Mechanisms of shear stress-induced endothelial nitric-oxide synthase phosphorylation and expression in ovine fetoplacental artery endothelial cells

Placental blood flow, nitric-oxide (NO) levels, and endothelial NO synthase (eNOS) expression increase during human and ovine pregnancy. Shear stress stimulates NO production and eNOS expression in ovine fetoplacental artery endothelial (OFPAE) cells. Because eNOS is the rate-limiting enzyme essential for NO synthesis, its activity and expression are both closely regulated. We investigated signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by OFPAE cells. The OFPAE cells were cultured at 3 dynes/cm2 shear stress, then exposed to 15 dynes/cm2 shear stress. Western blot analysis for phosphorylated ERK1/2, Akt, p38 mitogen activated protein kinase (MAPK), and eNOS showed that shear stress rapidly increased phosphorylation of ERK1/2 and Akt but not of p38 MAPK. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by the phosphatidyl inositol-3-kinase (PI-3K)-inhibitors wortmannin and LY294002 but not by the mitogen activated protein kinase kinase (MEK)-inhibitor UO126. Basic fibroblast growth factor (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels otherwise induced by the 15 dynes/cm2 shear stress. Blockade of either signaling pathway changed the shear stress-induced increase in eNOS protein levels. In conclusion, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibition of MEK. Prolonged shear stress-stimulated increases in eNOS protein were not affected by inhibition of MEK- or PI-3K-mediated pathways.     

Rap1 signaling in endothelial barrier control

The small G-protein Rap1 plays an important role in the regulation of endothelial barrier function, a process controlled largely by cell–cell adhesions and their connection to the actin cytoskeleton. During the various stages of barrier dynamics, different guanine nucleotide exchange factors (GEFs) control Rap1 activity, indicating that Rap1 integrates multiple input signals. Once activated, Rap1 induces numerous signaling cascades, together responsible for the increased endothelial barrier function. Most notably, Rap1 activation results in the inhibition of Rho to decrease radial stress fibers and the activation of Cdc42 to increase junctional actin. This implies that Rap regulates endothelial barrier function by dual control of cytoskeletal tension. The molecular details of the signaling pathways are becoming to be elucidated.     

Cyclic AMP potentiates vascular endothelial cadherin-mediated cell-cell contact to enhance endothelial barrier function through an Epac-Rap1 signaling pathway

Cyclic AMP (cAMP) is a well-known intracellular signaling molecule improving barrier function in vascular endothelial cells. Here, we delineate a novel cAMP-triggered signal that regulates the barrier function. We found that cAMP-elevating reagents, prostacyclin and forskolin, decreased cell permeability and enhanced vascular endothelial (VE) cadherin-dependent cell adhesion. Although the decreased permeability and the increased VE-cadherin-mediated adhesion by prostacyclin and forskolin were insensitive to a specific inhibitor for cAMP-dependent protein kinase, these effects were mimicked by 8-(4-chlorophenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate, a specific activator for Epac, which is a novel cAMP-dependent guanine nucleotide exchange factor for Rap1. Thus, we investigated the effect of Rap1 on permeability and the VE-cadherin-mediated cell adhesion by expressing either constitutive active Rap1 or Rap1GAPII. Activation of Rap1 resulted in a decrease in permeability and enhancement of VE-cadherin-dependent cell adhesion, whereas inactivation of Rap1 had the counter effect. Furthermore, prostacyclin and forskolin induced cortical actin rearrangement in a Rap1-dependent manner. In conclusion, cAMP-Epac-Rap1 signaling promotes decreased cell permeability by enhancing VE-cadherin-mediated adhesion lined by the rearranged cortical actin.     

Direct, real-time measurement of shear stress-induced nitric oxide produced from endothelial cells in vitro

Nitric oxide (NO) produced by the endothelium is involved in the regulation of vascular tone. Decreased NO production or availability has been linked to endothelial dysfunction in hypercholesterolemia and hypertension. Shear stress-induced NO release is a well-established phenomenon, yet the cellular mechanisms of this response are not completely understood. Experimental limitations have hindered direct, real-time measurements of NO under flow conditions. We have overcome these challenges with a new design for a parallel-plate flow chamber. The chamber consists of two compartments, separated by a Transwell® membrane, which isolates a NO recording electrode located in the upper compartment from flow effects. Endothelial cells are grown on the bottom of the membrane, which is inserted into the chamber flush with the upper plate. We demonstrate for the first time direct real-time NO measurements from endothelial cells with controlled variations in shear stress. Step changes in shear stress from 0.1 dyn/cm² to 6, 10, or 20 dyn/cm² elicited a transient decrease in NO followed by an increase to a new steady state. An analysis of NO transport suggests that the initial decrease is due to the increased removal rate by convection as flow increases. Furthermore, the rate at which the NO concentration approaches the new steady state is related to the time-dependent cellular response rather than transport limitations of the measurement configuration. Our design offers a method for studying the kinetics of the signaling mechanisms linking NO production with shear stress as well as pathological conditions involving changes in NO production or availability.     

Effects of pulsatile shear stress on nitric oxide production and endothelial cell nitric oxide synthase expression by ovine fetoplacental artery endothelial cells

Placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression increase during pregnancy. Shear stress, the frictional force exerted on endothelial cells by blood flow, stimulates vessel dilation, endothelial NO production, and eNOS expression. In order to study the effects of pulsatile flow/shear stress, we adapted Cellco CELLMAX artificial capillary modules to study ovine fetoplacental artery endothelial (OFPAE) cells for NO production and eNOS expression. OFPAE cells were grown in the artificial capillary modules at 3 dynes/cm2. Confluent cells were then exposed to 10, 15, or 25 dynes/cm2 for up to 24 h. NO production by OFPAE cells exposed to pulsatile shear stress was inhibited to nondetectable levels by the NOS inhibitor l-NMMA and reversed by excess NOS substrate l-arginine. NO production and expression of eNOS mRNA and protein by OFPAE cells were elevated by shear stress in a graded fashion (P < 0.05). The rise in NO production with 25 dynes/cm2 shear stress (8-fold) was greater (P < 0.05) than that observed for eNOS protein (3.6-fold) or eNOS mRNA (1.5-fold). The acute shear stress-induced rise in NO production by OFPAE cells was via eNOS activation, whereas the prolonged NO rise occurred by elevations in both eNOS expression and enzyme activation. Thus, elevations of placental blood flow and physiologic shear stress may be partly responsible for the increases in placental arterial endothelial eNOS expression and NO production during pregnancy.     

Modulation of pulmonary endothelial endothelin B receptor expression and signaling: implications for experimental hepatopulmonary syndrome

The hepatopulmonary syndrome (HPS) results from intrapulmonary vasodilation in the setting of cirrhosis and portal hypertension. In experimental HPS, pulmonary endothelial endothelin B (ET(B)) receptor overexpression and increased circulating endothelin-1 (ET-1) contribute to vasodilation through enhanced endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production. In both experimental cirrhosis and prehepatic portal hypertension, ET(B) receptor overexpression correlates with increased vascular shear stress, a known modulator of ET(B) receptor expression. We investigated the mechanisms of pulmonary endothelial ET(B) receptor-mediated eNOS activation by ET-1 in vitro and in vivo. The effect of shear stress on ET(B) receptor expression was assessed in rat pulmonary microvascular endothelial cells (RPMVECs). The consequences of ET(B) receptor overexpression on ET-1-dependent ET(B) receptor-mediated eNOS activation were evaluated in RPMVECs and in prehepatic portal hypertensive animals exposed to exogenous ET-1. Laminar shear stress increased ET(B) receptor expression in RPMVECs without altering mRNA stability. Both shear-mediated and targeted overexpression of the ET(B) receptor enhanced ET-1-mediated ET(B) receptor-dependent eNOS activation in RPMVECs through Ca(2+)-mediated signaling pathways and independent of Akt activation. In prehepatic portal hypertensive animals relative to control, ET-1 administration also activated eNOS independent of Akt activation and triggered HPS. These findings support that increased pulmonary microvascular endothelial ET(B) receptor expression modulates ET-1-mediated eNOS activation, independent of Akt, and contributes to the development of HPS.     

Urocortin increased LPS‐induced endothelial permeability by regulating the cadherin–catenin complex via corticotrophin‐releasing hormone receptor 2

Urocortin (Ucn1), a member of corticotrophin‐releasing hormone (CRH) family, has been reported to be upregulated in inflammatory diseases and function as an autocrine or paracrine inflammatory mediator. Growing evidence shows that Ucn1 increases the endothelial permeability in inflammatory conditions; however, the detailed mechanisms are not clear. In the present study, we investigated the mechanisms of increased endothelial permeability by Ucn1 in human umbilical vein endothelial cells (HUVECs) exposed to lipopolysaccharide (LPS). Pretreatment of HUVECs with Ucn1 increased the endothelial cell permeability, which was augmented by LPS synergistically. Significant downregulation of VE‐cadherin expression was also observed. Moreover, Ucn1 increased phosphorylation of protein kinase D (PKD) and heat shock protein 27 (HSP27) in a time‐ and CRHR₂‐dependent manner. Inhibition of PKD and HSP27 drastically attenuated Ucn1‐induced downregulation of VE‐cadherin expression. Further investigations demonstrated that Ucn1 phosphorylated β‐catenin at Ser552 to disrupt the cadherin–catenin complex and hence promote the disassociation of β‐catenin and VE‐cadherin. Disassociation of β‐catenin and VE‐cadherin resulted in decreased VE‐cadherin expression while on the contrary β‐catenin was increased, which may due to the inactivation of GSK‐3β. Increased β‐catenin translocated into the nucleus and subsequently bound to TCF/LEF site, contributing to the elevated expression of vascular endothelial growth factor (VEGF). The above effects of Ucn1 were completely reversed by CRHR₂ receptor blocker, antisauvagine‐30. Taken together, our data suggest that Ucn1 increase LPS‐induced endothelial permeability by disrupting the VE‐cadherin–β‐catenin complex via activation of CRHR₂ and PKD‐HSP27 signaling pathway. J. Cell. Physiol. 228: 1295–1303, 2013. © 2012 Wiley Periodicals, Inc.     

T‐cadherin modulates endothelial barrier function

T‐cadherin is an atypical member of the cadherin family, which lacks the transmembrane and intracellular domains and is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. Unlike canonical cadherins, it is believed to function primarily as a signaling molecule. T‐cadherin is highly expressed in endothelium. Using transendothelial electrical resistance measurements and siRNA‐mediated depletion of T‐cadherin in human umbilical vein endothelial cells, we examined its involvement in regulation of endothelial barrier. We found that in resting confluent monolayers adjusted either to 1% or 10% serum, T‐cadherin depletion modestly, but consistently reduced transendothelial resistance. This was accompanied by increased phosphorylation of Akt and LIM kinase, reduced phosphorylation of p38 MAP kinase, but no difference in tubulin acetylation and in phosphorylation of an actin filament severing protein cofilin and myosin light chain kinase. Serum stimulation elicited a biphasic increase in resistance with peaks at 0.5 and 4–5 h, which was suppressed by a PI3 kinase/Akt inhibitor wortmannin and a p38 inhibitor SB 239063. T‐cadherin depletion increased transendothelial resistance between the two peaks and reduced the amplitude of the second peak. T‐cadherin depletion abrogated serum‐induced Akt phosphorylation at Thr308 and reduced phosphorylation at Ser473, reduced phosphorylation of cofilin, and accelerated tubulin deacetylation. Adiponectin slightly improved transendothelial resistance irrespectively of T‐cadherin depletion. T‐cadherin depletion also resulted in a reduced sensitivity and delayed responses to thrombin. These data implicate T‐cadherin in regulation of endothelial barrier function, and suggest a complex signaling network that links T‐cadherin and regulation of barrier function. J. Cell. Physiol. 223: 94–102, 2010. © 2009 Wiley‐Liss, Inc.     

Cross talk of shear-induced production of prostacyclin and nitric oxide in endothelial cells

We tested the hypothesis that vessel homeostasis is maintained through the cross talk of shear-induced production of prostacyclin and nitric oxide (NO). Confluent human umbilical vein endothelial cells (HUVEC) were exposed to fluid shear stress at 15 dyn/cm(2) using a cone-plate device, and the concentrations of 6-keto-PGF(1alpha) and NO metabolites (nitrate and nitrite) in the medium were measured with radioimmunoassay and the Greiss method, respectively. Compared with static control, shear stress increased cumulative prostacyclin production by twofold after 90 min of exposure. Inhibition of NO synthase enhanced flow-induced prostacyclin production by twofold without affecting the baseline production. Guanylyl cyclase inhibitor enhanced flow-induced prostacyclin production to the same degree. In contrast, a stable agonist of cGMP attenuated the rapid early phase of flow-dependent prostacyclin production. Shear-induced NO metabolite production was unaffected even after indomethacin inhibited prostacyclin production. We conclude that NO shows an inhibitory effect on prostacyclin production under shear stress and that vessel homeostasis may be maintained through an increase in prostacyclin production when NO synthesis is impaired in endothelial cells.     

Vascular endothelial growth factor signals endothelial cell production of nitric oxide and prostacyclin through flk-1/KDR activation of c-Src.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mitogen that promotes angiogenesis, vascular hyperpermeability, and vasodilation by autocrine mechanisms involving nitric oxide (NO) and prostacyclin (PGI(2)) production. These experiments used immunoprecipitation and immunoassay procedures to characterize the signaling pathways by which VEGF induces NO and PGI(2) formation in cultured endothelial cells. The data showed that VEGF stimulates complex formation of the flk-1/kinase-insert domain-containing receptor (KDR) VEGF receptor with c-Src and that Src activation is required for VEGF induction of phospholipase C gamma1 activation and inositol 1,4,5-trisphosphate formation. Reporter cell assays showed that VEGF promotes a approximately 50-fold increase in NO formation, which peaks at 5-20 min. This effect is mediated by a signaling cascade initiated by flk-1/KDR activation of c-Src, leading to phospholipase C gamma1 activation, inositol 1,4,5-trisphosphate formation, release of [Ca(2+)](i) and nitric oxide synthase activation. Immunoassays of VEGF-induced 6-keto prostaglandin F(1alpha) formation as an indicator of PGI(2) production revealed a 3-4-fold increase that peaked at 45-60 min. The PGI(2) signaling pathway follows the NO pathway through release of [Ca(2+)](i), but diverges prior to NOS activation and also requires activation of mitogen-activated protein kinase. These results suggest that NO and PGI(2) function in parallel in mediating the effects of VEGF.     

Cyclosporin A inhibits flow-mediated activation of endothelial nitric-oxide synthase by altering cholesterol content in caveolae

Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow. Although cyclosporin A (CsA), an inhibitory ligand of cyclophilin A, is a widely used immunosuppressive drug, it causes arterial hypertension in part by impairing eNOS-dependent vasodilation. Here we show that CsA inhibits fluid shear stress-mediated eNOS activation in endothelial cells via decreasing cholesterol content in caveolae. Exposure of cultured bovine aortic endothelial cells to 1 mum CsA for 1 h significantly inhibited NO production and eNOS phosphorylation at Ser-1179 induced by flow (shear stress=dynes/cm2). The effect of CsA was not related to inhibition of two known eNOS kinases, protein kinase B (Akt) and protein kinase A, because CsA did not affect Akt or protein kinase A activation. In rabbit aorta perfused ex vivo, CsA also significantly inhibited flow-induced eNOS phosphorylation at Ser-1179 but had no effect on Akt measured by phosphorylation at Ser-473. However, CsA treatment decreased cholesterol content in caveolae and displaced eNOS from caveolae, which may be caused by CsA disrupting the association of caveolin-1 and cyclophilin A. The magnitude of the cholesterol depleting effect was similar to that of beta-cyclodextrin, a cholesterol-binding molecule, and beta-cyclodextrin had a similar inhibitory effect on flow-mediated eNOS activation. Treating bovine aortic endothelial cells for 24 h with 30 mug/ml cholesterol blocked the CsA effect and restored eNOS phosphorylation in response to flow. These data suggest that decreasing cholesterol content in caveolae by CsA is a potentially important pathogenic mechanism for CsA-induced endothelial dysfunction and hypertension.     

Oscillatory shear alters endothelial hydraulic conductivity and nitric oxide levels

This study addresses the role of nitric oxide (NO) and downstream signaling pathways in mediating the influences of oscillatory shear stress on the hydraulic conductivity (L(p)) of bovine aortic endothelial cell (BAEC) monolayers. Exposure of BAEC monolayers to 20 dyne/cm2 steady shear stress for 3 h induced a 3.3-fold increase in L(p). When an oscillatory shear amplitude of 10 dyne/cm2 was superimposed on a steady shear of 10 dyne/cm2 to produce a non-reversing oscillatory shear pattern (10+/-10 dyne/cm2), L(p) increased by 3.0-fold within 90 min. When the amplitude was increased to 15 dyne/cm2, resulting in a reversing oscillatory shear pattern (10+/-15 dyne/cm2), the increase in L(p) over 3 h was completely suppressed. Twenty and 10+/-10 dyne/cm2 induced 2.9- and 2.6-fold increases in NO production above non-sheared controls, respectively, whereas 10+/-15 dyne/cm2 stimulated a 14-fold increase in NO production. The inhibition of L(p) with reversing oscillatory shear may be associated with alterations in cyclic guanosine monophosphate (cGMP) production downstream of NO which is up-regulated by reversing oscillatory shear, but is unaffected by steady shear.     

Coordinated regulation of endothelial calcium signaling and shear stress-induced nitric oxide production by PKCβ and PKCη

BackgroundProtein Kinase C (PKC) is a promiscuous serine/threonine kinase regulating vasodilatory responses in vascular endothelial cells. Calcium-dependent PKCbeta (PKCβ) and calcium-independent PKCeta (PKCη) have both been implicated in the regulation and dysfunction of endothelial responses to shear stress and agonists.ObjectiveWe hypothesized that PKCβ and PKCη differentially modulate shear stress-induced nitric oxide (NO) production by regulating the transduced calcium signals and the resultant eNOS activation. As such, this study sought to characterize the contribution of PKCη and PKCβ in regulating calcium signaling and endothelial nitric oxide synthase (eNOS) activation after exposure of endothelial cells to ATP or shear stress.MethodsBovine aortic endothelial cells were stimulated in vitro under pharmacological inhibition of PKCβ with LY333531 or PKCη targeting with a pseudosubstrate inhibitor. The participation of PKC isozymes in calcium flux, eNOS phosphorylation and NO production was assessed following stimulation with ATP or shear stress.ResultsPKCη proved to be a robust regulator of agonist- and shear stress-induced eNOS activation, modulating calcium fluxes and tuning eNOS activity by multi-site phosphorylation. PKCβ showed modest influence in this pathway, promoting eNOS activation basally and in response to shear stress. Both PKC isozymes contributed to the constitutive and induced phosphorylation of eNOS. The observed PKC signaling architecture is intricate, recruiting Src to mediate a portion of PKCη's control on calcium entry and eNOS phosphorylation. Elucidation of the importance of PKCη in this pathway was tempered by evidence of a single stimulus producing concurrent phosphorylation at ser1179 and thr497 which are antagonistic to eNOS activity.ConclusionsWe have, for the first time, shown in a single species in vitro that shear stress- and ATP-stimulated NO production are differentially regulated by classical and novel PKCs. This study furthers our understanding of the PKC isozyme interplay that optimizes NO production. These considerations will inform the ongoing design of drugs for the treatment of PKC-sensitive cardiovascular pathologies.     

Endothelial basement membrane laminin 511 is essential for shear stress response

Shear detection and mechanotransduction by arterial endothelium requires junctional complexes containing PECAM‐1 and VE‐cadherin, as well as firm anchorage to the underlying basement membrane. While considerable information is available for junctional complexes in these processes, gained largely from in vitro studies, little is known about the contribution of the endothelial basement membrane. Using resistance artery explants, we show that the integral endothelial basement membrane component, laminin 511 (laminin α5), is central to shear detection and mechanotransduction and its elimination at this site results in ablation of dilation in response to increased shear stress. Loss of endothelial laminin 511 correlates with reduced cortical stiffness of arterial endothelium in vivo, smaller integrin β1‐positive/vinculin‐positive focal adhesions, and reduced junctional association of actin–myosin II. In vitro assays reveal that β1 integrin‐mediated interaction with laminin 511 results in high strengths of adhesion, which promotes p120 catenin association with VE‐cadherin, stabilizing it at cell junctions and increasing cell–cell adhesion strength. This highlights the importance of endothelial laminin 511 in shear response in the physiologically relevant context of resistance arteries.     

Adaptor protein Crk is required for ephrin-B1-induced membrane ruffling and focal complex assembly of human aortic endothelial cells

Endothelial cell migration is an essential step in vasculogenesis and angiogenesis, in which receptor tyrosine kinases play a pivotal role. We investigated the mechanism by which ephrin-B1 promotes membrane ruffling in human aortic endothelial cells, because membrane ruffling heralds cell body migration. We especially focused on the role of Crk adaptor protein in EphB-mediated signaling. Using DsRed-tagged Crk and a fluorescent time-lapse microscope, we showed that Crk was recruited to the nascent focal complex after ephrin-B1 stimulation. Furthermore, we found that p130(Cas), but not paxillin, recruited Crk to the nascent focal complex. The necessity of Crk in ephrin-B1-induced membrane ruffling was shown both by the overexpression of dominant negative Crk mutants and by the depletion of Crk by using RNA interference. Then, we examined the role of two major downstream molecules of Crk, Rac1 and Rap1. The dominant negative mutant of Rac1 completely inhibited ephrin-B1-induced membrane ruffling and focal complex assembly. In contrast, rap1GAPII, a negative regulator of Rap1, did not inhibit ephrin-B1-induced membrane ruffling. However, in rap1GAPII-expressing cells, ephrin-B1 did not induce membrane spreading, probably due to instability of the focal complex. These results indicated that Crk plays a critical role in Rac1-induced membrane ruffling and Rap1-mediated nascent focal complex stabilization contributing to ephrin-B1-induced human aortic endothelial cells migration.     

Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase

Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10-20 dyn/cm(2)) in conditionally immortalized human GEnC. Laminar shear stress caused an orientation of GEnC and stress fibers parallel to the direction of flow and induced Akt and eNOS phosphorylation along with NO production. Inhibition of the phophatidylinositol (PI)3-kinase/Akt pathway attenuated laminar shear stress-induced eNOS phosphorylation and NO production. Laminar shear stress of 10 dyn/cm(2) had a dramatic effect on GEnC permeability, reversibly decreasing the electrical resistance across GEnC monolayers. Finally, the laminar shear stress-induced reduction in electrical resistance was attenuated by the NOS inhibitors l-N(G)-monomethyl arginine (l-NMMA) and l-N(G)-nitroarginine methyl ester (l-NAME) and also by inhibition of the PI3-kinase/Akt pathway. Hence we have shown for GEnC in vitro that acute permeability responses to laminar shear stress are dependent on NO, produced via activation of the PI3-kinase/Akt pathway and increased eNOS phosphorylation. These results suggest the importance of laminar shear stress and NO in regulating the contribution of GEnC to the permeability properties of the glomerular capillary wall.     

Shear-induced endothelial mechanotransduction: the interplay between reactive oxygen species (ROS) and nitric oxide (NO) and the pathophysiological implications

Hemodynamic shear stress, the blood flow-generated frictional force acting on the vascular endothelial cells, is essential for endothelial homeostasis under normal physiological conditions. Mechanosensors on endothelial cells detect shear stress and transduce it into biochemical signals to trigger vascular adaptive responses. Among the various shear-induced signaling molecules, reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in vascular homeostasis and diseases. In this review, we explore the molecular, cellular, and vascular processes arising from shear-induced signaling (mechanotransduction) with emphasis on the roles of ROS and NO, and also discuss the mechanisms that may lead to excessive vascular remodeling and thus drive pathobiologic processes responsible for atherosclerosis. Current evidence suggests that NADPH oxidase is one of main cellular sources of ROS generation in endothelial cells under flow condition. Flow patterns and magnitude of shear determine the amount of ROS produced by endothelial cells, usually an irregular flow pattern (disturbed or oscillatory) producing higher levels of ROS than a regular flow pattern (steady or pulsatile). ROS production is closely linked to NO generation and elevated levels of ROS lead to low NO bioavailability, as is often observed in endothelial cells exposed to irregular flow. The low NO bioavailability is partly caused by the reaction of ROS with NO to form peroxynitrite, a key molecule which may initiate many pro-atherogenic events. This differential production of ROS and RNS (reactive nitrogen species) under various flow patterns and conditions modulates endothelial gene expression and thus results in differential vascular responses. Moreover, ROS/RNS are able to promote specific post-translational modifications in regulatory proteins (including S-glutathionylation, S-nitrosylation and tyrosine nitration), which constitute chemical signals that are relevant in cardiovascular pathophysiology. Overall, the dynamic interplay between local hemodynamic milieu and the resulting oxidative and S-nitrosative modification of regulatory proteins is important for ensuing vascular homeostasis. Based on available evidence, it is proposed that a regular flow pattern produces lower levels of ROS and higher NO bioavailability, creating an anti-atherogenic environment. On the other hand, an irregular flow pattern results in higher levels of ROS and yet lower NO bioavailability, thus triggering pro-atherogenic effects.     

Fluid shear stress induces differentiation of Flk-1-positive embryonic stem cells into vascular endothelial cells in vitro

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 G(2)-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 alpha-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.     

Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca²⁺/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases—protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases—are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation. shear stress; nitric oxide; endothelial cells; protein kinases     

Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A.

Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction between Akt and PKA may be an important mechanism by which eNOS activity is regulated in response to physiological stimuli such as shear stress.