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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.

Endothelial progenitor cells (EPCs) are adult stem cells that play a central role in neovascularization. EPCs are mobilized from bone marrow into peripheral blood, attach to existing endothelial cells, and then transmigrate across the endothelium into tissues, where they proliferate, differentiate, and form new blood vessels. In the process, EPCs are exposed to shear stress, a biomechanical force generated by flowing blood and tissue fluid flow. When cultured EPCs are exposed to controlled levels of shear stress in a flow-loading device, their bioactivities in terms of proliferation, anti-apoptosis, migration, production of bioactive substances, anti-thrombosis, and tube formation increase markedly. Expression of endothelial marker genes and proteins by EPCs also increases in response to shear stress, and they differentiate into mature endothelial cells. Great advances have been made in elucidating the mechanisms by which mature endothelial cells sense and respond to shear stress, but not in EPCs. Further study of EPC responses to shear stress will be necessary to better understand the physiological and pathophysiological roles of EPCs and to apply EPCs to new therapies in the field of regenerative medicine.

Blood vessels alter their morphology and function in response to changes in blood flow, and their responses are based on blood flow detection by the vascular endothelium. Endothelial cells (ECs) covering the inner surface of blood vessels sense shear stress generated by flowing blood and transmit the signal into the interior of the cell, which evokes a cellular response. The EC response to shear stress is closely linked to the regulation of vascular tone, blood coagulation and fibrinolysis, angiogenesis, and vascular remodelling, and it plays an important role in maintaining the homoeostasis of the circulatory system. Impairment of the EC response to shear stress leads to the development of vascular diseases such as hypertension, thrombosis, aneurysms, and atherosclerosis. Rapid progress has been made in elucidating shear stress mechanotransduction by using in vitro methods that apply controlled levels of shear stress to cultured ECs in fluid-dynamically designed flow-loading devices. The results have revealed that shear stress is converted into intracellular biochemical signals that are mediated by a variety of membrane molecules and microdomains, including ion channels, receptors, G-proteins, adhesion molecules, the cytoskeleton, caveolae, the glycocalyx, and primary cilia, and that multiple downstream signalling pathways become activated almost simultaneously. Nevertheless, neither the shear-stress-sensing mechanisms nor the sensor molecules that initially sense shear stress are yet known. Their identification would contribute to a better understanding of the pathophysiology of the vascular diseases that occur in a blood flow-dependent manner and to the development of new treatments for them.

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.

Endothelial progenitor cells (EPCs) are mobilized from bone marrow to peripheral blood, and contribute to angiogenesis in tissue. In the process, EPCs are exposed to shear stress generated by blood flow and tissue fluid flow. Our previous study showed that shear stress induces differentiation of mature EPCs in adhesive phenotype into mature endothelial cells and, moreover, arterial endothelial cells. In this study we investigated whether immature EPCs in a circulating phenotype differentiate into mature EPCs in response to shear stress. When floating-circulating phenotype EPCs derived from ex vivo expanded human cord blood were exposed to controlled levels of shear stress in a flow-loading device, the bioactivities of adhesion, migration, proliferation, antiapoptosis, tube formation, and differentiated type of EPC colony formation increased. The surface protein expression rate of the endothelial markers VEGF receptor 1 (VEGF-R1) and -2 (VEGF-R2), VE-cadherin, Tie2, VCAM1, integrin αv/β3, and E-selectin increased in shear-stressed EPCs. The VEGF-R1, VEGF-R2, VE-cadherin, and Tie2 protein increases were dependent on the magnitude of shear stress. The mRNA levels of VEGF-R1, VEGF-R2, VE-cadherin, Tie2, endothelial nitric oxide synthase, matrix metalloproteinase 9, and VEGF increased in shear-stressed EPCs. Inhibitor analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signal transduction pathway is a potent activator of adhesion, proliferation, tube formation, and differentiation in response to shear stress. Western blot analysis revealed that shear stress activated the VEGF-R2 phosphorylation in a ligand-independent manner. These results indicate that shear stress increases differentiation, adhesion, migration, proliferation, antiapoptosis, and vasculogenesis of circulating phenotype EPCs by activation of VEGF-R2 and the PI3K/Akt/mTOR signal transduction pathway.

Our ongoing studies show that vascular endothelial cell growth factor (VEGF)-bound surfaces selectively capture endothelial progenitor cells (EPCs) in vitro and in vivo, and that surface-bound VEGF stimulates intracellular signal transduction pathways over prolonged culture periods, resulting in inductive differentiation of EPCs. In this article, we investigated whether simulated arterial shear stress augments the differentiation of EPCs adhered to a VEGF-bound surface. Human peripheral blood-derived mononuclear cells adhered to a VEGF-bound surface were exposed to 1 day of shear stress (15 dynes/cm(2), corresponding to shear load in arteries). Shear stress suppressed the expression of mRNAs encoding CD34 and CD133, which are markers for EPCs, and augmented the expression of mRNAs encoding CD31 and von Willebrand factor (vWF) as well as vWF protein, which are markers for endothelial cells (ECs). Shear stress enhanced expression of ephrinB2 mRNA, a marker for arterial ECs, but did not significantly change expression of EphB4 mRNA, a marker for venous ECs. Focused protein array analysis showed that mechanotransduction by shear stress activated the p38 and MAPK pathways in EPCs. Thus, arterial shear stress, in concert with surface-bound VEGF, augments the differentiation of EPCs. These results strongly support previous observation of rapid differentiation of EPCs captured on VEGF-bound stents in a porcine model.

BACKGROUND AND PURPOSE Cerebral aneurysm is a frequent cerebrovascular event and a major cause of fatal subarachnoid haemorrhage, but there is no medical treatment for this condition. Haemodynamic stress and, recently, chronic inflammation have been proposed as major causes of cerebral aneurysm. Nevertheless, links between haemodynamic stress and chronic inflammation remain ill-defined, and to clarify such links, we evaluated the effects of prostaglandin E2 (PGE2), a mediator of inflammation, on the formation of cerebral aneurysms. EXPERIMENTAL APPROACH Expression of COX and prostaglandin E synthase (PGES) and PGE receptors were examined in human and rodent cerebral aneurysm. The incidence, size and inflammation of cerebral aneurysms were evaluated in rats treated with COX-2 inhibitors and mice lacking each prostaglandin receptor. Effects of shear stress and PGE receptor signalling on expression of pro-inflammatory molecules were studied in primary cultures of human endothelial cells (ECs). KEY RESULTS COX-2, microsomal PGES-1 and prostaglandin E receptor 2 (EP2) were induced in ECs in the walls of cerebral aneurysms. Shear stress applied to primary ECs induced COX-2 and EP2. Inhibition or loss of COX-2 or EP2in vivo attenuated each other's expression, suppressed nuclear factor κB (NF-κB)-mediated chronic inflammation and reduced incidence of cerebral aneurysm. EP2 stimulation in primary ECs induced NF-κB activation and expression of the chemokine (C-C motif) ligand 2, essential for cerebral aneurysm. CONCLUSIONS AND IMPLICATIONS These results suggest that shear stress activated PGE2-EP2 pathway in ECs and amplified chronic inflammation via NF-κB. We propose EP2 as a therapeutic target in cerebral aneurysm.

Endothelial cells (ECs) release ATP in response to shear stress, a fluid mechanical force generated by flowing blood but, although its release has a crucial role in controlling a variety of vascular functions by activating purinergic receptors, the mechanism of ATP release has never been established. To analyze the dynamics of ATP release, we developed a novel chemiluminescence imaging method by using cell-surface-attached firefly luciferase and a CCD camera. Upon stimulation of shear stress, cultured human pulmonary artery ECs simultaneously released ATP in two different manners, a highly concentrated, localized manner and a less concentrated, diffuse manner. The localized ATP release occurred at caveolin-1-rich regions of the cell membrane, and was blocked by caveolin-1 knockdown with siRNA and the depletion of plasma membrane cholesterol with methyl-β-cyclodexrin, indicating involvement of caveolae in localized ATP release. Ca2+ imaging with Fluo-4 combined with ATP imaging revealed that shear stress evoked an increase in intracellular Ca2+ concentration and the subsequent Ca2+ wave that originated from the same sites as the localized ATP release. These findings suggest that localized ATP release at caveolae triggers shear-stress-dependent Ca2+ signaling in ECs.

The structure and function of blood vessels adapt to environmental changes, for example, physical development and exercise. This phenomenon is based on the ability of endothelial cells (ECs) to sense and respond to blood flow. ECs are in direct contact with blood flow and exposed to shear stress. A number of recent studies have revealed that ECs recognize changes in shear stress and transmit signals to the interior of the cell, which leads to cellular responses that involve changes in cell morphology, cell function, and gene expression. Cultured human pulmonary artery ECs (HPAECs) showed Ca²(+) influx via an ATP-operated cation channel, P2X4, in response to shear stress. We have recently found that shear-induced activation of P2X4 requires endogenously released ATP and that shear stress induced HPAECs to release ATP, which was mediated by cell-surface ATP synthase located in caveolae. To gain insight into its significance, we generated a P2X4-deficient mouse. P2X4(-/-) mice do not exhibit normal EC responses to flow, such as Ca²(+) influx and subsequent production of NO, a potent vasodilator. Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2X4(-/-) mice. Furthermore, P2X4(-/-) mice have higher blood pressure than wild-type mice. Moreover, no adaptive vascular remodeling is observed in the P2X4(-/-) mice. Thus, P2X4-mediated shear stress mechanotransduction plays an important role in the vascular homeostasis, including the control of blood pressure and vascular remodeling.

We investigate the sliding friction of HUVEC monolayers cultured on PNaSS gel, intending to elucidate the role of glycocalyx on the surface of ECs in friction reduction. Three sets of HUVEC monolayers are investigated: an as-cultured HUVEC monolayer; a HUVEC monolayer treated with TGF-β 1 , which increases the glycocalyx by 148%; and a HUVEC monolayer treated with heparinase I, which reduces the glycocalyx by 57%. When being slid on a flat, glass surface, the frictional stress of the HUVEC monolayer decreases in the order: heparinase-I-treated > as-cultured > TGF-β 1 -treated samples. The results suggest that glycocalyx may play a role in reducing the friction of endothelial cell monolayer.

Vascular functions are regulated not only by chemical mediators, such as hormones, cytokines, and neurotransmitters, but by mechanical hemodynamic forces generated by blood flow and blood pressure. The mechanical force-mediated regulation is based on the ability of vascular cells, including endothelial cells and smooth muscle cells, to recognize fluid mechanical forces, i.e., the shear stress produced by flowing blood and the cyclic strain generated by blood pressure, and to transmit the signals into the cell interior, where they trigger cell responses that involve changes in cell morphology, cell function, and gene expression. Recent studies have revealed that immature cells, such as endothelial progenitor cells (EPCs) and embryonic stem (ES) cells, as well as adult vascular cells, respond to fluid mechanical forces. Shear stress and cyclic strain promote the proliferation and differentiation of EPCs and ES cells into vascular cells and enhance their ability to form new vessels. Even more recently, attempts have been made to apply fluid mechanical forces to EPCs and ES cells cultured on polymer tubes and develop tissue-engineered blood vessel grafts that have a structure and function similar to that of blood vessels in vivo. This review summarizes the current state of knowledge concerning the mechanobiological responses of stem/progenitor cells and its potential applications to tissue engineering.

Objective— Arterial-venous specification in the embryo has been assumed to depend on the influence of fluid mechanical forces, but its cellular and molecular mechanisms are still poorly understood. Our previous in vitro study revealed that fluid shear stress induces endothelial cell (EC) differentiation by murine embryonic stem (ES) cells. In the present study we investigated whether shear stress regulates the arterial-venous specification of ES-cell-derived ECs. Methods and Results— When murine ES cell-derived VEGFR2 + cells were exposed to shear stress, expression of the arterial EC marker protein ephrinB2 increased dose-dependently. The ephrinB2 mRNA levels also increased in response to shear stress, whereas the mRNA levels of the venous EC marker EphB4 decreased. Notch cleavage and translocation of the Notch intracellular domain (NICD) into the nucleus occurred as early as 30 minutes after the start of shear stress and increased with time. Gamma-Secretase inhibitors (DAPT and L685 458) and the recombinant extracellular domain of the Notch ligand DLL4 abolished the shear stress-induced NICD translocation, and that, in turn, blocked the shear stress-induced upregulation of ephrinB2 expression. In addition, the VEGF receptor kinase inhibitor SU1498 was found to suppress both the shear-stress-induced Notch cleavage and up-regulation of ephrinB2 expression. Conclusion— Exposure to shear stress induces an increase in expression of ephrinB2 in murine ES cells via VEGF-Notch signaling pathways.

Autologous vascular tissues with a small diameter, "biotubes," were developed in vivo using a novel concept in regenerative medicine, "in-body tissue architecture technology." The effect of pulsatile flow in vitro was investigated on the structural and functional properties of the biotubes. Silicone rods (diameter, 3.0 mm; length, 35.0 mm), used as molds, were embedded into dorsal subcutaneous spaces of Wister rats. After 4 weeks, the autologous tubular tissues formed around the rods were harvested. Some tissues were incubated for 2 days under pulsatile flow simulating conditions in the human arteries with small caliber (wall shear stress (WSS), 15.5-77.3 dyn/cm(2); circumferential stress (CS), 0.6-4.5 x 10(5) dyn/cm(2)). Upon flow loading, the sparse, randomly oriented collagen fibers in the biotubes became dense and oriented in the regular circumferential direction. Compliances (beta values) of the control (ca. 30) and flow-loaded (ca. 20) biotubes were equivalent to that of the human coronary arteries and femoral arteries, respectively. Further, upon flow loading, the burst pressure significantly increased from ca. 1000 mmHg to ca. 1800 mmHg, along with the alpha-SMA-positive cell ratio. Pulsatile flow loading in vitro for 2 days could induce biotube maturation in terms of collagen structures and mechanical properties.

Endothelial cells (ECs) lining blood vessel walls respond to shear stress, a fluid mechanical force generated by flowing blood, and the EC responses play an important role in the homeostasis of the circulatory system. Abnormal EC responses to shear stress impair various vascular functions and lead to vascular diseases, including hypertension, thrombosis, and atherosclerosis. Bioengineering approaches in which cultured ECs are subjected to shear stress in fluid-dynamically designed flow-loading devices have been widely used to analyze EC responses at the cellular and molecular levels. Remarkable progress has been made, and the results have shown that ECs alter their morphology, function, and gene expression in response to shear stress. Shear stress affects immature cells, as well as mature ECs, and promotes differentiation of bone-marrow-derived endothelial progenitor cells and embryonic stem cells into ECs. Much research has been done on shear stress sensing and signal transduction, and their molecular mechanisms are gradually coming to be understood. However, much remains uncertain, and many candidates have been proposed for shear stress sensors. More extensive studies of vascular mechanobiology should increase our understanding of the molecular basis of the blood-flow-mediated control of vascular functions.

Embryonic stem (ES) cells are exposed to fluid-mechanical forces, such as cyclic strain and shear stress, during the process of embryonic development but much remains to be elucidated concerning the role of fluid-mechanical forces in ES cell differentiation. Here, we show that cyclic strain induces vascular smooth muscle cell (VSMC) differentiation in murine ES cells. Flk-1-positive (Flk-1+) ES cells seeded on flexible silicone membranes were subjected to controlled levels of cyclic strain and examined for changes in cell proliferation and expression of various cell lineage markers. When exposed to cyclic strain (4–12% strain, 1 Hz, 24 h), the Flk-1+ES cells significantly increased in cell number and became oriented perpendicular to the direction of strain. There were dose-dependent increases in the VSMC markers smooth muscle α-actin and smooth muscle-myosin heavy chain at both the protein and gene expression level in response to cyclic strain, whereas expression of the vascular endothelial cell marker Flk-1 decreased, and there were no changes in the other endothelial cell markers (Flt-1, VE-cadherin, and platelet endothelial cell adhesion molecule 1), the blood cell marker CD3, or the epithelial marker keratin. The PDGF receptor β (PDGFRβ) kinase inhibitor AG-1296 completely blocked the cyclic strain-induced increase in cell number and VSMC marker expression. Cyclic strain immediately caused phosphorylation of PDGFRβ in a dose-dependent manner, but neutralizing antibody against PDGF-BB did not block the PDGFRβ phosphorylation. These results suggest that cyclic strain activates PDGFRβ in a ligand-independent manner and that the activation plays a critical role in VSMC differentiation from Flk-1+ES cells.

We developed a compliant tube-type flow-loading apparatus that allows simultaneous application of physiological levels of shear stress and cyclic stretch to cultured cells and examined gene responses to a combination of the two forces. Human umbilical vein endothelial cells were exposed to shear stress and/or cyclic stretch for 24 h, and changes in the mRNA levels of endothelin-1 (ET-1), a potent vasoconstrictor, and endothelial nitric oxide synthase (eNOS), which catalyzes the production of a potent vasodilator, NO, were determined by reverse transcriptase/PCR. Cyclic stretch (10%, 1 Hz) alone increased ET-1 mRNA levels approximately 1.6-fold, but had no effect on eNOS mRNA levels. A shear stress of 7 dynes/cm 2 and 15 dynes/cm 2 alone decreased ET-1 mRNA levels to around 83% and 61%, respectively, of the basal level, but increased the eNOS mRNA level to around 2.2-fold and 3.2-fold, respectively. When cyclic stretch and shear stress were applied simultaneously, ET-1 mRNA levels did not change significantly, but the eNOS mRNA level increased to a level equivalent to the increase in response to shear stress alone. These results indicate that the response of endothelial genes to shear stress or cyclic stretch depends on whether the two forces are applied separately or together.

Objective This study aimed to develop an autologous heart valve without using traditional in vitro tissue-engineering methods, which necessitate complicated cell management protocols under exceptionally clean laboratory facilities. Methods An autologous heart valve construct composed of trileaflets was prepared using a specially designed mold. The mold was prepared by covering a silicone rod with a crown-shaped tubular polyurethane scaffold containing 3 horns. The mold was implanted in the dorsal subcutaneous space in Japan White rabbits for 4 weeks. After harvesting, the implanted trileaflet valve-shaped structure with an internal diameter of either 5 or 20 mm was obtained by trimming the membranous tissue formed between the horns located around the silicone rod. The valve substitute was examined both macroscopically and histologically. The tensile strength of the leaflets was measured to rupture. The degree of regurgitation in valve function was evaluated using a flow circuit by calculating the ratio of the regurgitation volume to the forward flow volume. Results After implantation, the mold was completely covered with connective tissue consisting mostly of collagen and fibroblasts. Harvesting of the mold was straightforward, because there was little adhesion between the formed tissue and the native skin tissue. The trileaflet heart valve construct was obtained after withdrawing the inserted rods and trimming the membranous tissues formed between the horns of the scaffold. It was firmly attached to the scaffold, the interstices and surface of which revealed connective tissues composed of components similar to those of the leaflet tissue. Although the mechanical properties of the leaflet tissue were less efficient than those of the native porcine aortic valve leaflets, satisfactory valvular functions were demonstrated under pulsatile conditions using a flow circuit. No regurgitation was observed under retrograde hydrostatic pressures of up to 60 mm Hg, the physiologic pressure acting on the aortic valves during retrograde aortic flow. Conclusions The biovalve, an autologous, in vivo tissue-engineered, trileaflet, valve-shaped construct, was developed using our novel in-body tissue architecture technology. The biovalve has the potential to be an ideal prosthetic heart valve, with excellent biocompatibility to the growth of the recipient's heart.

Results of the previous studies on working memory processing of natural scenes in the human brain using fMRI have shown activation of a distributed network involving mainly medial temporal lobe and prefrontal sources. In spite of its relative high spatial accuracy in localizing the involved areas, possible interactions between the localized source activities in time are played in a finer temporal scale. In this study we used multichannel whole-head magnetoencephalography (MEG) which possesses both a relative good spatial and temporal resolution, to shed light on the possible mechanisms of interactions between the involved sources in the brain, after localizing them properly. 12 healthy human subjects were presented with a small set of natural scenes from the seaside. The memory task was a continuous 1-back comparison task, and the control was an automatic visuomotor response task involving the same sequence of trials (the order of task performance was random). For source estimation a weighted L2 minimum-norm algorithm was used. Focusing in a time interval between 300–600 ms when the impact of deep encoding of the presented stimuli during the 1-back task performance is more prominent and comparable, the interaction between the localized occipital, inferior medial temporal, posterior parietal, and prefrontal sources, was found to be important for deep (elaborative) encoding of these stimuli in working memory.

Partial hepatectomy causes hemodynamic changes that increase portal blood flow in the remaining lobe, where the expression of immediate-early genes, including plasminogen activator inhibitor-1 (PAI-1), is induced. We hypothesized that a hyperdynamic circulatory state occurring in the remaining lobe induces immediate-early gene expression. In this study, we investigated whether the mechanical force generated by flowing blood, shear stress, induces PAI-1 expression in hepatocytes. When cultured rat hepatocytes were exposed to flow, PAI-1 mRNA levels began to increase within 3 h, peaked at levels significantly higher than the static control levels, and then gradually decreased. The flow-induced PAI-1 expression was shear stress dependent rather than shear rate dependent and accompanied by increased hepatocyte production of PAI-1 protein. Shear stress increased PAI-1 transcription but did not affect PAI-1 mRNA stability. Functional analysis of the 2.1-kb PAI-1 5′-promoter indicated that a 278-bp segment containing transcription factor Sp1 and Ets-1 consensus sequences was critical to the shear stress-dependent increase of PAI-1 transcription. Mutations of both the Sp1 and Ets-1 consensus sequences, but not of either one alone, markedly prevented basal PAI-1 transcription and abolished the response of the PAI-1 promoter to shear stress. EMSA and chromatin immunoprecipitation assays showed binding of Sp1 and Ets-1 to each consensus sequence under static conditions, which increased in response to shear stress. In conclusion, hepatocyte PAI-1 expression is flow sensitive and transcriptionally regulated by shear stress via cooperative interactions between Sp1 and Ets-1.

Primary fibroblasts isolated from foetal mouse cornea, skin and tendon were subjected to linear shear stress and analysed for morphological parameters and by microarray, as compared with unstimulated controls. Approx. 350 genes were either up- or down-regulated by a significant amount, with 51 of these being common to all three cell types. Approx. 50% of altered genes in tendon and cornea fibroblasts were changed in common with one of the other cell types, with the remaining approx. 50% being specific to tendon or cornea. In skin fibroblasts, however, less than 25% of genes whose transcription was altered were specific only to skin. The functional spectrum of genes that were up- or down-regulated was diverse, with apparent house-keeping genes forming the major category of up-regulated genes. However, a significant number of genes associated with cell adhesion, extracellular matrix and matrix remodelling, as well as cytokines and other signalling factors, were also affected. Somewhat surprisingly, in these latter categories the trend was towards a reduction in mRNA levels. Verification of the mRNA quantity of a subset of these genes was performed by reverse transcriptase PCR and was found to be in agreement with the microarray analysis. These findings provide the first in-depth analysis of phenotypic differences between fibroblast cells from different tissue sources and reveal the responses of these cells to mechanical stress.

The structure and function of blood vessels adapt to environmental changes such as physical development and exercise. This phenomenon is based on the ability of the endothelial cells to sense and respond to blood flow; however, the underlying mechanisms remain unclear. Here we show that the ATP-gated P2X4 ion channel, expressed on endothelial cells and encoded by P2rx4 in mice, has a key role in the response of endothelial cells to changes in blood flow. P2rx4(-/-) mice do not have normal endothelial cell responses to flow, such as influx of Ca(2+) and subsequent production of the potent vasodilator nitric oxide (NO). Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2rx4(-/-) mice. Furthermore, P2rx4(-/-) mice have higher blood pressure and excrete smaller amounts of NO products in their urine than do wild-type mice. Moreover, no adaptive vascular remodeling, that is, a decrease in vessel size in response to a chronic decrease in blood flow, was observed in P2rx4(-/-) mice. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodeling.

This study evaluated the possibility of differentiation from embryonic stem (ES) cells to vascular wall cells by physical (mechanical) stress loading in vitro. A cell mixture containing Flk1-positive cells (ca. 30%) derived from murine ES cells was added to a compliant microporous tube made of segmented polyurethane. The compliance of the tube was close to that of the human artery [the stiffness parameter (beta) = 57.2 (n = 5, SD5%)]. The luminal surface of the tube was fully covered with the cells by preincubation for two days in the presence of vascular endothelial growth factor (VEGF). After 2 days of additional incubation without VEGF under static conditions, layering of the grown cells, mostly smooth muscle actin (SMA)-positive cells, was observed only on the luminal surface of the tube. The cells were flat, polygonal, and randomly oriented. On the other hand, after a 2-day incubation under a weak pulsatile flow simulating the human venous systems [wall shear stress (WSS) from -0.98 to 2.2 dyn/cm(2); circumferential strain (CS) 4.6-9.6 x 10(4) dyn/cm(2)] without VEGF, cells in the superficial layer were regularly oriented in the direction of the pulsatile flow. The oriented cells exhibited endothelial-like appearance, indicating that they were platelet endothelial cell adhesion molecule 1 (PECAM1)-positive. In addition, the cells growing into the interstices in the deeper layer showed smooth muscle-like appearance, indicating that they were SMA-positive. Differentiation to two different cell types and segregation of incorporated ES cells may be simultaneously encouraged by the combination of WSS and CS. It is expected that the monobloc building of hierarchically structured hybrid vascular prostheses composed of several vascular wall cell types is possible by physically synchronized differentiation of ES cells.

Various hydrogels without modification by any cell adhesive proteins have been investigated as cell scaffolds. The present study shows that bovine fetal aorta endothelial cells can adhere, spread, proliferate, and reach confluence on poly(acrylic acid), poly(sodium p-styrene sulfonate), and poly(2-acrylamido-2- methyl-1-propanesulfonic sodium) gels, whereas cells reach subconfluence on poly(vinyl alcohol) and poly(methacrylic acid) gels. The proliferation behavior was sensitive to both hydrogel charge density and crosslinker concentration. The relationship between cell proliferation and zeta potential of gels was discussed. It was found that hydrogels with a negative zeta potential higher than about 20 mV facilitates cell proliferation.

Pluripotent embryonic stem (ES) cells are capable of differentiating into all cell lineages, but the molecular mechanisms that regulate ES cell differentiation have not been sufficiently explored. In this study, we report that shear stress, a mechanical force generated by fluid flow, can induce ES cell differentiation. When Flk-1-positive (Flk-1+) mouse ES cells were subjected to shear stress, their cell density increased markedly, and a larger percentage of the cells were in the S and G2-M phases of the cell cycle than Flk-1+ES cells cultured under static conditions. Shear stress significantly increased the expression of the vascular endothelial cell-specific markers Flk-1, Flt-1, vascular endothelial cadherin, and PECAM-1 at both the protein level and the mRNA level, but it had no effect on expression of the mural cell marker smooth muscle α-actin, blood cell marker CD3, or the epithelial cell marker keratin. These findings indicate that shear stress selectively promotes the differentiation of Flk-1+ES cells into the endothelial cell lineage. The shear stressed Flk-1+ES cells formed tubelike structures in collagen gel and developed an extensive tubular network significantly faster than the static controls. Shear stress induced tyrosine phosphorylation of Flk-1 in Flk-1+ES cells that was blocked by a Flk-1 kinase inhibitor, SU1498, but not by a neutralizing antibody against VEGF. SU1498 also abolished the shear stress-induced proliferation and differentiation of Flk-1+ES cells, indicating that a ligand-independent activation of Flk-1 plays an important role in the shear stress-mediated proliferation and differentiation by Flk-1+ES cells.

The mouse is currently recognized as the most advantageous experimental animal amongst the muridae because of its small size, universal availability, and the large variety of lines available including disease models. We propose a new axial-pattern double flap model which allows symmetrical preparation of two flaps, reducing difficulties regarding inter-animal variation. Two symmetrical island flaps were harvested based on the bilateral deep circumflex iliac arteries (DCIAs) and subjected to ischaemia followed by reperfusion. On postoperative day 7, the flap survival area was measured. Comparison between the right and left flap in each animal showed an almost identical survival area. The model provides a reliable experimental means for research in flap pathophysiology. It could be especially useful for estimating effects of topical therapeutic manipulation to individual flaps including local application of agents and cell and/or gene transplantation.

The effect of the shear flow on the thickness change of a polyelectrolyte membrane grafted onto a glass substrate was directly investigated with a flow cell combined with a confocal laser scanning microscope. The membrane thickness decreased proportionally to an increase in the shear stress of the flow when the shear rate exceeded a critical value of 1 s−1. The higher the ionic strength was of the fluid, the greater the thinning effect was. The correlation between the critical shear rate and the relaxation of the polymer in the gel membrane was examined. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2808–2815, 2003

DNA microarray gene expression analysis was conducted in human umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (HCAECs) exposed to laminar or turbulent shear stress. Approximately 3% of the total 5600 gene in HUVECs and HCAECs increased their expression more than two-fold or decreased it to less than half the static control in response to an arterial level of laminar shear stress (15 dynes/cm(2) for 24 hours). The proportions of shear-stress-responsive genes decreased to around 2% under the venous level of laminar shear stress (1.5 dynes/cm(2)) in both cell lines. Turbulent shear stress of 1.5 dynes/cm(2) altered the expression of 1.1% of all genes in the HCAECs. Laminar shear stress, but not turbulent shear stress, decreased the expression of a number of genes involved in DNA synthesis and the cell cycle in both HUVECs and HCAECs. Clustering analysis showed a variety of temporal profiles of gene expression in HUVECs exposed to laminar shear stress of 15 dynes/cm(2) for 3, 6, 12, 24, and 48 hours. Turbulent shear stress affected expression of many genes that play a role in vascular remodeling, including genes encoding plasminogen activators and their inhibitor, endothelin-1, transforming growth factor-beta, collagen type IV, and ephrin A1.

The electric control of cellular functions via Ca 2+ was formerly suggested. From this viewpoint, the involvement of a Ca 2+ channel was studied using bovine fetal arterial endothelial (BFAE) cells in which P2X4, an ATP-operated and fluid shear stress sensitive Ca 2+ channel, exists predominantly. An electric stimulus (sine wave, 10 Hz, 10 Vpp, 30 s) caused a marked influx of Ca 2+ into BFAE cells from an extracellular solution. The magnitude of the [Ca 2+ ] i change increased with a decrease in the frequency in the range from 100 Hz to 5 Hz. Regarding the pathway of this Ca 2+ influx, single-cell imaging and an ATP depletion experiment strongly suggested the involvement of a pathway different from P2X4. This pathway was thought to be a non-specific one, because typical Ca 2+ channel blockers, such as verapamil, Gd 3+ , and Co 2+ , could not inhibit the Ca 2+ influx.

The caveola is a membrane domain that compartmentalizes signal transduction at the cell surface. Normally in endothelial cells, groups of caveolae are found clustered along stress fibers or at the lateral margins in all regions of the cell. Subsets of these clusters appear to contain the signaling machinery for initiating Ca2+ wave formation. Here we report that induction of cell migration, either by wounding a cell monolayer or by exposing cells to laminar shear stress, causes caveolae to move to the trailing edge of the cell. Concomitant with the relocation of the caveolae,sites of Ca2+ wave initiation move to the same location. In as much as the relocated caveolae contain elements of the signaling machinery required for ATP-stimulated release of Ca2+ from the ER, these results suggest that caveolae function as containers that carry this machinery to different cellular locations.

Since ischemia-reperfusion injury continues to be a major problem in reconstructive microsurgery, improvement of experimental models is still desirable. We developed a model that allows direct visualization of flap microcirculation in mice by intravital microscopic techniques. A newly designed skinfold chamber was installed on the dorsum of mice, and microcirculation was inspected with an intravital microscope. An island flap, nourished by the deep circumflex iliac arteries, was elevated after implantation of the chamber, allowing visualization of the microcirculation in the island flap. The island flap was exposed to global ischemia by clamping the pedicles, and the clamps were then released to allow reperfusion. Various microcirculatory responses induced by ischemia-reperfusion were visualized. This model accurately simulated the clinical situation in reconstructive surgery and successfully realized chronic visualization of the flap microcirculation in vivo.

To investigate whether hyperthermic preconditioning can actually protect skin flaps against ischemia/reperfusion injury, the authors first developed a new skin-flap model in 15 mice, a dorsal bipedicle island skin-flap model. Then, another 75 mice were separated into five groups. Mice in Groups 1 to 4 received the same hyperthermic preconditioning, but had different recovery times of 6 hr, 24 hr, 48 hr, and 72 hr, respectively. Mice in Group 5 served as control. Island skin flaps were elevated in all groups, and then were subjected to 8 hr of ischemia and subsequent reperfusion. Flap survival was statistically significantly higher than in controls in animals in Groups 1 and 3, with recovery times of 6 hr and 48 hr, respectively. Mice in Groups 2 and 4 had recovery times of 24 hr and 72 hr, respectively. Hyperthermic preconditioning could thus protect skin flaps against ischemia/reperfusion injury, and there were two optimal periods for such a protective effect.

[Objective] Our study using an animal model of experimentally induced cerebral aneurysms suggests that perception of an excessive increase in wall shear stress (WSS) by endothelial cells initiates the arterial wall degeneration caused by expression of various enzymes, leading to aneurysm development. Our computational fluid dynamics analysis also demonstrated that the magnitude of WSS is enhanced at the site where human aneurysms develop. In this study, we examine the role of WSS on cerebral aneurysm formation, using a shear sensor, P2X4 purinoceptor deficient mice (P2X4 KO). [Methods] P2X4 KO (n = 19) and wild types (n = 21) were used for aneurysm-inducing surgery as described before. Aneurysm as defined here refers to an outward bulging detected by light microscopy (more than 10 um). Damage to internal elastic lamina as early aneurysm changes was classified into 3 grades according to microscopic pathological changes. [Result] The grade of damage in P2X4 KO group was significantly lower than that in wild...

Endothelial progenitor cells (EPCs) have been demonstrated to be effective for the treatment of cardiovascular diseases. However, the differentiation process from circulation to adhesion has not been clarified because circulating EPCs rarely attached to dishes in EPC cultures previously. Here we investigated whether immature circulating EPCs differentiate into mature adhesive EPCs in response to dextran. When floating-circulating EPCs derived from ex vivo expanded human cord blood were cultured with 5% and 10% dextran, they attached to fibronectin-coated dishes and grew exponentially. The bioactivities of adhesion, proliferation, migration, tube formation, and differentiated type of EPC colony formation increased in EPCs exposed to dextran. The surface protein expression rate of the endothelial markers vascular endothelial growth factor (VEGF)-R1/2, VE-cadherin, Tie2, ICAM1, VCAM1, and integrin αv/β3 increased in EPCs exposed to dextran. The mRNA levels of VEGF-R1/2, VE-cadherin, Tie2, endothelial nitric oxide synthase, MMP9, and VEGF increased in EPCs treated with dextran. Those of endothelium-related transcription factors ID1/2, FOXM1, HEY1, SMAD1, FOSL1, NFkB1, NRF2, HIF1A, EPAS1 increased in dextran-treated EPCs; however, those of hematopoietic- and antiangiogenic-related transcription factors TAL1, RUNX1, c-MYB, GATA1/2, ERG, FOXH1, HHEX, SMAD2/3 decreased in dextran-exposed EPCs. Inhibitor analysis showed that PI3K/Akt, ERK1/2, JNK, and p38 signal transduction pathways are involved in the differentiation in response to dextran. In conclusion, dextran induces differentiation of circulating EPCs in terms of adhesion, migration, proliferation, and vasculogenesis. The differentiation mechanism in response to dextran is regulated by multiple signal transductions including PI3K/Akt, ERK1/2, JNK, and p38. These findings indicate that dextran is an effective treatment for EPCs in regenerative medicines.

Vascular endothelial cells (ECs) sense shear stress generated by flowing blood and alter their functions that play important roles in vascular homeostasis and pathophysiology. A unique feature of shear-stress-sensing is the rapid activation of many different types of membrane–bound molecules, including receptors, ion channels, and adhesion proteins, but the mechanisms remain unknown. One hypothesis is that shear stress might alter the physical properties of EC membranes, which lead to the activation of various membrane-bound molecules. To determine how shear stress influences the cell membrane, cultured human pulmonary artery ECs were exposed to shear stress in a flow-loading apparatus and examined for changes in membrane lipid order and fluidity by means of Laurdan two-photon imaging and FRAP measurements. Upon shear stress stimulation, the lipid order of EC membranes rapidly decreased in an intensity-dependent manner, and caveolar membrane domains changed form the liquid-ordered state to the liquid-disordered state. Notably, a similar decrease in lipid order occurred when artificial membranes of giant unilamellar vesicles composed of cholesterol and phospholipids were exposed to shear stress, suggesting that this is a physical phenomenon. Membrane fluidity increased over the entire EC membranes in response to shear stress. Addition of cholesterol to ECs abolished the effects of shear stress on the membrane lipid order and fluidity, and it markedly suppressed ATP release which is a well-known EC response to shear stress and is involved in shear stress Ca2 + signaling. These findings indicate that EC membranes, especially caveolar membrane domains, rapidly respond to shear stress by changing their physical properties, including their lipid phase order and fluidity, and that these changes may be linked to shear-stress-sensing and response mechanisms.