotting. Although immunolabeling densities for COX-2, iNOS, and MCP-1 were not readily observed in the nonsurgical mouse groups, such densities were clearly seen in the arterial wall of P2X4+/+ mice after aneurysm-inducing surgery. In contrast, in the P2X4−/− mice after the surgery, immunolabeling of COX-2 and iNOS was not observed in the arterial wall, whereas that of MCP-1 was readily observed in the adventitia, but not the intima.CONCLUSIONSThese data suggest that P2X4 is required for the inflammation that contributes to both cerebral aneurysm formation and growth. Enhanced shear stress–associated hemodynamic stress on the vascular endothelium may trigger cerebral aneurysm development. Paroxetine may have potential for the clinical treatment of cerebral aneurysms, given that this agent exhibits efficacy as a clinical antidepressant.

Significance The mechanotransduction of shear stress in vascular endothelial cells is still not completely understood. We show a pathway of shear stress signal transduction mediated by plasma membrane cholesterol-dependent mitochondrial oxidative phosphorylation. The latest imaging technology using domain 4 mutant-derived cholesterol biosensors and a fluorescence resonance energy transfer-based adenosine triphosphate (ATP) biosensor revealed that shear stress rapidly decreases cholesterol levels in the plasma membrane via both efflux and internalization, and reduction in plasma membrane cholesterol was linked to the activation of mitochondrial ATP production. The addition of cholesterol blocked these shear stress effects. Increased mitochondrial ATP production led to ATP release from the endothelial cells, thereby activating purinoceptors in the plasma membrane and leading to purinergic Ca2+ signaling in response to shear stress., Vascular endothelial cells (ECs) sense and respond to hemodynamic shear stress, which is critical for circulatory homeostasis and the pathophysiology of vascular diseases. The mechanisms of shear stress mechanotransduction, however, remain elusive. We previously demonstrated a direct role of mitochondria in the purinergic signaling of shear stress: shear stress increases mitochondrial adenosine triphosphate (ATP) production, triggering ATP release and Ca2+ signaling via EC purinoceptors. Here, we showed that shear stress rapidly decreases cholesterol in the plasma membrane, thereby activating mitochondrial ATP production. Imaging using domain 4 mutant-derived cholesterol biosensors showed that the application of shear stress to cultured ECs markedly decreased cholesterol levels in both the outer and inner plasma membrane bilayers. Flow cytometry showed that the cholesterol levels in the outer bilayer decreased rapidly after the onset of shear stress, reached a minimum (around 60% of the control level) at 10 min, and plateaued thereafter. After the shear stress ceased, the decreased cholesterol levels returned to those seen in the control. A biochemical analysis showed that shear stress caused both the efflux and the internalization of plasma membrane cholesterol. ATP biosensor imaging demonstrated that shear stress significantly increased mitochondrial ATP production. Similarly, the treatment of cells with methyl-β-cyclodextrin (MβCD), a membrane cholesterol-depleting agent, increased mitochondrial ATP production. The addition of cholesterol to cells inhibited the increasing effects of both shear stress and MβCD on mitochondrial ATP production in a dose-dependent manner. These findings indicate that plasma membrane cholesterol dynamics are closely coupled to mitochondrial oxidative phosphorylation in ECs.

Vascular endothelial cells (ECs) sense and transduce hemodynamic shear stress into intracellular biochemical signals, and Ca2+signaling plays a critical role in this mechanotransduction, i.e., ECs release ATP in the caveolae in response to shear stress and, in turn, the released ATP activates P2 purinoceptors, which results in an influx into the cells of extracellular Ca2+. However, the mechanism by which the shear stress evokes ATP release remains unclear. Here, we demonstrated that cellular mitochondria play a critical role in this process. Cultured human pulmonary artery ECs were exposed to controlled levels of shear stress in a flow-loading device, and changes in the mitochondrial ATP levels were examined by real-time imaging using a fluorescence resonance energy transfer-based ATP biosensor. Immediately upon exposure of the cells to flow, mitochondrial ATP levels increased, which was both reversible and dependent on the intensity of shear stress. Inhibitors of the mitochondrial electron transport chain and ATP synthase as well as knockdown of caveolin-1, a major structural protein of the caveolae, abolished the shear stress-induced mitochondrial ATP generation, resulting in the loss of ATP release and influx of Ca2+into the cells. These results suggest the novel role of mitochondria in transducing shear stress into ATP generation: ATP generation leads to ATP release in the caveolae, triggering purinergic Ca2+signaling. Thus, exposure of ECs to shear stress seems to activate mitochondrial ATP generation through caveola- or caveolin-1-mediated mechanisms.NEW & NOTEWORTHY The mechanism of how vascular endothelial cells sense shear stress generated by blood flow and transduce it into functional responses remains unclear. Real-time imaging of mitochondrial ATP demonstrated the novel role of endothelial mitochondria as mechanosignaling organelles that are able to transduce shear stress into ATP generation, triggering ATP release and purinoceptor-mediated Ca2+signaling within the cells.

Vascular endothelial cells (ECs) maintain circulatory system homeostasis by changing their functions in response to changes in hemodynamic forces, including shear stress and stretching. However, it is unclear how ECs sense changes in shear stress and stretching and transduce these changes into intracellular biochemical signals. The plasma membranes of ECs have recently been shown to respond to shear stress and stretching differently by rapidly changing their lipid order, fluidity, and cholesterol content. Such changes in the membranes' physical properties trigger the activation of membrane receptors and cell responses specific to each type of force. Artificial lipid-bilayer membranes show similar changes in lipid order in response to shear stress and stretching, indicating that they are physical phenomena rather than biological reactions. These findings suggest that the plasma membranes of ECs act as mechanosensors; in response to mechanical forces, they first alter their physical properties, modifying the conformation and function of membrane proteins, which then activates downstream signaling pathways. This new appreciation of plasma membranes as mechanosensors could help to explain the distinctive features of mechanotransduction in ECs involving shear stress and stretching, which activate a variety of membrane proteins and multiple signal transduction pathways almost simultaneously.

Background and purpose: It is thought that the cerebral aneurysm is caused by chronic inflammation of the arterial wall. However, it is still not clear what triggers the inflammation. We hypothesize that shear stress-related hemodynamics initiates cerebral aneurysm formation, and have reported that disruption of the endothelial shear stress sensor, P2X4 purinoceptor, significantly reduces cerebral aneurysm formation using knockout mice of the sensor. We examined here whether inhibition of P2X4 purinoceptor has any influence on expressions of inflammatory contributors to aneurysm formation or not. Methods: Sprague-Dawley rats (n = 61) were subjected to cerebral aneurysm-generating surgery with ligation of unilateral common carotid artery and renal hypertension, and the P2X4 purinoceptor inhibitor, paroxetine (8 mg/kg/day) was administered after the surgery for 3 weeks. The incidence of aneurysm formation was examined with light microscopy and expressions of inflammatory contributors to aneurysm formation using a quantitative real-time PCR analysis. Results: Paroxetine significantly attenuated the incidence of induced aneurysms in rats after aneurysm-generating surgery (p = 0.031, Wilcoxon/Kruskal-Wallis test). Expression levels of known inflammatory contributors to aneurysm formation, COX-2, TNFα, MCP-1, IL1β, and iNOS, were significantly increased in rats after aneurysm-inducing surgery than control non-operated animals by a quantitative real-time PCR analysis. Paroxetine significantly reduced the expression levels of COX-2, TNFα, MCP-1, IL1β, and iNOS (p = 0.0019, 0.0101, 0.0019, 0.0239, 0.0101, respectively). Conclusions: These data suggest that shear sensing on the arterial endothelium initiates chronic inflammation in the arterial wall during cerebral aneurysm formation. Because paroxetine has been used for human as an antidepressant, clinical use of this P2X4 purinoceptor inhibitor is expected as a novel therapy for cerebral aneurysms.

Vascular endothelial cells (ECs) respond to the hemodynamic forces stretch and shear stress by altering their morphology, functions, and gene expression. However, how they sense and differentiate between these two forces has remained unknown. Here we report that the plasma membrane itself differentiates between stretch and shear stress by undergoing transitions in its lipid phases. Uniaxial stretching and hypotonic swelling increased the lipid order of human pulmonary artery EC plasma membranes, thereby causing a transition from the liquid-disordered phase to the liquid-ordered phase in some areas, along with a decrease in membrane fluidity. In contrast, shear stress decreased the membrane lipid order and increased membrane fluidity. A similar increase in lipid order occurred when the artificial lipid bilayer membranes of giant unilamellar vesicles were stretched by hypotonic swelling, indicating that this is a physical phenomenon. The cholesterol content of EC plasma membranes significantly increased in response to stretch but clearly decreased in response to shear stress. Blocking these changes in the membrane lipid order by depleting membrane cholesterol with methyl-β-cyclodextrin or by adding cholesterol resulted in a marked inhibition of the EC response specific to stretch and shear stress, i.e., phosphorylation of PDGF receptors and phosphorylation of VEGF receptors, respectively. These findings indicate that EC plasma membranes differently respond to stretch and shear stress by changing their lipid order, fluidity, and cholesterol content in opposite directions and that these changes in membrane physical properties are involved in the mechanotransduction that activates membrane receptors specific to each force.

Background and purpose: Although the assumption that wall shear stress-related hemodynamics is closely involved in cerebral aneurysm formation is now widely accepted, direct evidence still remains to be presented. Here, we examine a role of shear stress in cerebral aneurysm development, focused on a vascular endothelial shear sensor, P2X4 purinoceptor. Methods: P2X4 knockout mice and wild type mice were subjected to cerebral aneurysm-generating surgery with ligation of unilateral common carotid artery and renal hypertension. Damage to the internal elastic lamina, early phase aneurysm development, and the incidence of aneurysm induction were examined by light microscope. The expression of biochemical contributors to aneurysm formation was immunohistochemically examined. Additionally, the P2X4 purinoceptor inhibitor, paroxetine (10mg/kg/day) was administered to Sprague-Dawley rats after aneurysm-inducing surgery, and the incidence of aneurysm formation was examined. Results: Damage to the internal elastic lamina and the frequency of CA induction were significantly diminished in P2X4 knockout mice compared to control wild-type mice after CA-inducing surgery ( p =0.027 and 0.018, respectively). Expression levels of known contributors to aneurysm formation, iNOS, MCP-1, cathepsin L, and COX-2, were immunohistochemically weakened in P2X4 KO mice compared to wild-type mice. Moreover, paroxetine significantly attenuated the incidence of induced aneurysms in rats ( p =0.031). Conclusions: Disruption of the endothelial shear stress sensor, P2X4 purinoceptor drastically reduces cerebral aneurysm formation, suggesting that shear stress-related hemodynamics initiates cerebral aneurysm formation. The P2X4 purinoceptor inhibitor paroxetine, which is also prescribed to human as an antidepressant, may be a potential clinical prophylactic agent against cerebral aneurysm formation.

Vascular endothelial cells(ECs)play a critical role in controlling a variety of vascular functions including maintenance of the vascular tone, blood coagulation and fibrinolysis, and selective permeability of proteins. It has recently become apparent that ECs respond to hemodynamic forces, namely, shear stress and stretch, by altering their morphology, functions and gene expression profile. These responses also play important roles in maintaining normal circulatory system functions and homeostasis, and their impairment leads to various vascular diseases, including hypertension, aneurysm and atherosclerosis. The mechanisms underlying the mechanotransduction, however, are not yet clearly understood. In this article, we review the literature on the EC responses to mechanical forces and their roles in the regulation of the circulatory system, while also discussing the mechanosensing mechanisms of ECs.