Flow-induced expression of endothelial Na-K-Cl cotransport: dependence on K(+) and Cl(-) channels

Steady laminarshear stress has been shown previously to markedly increase Na-K-Clcotransporter mRNA and protein in human umbilical vein endothelialcells and also to rapidly increase endothelial K⁺ andCl channel conductances. The present study was done toevaluate the effects of shear stress on Na-K-Cl cotransporter activity and protein expression in bovine aortic endothelial cells (BAEC) and todetermine whether changes in cotransporter expression may be dependenton early changes in K⁺ and Cl channelconductances. Confluent BAEC monolayers were exposed in aparallel-plate flow chamber to either steady shear stress (19 dyn/cm²) or purely oscillatory shear stress (0 ± 19 dyn/cm²) for 6-48 h. After shearing, BAEC monolayerswere assessed for Na-K-Cl cotransporter activity or were subjected toWestern blot analysis of cotransporter protein. Steady shear stress ledto a 2- to 4-fold increase in BAEC cotransporter protein levels and a1.5- to 1.8-fold increase in cotransporter activity, increases thatwere sustained over the longest time periods studied. Oscillatory flow,in contrast, had no effect on cotransporter protein levels. In thepresence of flow-sensitive K⁺ and Cl channelpharmacological blockers, the steady shear stress-induced increase incotransporter protein was virtually abolished. These results suggestthat shear stress modulates the expression of the BAEC Na-K-Clcotransporter by mechanisms that are dependent on flow-activated ion channels.

     

Flow-activated chloride channels in vascular endothelium. Shear stress sensitivity, desensitization dynamics, and physiological implications

Although activation of outward rectifying Cl(-) channels is one of the fastest responses of endothelial cells (ECs) to shear stress, little is known about these channels. In this study, we used whole-cell patch clamp recordings to characterize the flow-activated Cl(-) current in bovine aortic ECs (BAECs). Application of shear stress induced rapid development of a Cl(-) current that was effectively blocked by the Cl(-) channel antagonist 5-nitro-2-(3-phenopropylamino)benzoic acid (100 microM). The current initiated at a shear stress as low as 0.3 dyne/cm(2), attained its peak within minutes of flow onset, and saturated above 3.5 dynes/cm(2) approximately 2.5-3.5-fold increase over pre-flow levels). The Cl(-) current desensitized slowly in response to sustained flow, and step increases in shear stress elicited increased current only if the shear stress levels were below the 3.5 dynes/cm(2) saturation level. Oscillatory flow with a physiological oscillation frequency of 1 Hz, as occurs in disturbed flow zones prone to atherosclerosis, failed to elicit the Cl(-) current, whereas lower oscillation frequencies led to partial recovery of the current. Nonreversing pulsatile flow, generally considered protective of atherosclerosis, was as effective in eliciting the current as steady flow. Measurements using fluids of different viscosities indicated that the Cl(-) current is responsive to shear stress rather than shear rate. Blocking the flow-activated Cl(-) current abolished flow-induced Akt phosphorylation in BAECs, whereas blocking flow-sensitive K(+) currents had no effect, suggesting that flow-activated Cl(-) channels play an important role in regulating EC flow signaling.     

Flow-activated ion channels in vascular endothelium

The ability of vascular endothelial, cells (ECs) to respond to fluid mechanical forces associated with blood flow is essential for flow-mediated vasoregulation and arterial wall remodeling. Abnormalities in endothelial responses to flow also play a role in the development of atherosclerosis. Although our understanding of the endothelial signaling pathways stimulated by flow has greatly increased over the past two decades, the mechanisms by which ECs sense flow remain largely unknown. Activation of flow-sensitive ion channels is among the fastest known endothelial responses to flow; therefore, these ion channels have been proposed as candidate flow sensors. This review focuses on: 1) describing the various types of flow-sensitive ion channels that have been reported in ECs, 2) discussing the implications of activation of these ion channels for endothelial function, and 3) proposing candidate mechanisms for activation of flow-sensitive ion channels.     

Dual effect of fluid shear stress on volume-regulated anion current in bovine aortic endothelial cells

The key mechanism responsible formaintaining cell volume homeostasis is activation ofvolume-regulated anion current (VRAC). The role of hemodynamicshear stress in the regulation of VRAC in bovine aortic endothelialcells was investigated. We showed that acute changes in shear stresshave a biphasic effect on the development of VRAC. A shear stress stepfrom a background flow (0.1 dyn/cm²) to 1 dyn/cm² enhanced VRAC activation induced by an osmoticchallenge. Flow alone, in the absence of osmotic stress, did not induceVRAC activation. Increasing the shear stress to 3 dyn/cm²,however, resulted in only a transient increase of VRAC activity followed by an inhibitory phase during which VRAC was gradually suppressed. When shear stress was increased further (5-10dyn/cm²), the current was immediately strongly suppressed.Suppression of VRAC was observed both in cells challenged osmoticallyand in cells that developed spontaneous VRAC under isotonic conditions. Our findings suggest that shear stress is an important factor inregulating the ability of vascular endothelial cells to maintain volume homeostasis.

     

Vascular endothelial wound closure under shear stress: role of membrane fluidity and flow-sensitive ion channels.

Sufficiently rapid healing of vascular endothelium following injury is essential for preventing further pathological complications. Recent work suggests that fluid dynamic shear stress regulates endothelial cell (EC) wound closure. Changes in membrane fluidity and activation of flow-sensitive ion channels are among the most rapid endothelial responses to flow and are thought to play an important role in EC responsiveness to shear stress. The goal of the present study was to probe the role of these responses in bovine aortic EC (BAEC) wound closure under shear stress. BAEC monolayers were mechanically wounded and subsequently subjected to either "high" (19 dyn/cm(2)) or "low" (3 dyn/cm(2)) levels of steady shear stress. Image analysis was used to quantify cell migration and spreading under both flow and static control conditions. Our results demonstrate that, under static conditions, BAECs along both wound edges migrate at similar velocities to cover the wounded area. Low shear stress leads to significantly lower BAEC migration velocities, whereas high shear stress results in cells along the upstream edge of the wound migrating significantly more rapidly than those downstream. The data also show that reducing BAEC membrane fluidity by enriching the cell membrane with exogenous cholesterol significantly slows down both cell spreading and migration under flow and hence retards wound closure. Blocking flow-sensitive K and Cl channels reduces cell spreading under flow but has no impact on cell migration. These findings provide evidence that membrane fluidity and flow-sensitive ion channels play distinct roles in regulating EC wound closure under flow.     

Macrorheology and adaptive microrheology of endothelial cells subjected to fluid shear stress

Vascular endothelial cells (ECs) respond to temporal and spatial characteristics of hemodynamic forces by alterations in their adhesiveness to leukocytes, secretion of vasodilators, and permeability to blood-borne constituents. These physiological and pathophysiological changes are tied to adaptation of cell mechanics and mechanotransduction, the process by which cells convert forces to intracellular biochemical signals. The exact time scales of these mechanical adaptations, however, remain unknown. We used particle-tracking microrheology to study adaptive changes in intracellular mechanics in response to a step change in fluid shear stress, which simulates both rapid temporal and steady features of hemodynamic forces. Results indicate that ECs become significantly more compliant as early as 30 s after a step change in shear stress from 0 to 10 dyn/cm² followed by recovery of viscoelastic parameters within 4 min of shearing, even though shear stress was maintained. After ECs were sheared for 5 min, return of shear stress to 0 dyn/cm² in a stepwise manner did not result in any further rheological adaptation. Average vesicle displacements were used to determine time-dependent cell deformation and macrorheological parameters by fitting creep function to a linear viscoelastic liquid model. Characteristic time and magnitude for shear-induced deformation were 3 s and 50 nm, respectively. We conclude that ECs rapidly adapt their mechanical properties in response to shear stress, and we provide the first macrorheological parameters for time-dependent deformations of ECs to a physiological forcing function. Such studies provide insight into pathologies such as atherosclerosis, which may find their origins in EC mechanics. viscoelasticity; atherosclerosis; cell mechanics; particle tracking; mechanotransduction     

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease−2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm²) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm²) (P < 0.01). ³H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.     

Short-Term Shear Stress Induces Rapid Actin Dynamics in Living Endothelial Cells

Hemodynamic shear stress guides a variety of endothelial phenotype characteristics, including cell morphology, cytoskeletal structure, and gene expression profile. The sensing and processing of extracellular fluid forces may be mediated by mechanotransmission through the actin cytoskeleton network to intracellular locations of signal initiation. In this study, we identify rapid actin-mediated morphological changes in living subconfluent and confluent bovine aortic endothelial cells (ECs) in response to onset of unidirectional steady fluid shear stress (15 dyn/cm²). After flow onset, subconfluent cells exhibited dynamic edge activity in lamellipodia and small ruffles in the downstream and side directions for the first 12 min; activity was minimal in the upstream direction. After 12 min, peripheral edge extension subsided. Confluent cell monolayers that were exposed to shear stress exhibited only subtle increases in edge fluctuations after flow onset. Addition of cytochalasin D to disrupt actin polymerization served to suppress the magnitude of flow-mediated actin remodeling in both subconfluent confluent EC monolayers. Interestingly, when subconfluent ECs were exposed to two sequential flow step increases (1 dyn/cm² followed by 15 dyn/cm² 12 min later), actin-mediated edge activity was not additionally increased after the second flow step. Thus, repeated flow increases served to desensitize mechanosensitive structural dynamics in the actin cytoskeleton.     

Shear-induced reactive nitrogen species inhibit mitochondrial respiratory complex activities in cultured vascular endothelial cells

There is evidence that nitric oxide (NO), superoxide (O₂^–), and their associated reactive nitrogen species (RNS) produced by vascular endothelial cells (ECs) in response to hemodynamic forces play a role in cell signaling. NO is known to impair mitochondrial respiration. We sought to determine whether exposure of human umbilical vein ECs (HUVECs) to steady laminar shear stress and the resultant NO production modulate electron transport chain (ETC) enzymatic activities. The activities of respiratory complexes I, II/III, and IV were dependent on the presence of serum and growth factor supplement in the medium. EC exposure to steady laminar shear stress (10 dyn/cm²) resulted in a gradual inhibition of each of the complexes starting as early as 5 min from the flow onset and lasting up to 16 h. Ramp flow resulted in inhibition of the complexes similar to that of step flow. When ECs were sheared in the presence of the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 100 µM), the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; 100 µM), or the peroxynitrite (ONOO^–) scavenger uric acid (UA; 50 µM), the flow-inhibitory effect on mitochondrial complexes was attenuated. In particular, L-NAME and UA abolished the flow effect on complex IV. Increased tyrosine nitration was observed in the mitochondria of sheared ECs, and UA blocked the shear-induced nitrotyrosine staining. In summary, shear stress induces mitochondrial RNS formation that inhibits the electron flux of the ETC at multiple sites. This may be a critical mechanism by which shear stress modulates EC signaling and function. oxidative stress; mitochondria; endothelium     

Rac1 mediates laminar shear stress-induced vascular endothelial cell migration

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. ECs are constantly subjected to shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular behaviors and functions. The aim of this study is to elucidate the effects of Rac1, which is the member of small G protein family, on EC migration under different laminar shear stress (5.56, 10.02, and 15.27 dyn/cm²). The cell migration distance under laminar shear stress increased significantly than that under the static culture condition. Especially, under relative high shear stress (15.27 dyn/cm²) there was a higher difference at 8 h (P < 0.01) and 2 h (P < 0.05) compared with static controls. RT-PCR results further showed increasing mRNA expression of Rac1 in ECs exposed to laminar shear stress than that exposed to static culture. Using plasmids encoding the wild-type (WT), an activated mutant (Q61L), and a dominant-negative mutant (T17N), plasmids encoding Rac1 were transfected into EA.hy 926 cells. The average net migration distance of Rac1Q61L group increased significantly, while Rac1T17N group decreased significantly in comparison with the static controls. These results indicated that Rac1 mediated shear stress-induced EC migration. Our findings conduce to elucidate the molecular mechanisms of EC migration induced by shear stress, which is expected to understand the pathophysiological basis of wound healing in health and diseases.     

Shear stress increases expression of a KATP channel in rat and bovine pulmonary vascular endothelial cells

We have shown previously that acute ischemia leads to depolarization of pulmonary microvascular endothelial cells that is prevented with cromakalim, suggesting the presence of ATP-sensitive K⁺ (K_ATP) channels in these cells. Thus K_ATP channel expression and activity were evaluated in rat pulmonary microvascular endothelial cells (RPMVEC) by whole cell current measurements, dot blot (mRNA), and immunoblot (protein) for the inwardly rectifying K⁺ channel (K_IR) 6.2 subunit and fluorescent ligand binding for the sulfonylurea receptor (SUR). Low-level expression of a K_ATP channel was detected in endothelial cells in routine (static) culture and led us to examine whether its expression is inducible when endothelial cells are adapted to flow. Channel expression (mRNA and both K_IR6.2 and SUR proteins) and inwardly rectified membrane current by patch clamp increased significantly when RPMVEC were adapted to flow at 10 dyn/cm² for 24 h in either a parallel plate flow chamber or an artificial capillary system. Induction of the K_ATP channel with flow adaptation was also observed in bovine pulmonary artery endothelial cells. Flow-adapted but not static RPMVEC showed cellular plasma membrane depolarization upon stop of flow that was inhibited by a K_ATP channel opener and prevented by addition of cycloheximide to the medium during the flow adaptation period. These studies indicate the induction of K_ATP channels by flow adaptation in pulmonary endothelium and that the expression and activity of this channel are essential for the endothelial cell membrane depolarization response with acute decrease in shear stress. flow adaptation; K_IR 6.2; sulfonylurea receptor; fluorescent glyburide; pulmonary microvascular endothelial cells     

Plaque-prone hemodynamics impair endothelial function in pig carotid arteries

Hemodynamic forces play an active role in vascular pathologies, particularly in relation to the localization of atherosclerotic lesions. It has been established that low shear stress combined with cyclic reversal of flow direction (oscillatory shear stress) affects the endothelial cells and may lead to an initiation of plaque development. The aim of the study was to analyze the effect of hemodynamic conditions in arterial segments perfused in vitro in the absence of other stimuli. Left common porcine carotid segments were mounted into an ex vivo arterial support system and perfused for 3 days under unidirectional high and low shear stress (6 +/- 3 and 0.3 +/- 0.1 dyn/cm(2)) and oscillatory shear stress (0.3 +/- 3 dyn/cm(2)). Bradykinin-induced vasorelaxation was drastically decreased in arteries exposed to oscillatory shear stress compared with unidirectional shear stress. Impaired nitric oxide-mediated vasodilation was correlated to changes in both endothelial nitric oxide synthase (eNOS) gene expression and activation in response to bradykinin treatment. This study determined the flow-mediated effects on native tissue perfused with physiologically relevant flows and supports the hypothesis that oscillatory shear stress is a determinant factor in early stages of atherosclerosis. Indeed, oscillatory shear stress induces an endothelial dysfunction, whereas unidirectional shear stress preserves the function of endothelial cells. Endothelial dysfunction is directly mediated by a downregulation of eNOS gene expression and activation; consequently, a decrease of nitric oxide production and/or bioavailability occurs.     

Ionic mechanisms of depolarizing and hyperpolarizing quinine receptor potentials in Paramecium caudatum

The ionic mechanisms of the depolarizing and the hyperpolarizing quinine receptor potentials in the ciliate Paramecium caudatum were examined by using a behavioral mutant strain. The depolarizing receptor potential was induced by stimulating the anterior end of the specimen, and the hyperpolarizing receptor potential by stimulating the posterior end. The amplitude of both the depolarizing and the hyperpolarizing receptor potentials increased linearly with logarithmic increase in quinine concentration applied. Threshold concentration for inducing the depolarizing receptor potential was lower than that for the hyperpolarizing one. The peak level of the depolarizing receptor potential shifted towards the depolarizing direction with increasing external Ca²⁺ concentration while that of the hyperpolarizing receptor potential shifted in the depolarizing direction with increasing external K⁺ concentration. Under voltage-clamp conditions, the specimen produced an inward current in response to anterior stimulation, and an outward current in response to posterior stimulation. Both the peak inward and the peak outward currents showed a linear relationship with membrane potential. Current-voltage relationships of the receptor currents indicated conductance increase during the application of quinine. The depolarizing quinine receptor potential appears to be produced by an activation of Ca²⁺ channels, and the hyperpolarizing quinine receptor potential by an activation of K⁺ channels. Accepted: 3 October 1997     

Shear-stress effect on mitochondrial membrane potential and albumin uptake in cultured endothelial cells

Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to shear stress induced by blood flow in vivo, and shear stress affects ATP-dependent macromolecular transport in ECs. However, the relationship between the ATP production and shear stress is still unclear. We, therefore, evaluated mitochondrial ATP synthesis activity in cultured endothelial cells exposed to shear stress, using a confocal laser scanning microscope (CLSM) and a mitochondrial membrane potential probe (5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethyl-benzimidazolycarbocyanine iodide, JC-1). Low shear stress (10 dyn/cm(2)) increased mitochondrial membrane potential by 30%. On the contrary, high shear stress (60 dyn/cm(2)) decreased it by 20%. This observation was consistent with the ATP-dependent albumin uptake into endothelial cells. Our results indicate that ATP synthetic activity is related to the albumin uptake into endothelial cells.     

Differential responsiveness of vascular endothelial cells to different types of fluid mechanical shear stress

Early atherosclerotic lesions localize preferentially, in arterial regions exposed to low flow, oscillatory flow, or both; however, the cellular basis of this observation remains to be determined. Atherogenesis involves dysfunction of the vascular endothelium, the cellular monolayer lining the inner surfaces of blood vessels. How low flow, oscillatory flow, or both may lead to endothelial dysfunction remains unknown. Over the past two decades, fluid mechanical shear (or frictional) stress has been shown to intricately regulate the structure and function of vascular endothelial cells (ECs). Furthermore, recent data indicate that beyond being merely responsive to shear stress, ECs are able to distinguish among and respond differently to different types of shear stress. This review focuses on EC differential responses to different types of steady and unsteady shear stress and discusses the implications of these responses for the localization of early atherosclerotic lesions. The mechanisms by which endothelial differential responsiveness to different types of flow may occur are also discussed.     

Hemodynamic force dictates endothelial angiogenesis through MIEN1-ERK/MAPK-signaling axis

It is well-recognized that blood flow at branches and bends of arteries generates disturbed shear stress, which plays a crucial in driving atherosclerosis. Flow-generated fluid shear stress (FSS), as one of the key hemodynamic factors, is appreciated for its critical involvement in regulating angiogenesis to facilitate wound healing and tissue repair. Endothelial cells can directly sense FSS but the mechanobiological mechanism by which they decode different patterns of FSS to trigger angiogenesis remains unclear. In the current study, laminar shear stress (LSS, 15 dyn/cm²) was employed to mimic physiological blood flow, while disturbed shear stress (DSS, ranging from 0.5 ± 4 dyn/cm²) was applied to simulate pathological conditions. The aim was to investigate how these distinct types of blood flow regulated endothelial angiogenesis. Initially, we observed that DSS impaired angiogenesis and downregulated endogenous vascular endothelial growth factor B (VEGFB) expression compared to LSS. We further found that the changes in membrane protein, migration and invasion enhancer 1 (MIEN1) play a role in regulating ERK/MAPK signaling, thereby contributing to endothelial angiogenesis in response to FSS. We also showed the involvement of MIEN1-directed cytoskeleton organization. These findings suggest the significance of shear stress in endothelial angiogenesis, thereby enhancing our understanding of the alterations in angiogenesis that occur during the transition from physiological to pathological blood flow.     

Electrophysiological characterization of murine HL-5 atrial cardiomyocytes

HL-5 cells are cultured murine atrial cardiomyocytes and have been used in studies to address important cellular and molecular questions. However, electrophysiological features of HL-5 cells have not been characterized. In this study, we examined such properties using whole cell patch-clamp techniques. Membrane capacitance of the HL-5 cells was from 8 to 62 pF. The resting membrane potential was –57.8 ± 1.4 mV (n = 51). Intracellular injection of depolarizing currents evoked action potentials (APs) with variable morphologies in 71% of the patched cells. Interestingly, the incidence of successful, current-induced APs positively correlated with the hyperpolarizing degrees of resting membrane potentials (r = 0.99, P < 0.001). Only a few of the patched cells (4 of 51, 7.8%) exhibited spontaneous APs. The muscarinic agonist carbachol activated the acetylcholine-activated K⁺ current and significantly shortened the duration of APs. Immunostaining confirmed the presence of the muscarinic receptor type 2 in HL-5 cells. The hyperpolarization-activated cation current (I_f) was detected in 39% of the patched cells. The voltage to activate 50% of I_f channels was –73.4 ± 1.2 mV (n = 12). Voltage-gated Na⁺, Ca²⁺, and K⁺ currents were observed in the HL-5 cells with variable incidences. Compared with the adult mouse cardiomyocytes, the HL-5 cells had prolonged APs and small outward K⁺ currents. Our data indicate that HL-5 cells display significant electrophysiological heterogeneity of morphological appearance of APs and expression of functional ion channels. Compared with adult murine cardiomyocytes, HL-5 cells show an immature phenotype of cardiac AP morphology. action potential; ion channel; muscarinic receptor     

A model for shear stress-induced deformation of a flow sensor on the surface of vascular endothelial cells

Fluid mechanical shear stress elicits humoral, metabolic, and structural responses in vascular endothelial cells (ECs); however, the mechanisms involved in shear stress sensing and transduction remain incompletely understood. Beyond being responsive to shear stress, ECs distinguish among and respond differently to different types of shear stress. Recent observations suggest that endothelial shear stress sensing may occur through direct interaction of the flow with cell-surface structures that act as primary flow sensors. This paper presents a mathematical model for the shear stress-induced deformation of a flow sensor on the EC surface. The sensor is modeled as a cytoskeleton-coupled viscoelastic structure exhibiting standard linear solid behavior. Since ECs respond differently to different types of flow, the deformation and resulting velocity of the sensor in response to steady, non-reversing pulsatile, and oscillatory flow have been studied. Furthermore, the sensitivity of the results to changes in various model parameters including the magnitude of applied shear stress, the constants that characterize the viscoelastic behavior, and the pulsatile flow frequency (f) has been investigated. The results have demonstrated that in response to a suddenly applied shear stress, the sensor exhibits a level of instantaneous deformation followed by gradual creeping to the long-term response. The peak deformation increases linearly with the magnitude of the applied shear stress and decreases for viscoelastic constants that correspond to stiffer sensors. While the sensor deformation depends on f for low f values, the deformation becomes f -independent above a critical threshold frequency. Finally, the peak sensor deformation is considerably larger for steady and non-reversing pulsatile flow than for oscillatory flow. If the extent of sensor deformation correlates with the intensity of flow-mediated endothelial signaling, then our results suggest possible mechanisms by which ECs distinguish among steady, non-reversing pulsatile, and oscillatory shear stress.     

Association of SIRT1 expression with shear stress induced endothelial progenitor cell differentiation

Shear stress imposed by blood flow is crucial for differentiation of endothelial progenitor cells (EPCs). Histone deacetylase SIRT1 has been shown to play a pivotal role in many physiological processes. However, association of SIRT1 expression with shear stress‐induced EPC differentiation remains to be elucidated. The present study was designed to determine the effect of SIRT1 on EPC differentiation induced by shear stress, and to seek the underlying mechanisms. Human umbilical cord blood‐derived EPCs were exposed to laminar shear stress of 15 dyn/cm² by parallel plate flow chamber system. Shear stress enhanced EPC differentiation toward endothelial cells (ECs) while inhibited to smooth muscle cells (SMCs). The expressions of phospho‐Akt, SIRT1 and histone H3 acetylation (Ac‐H3) in EPCs were detected after exposure to shear stress for 2, 6, 12, and 24 h, respectively. Shear stress significantly activated Akt phosphorylation, augmented SIRT1 expression and downregulated Ac‐H3. SIRT1 siRNA in EPCs diminished the expression of EC markers, but increased the expression of SMC markers, and resulted in upregulation of Ac‐H3. Whereas, resveratrol, an activator of SIRT1, had the opposite effects on both EPC differentiation and histone H3 acetylation. Wortmannin, an inhibitor of PI3‐kinase, suppressed endothelial differentiation of EPCs, decreased SIRT1, and upregulated Ac‐H3 expression. In addition, SIRT1 promoted tube formation of EPCs in matrix gels. These results provided a mechanobiological basis of shear stress‐induced EPC differentiation into ECs and suggest that PI3k/Akt‐SIRT1‐Ac‐H3 pathway is crucial in such a process. J. Cell. Biochem. 113: 3663–3671, 2012. © 2012 Wiley Periodicals, Inc.     

Cooperative Effects of Matrix Stiffness and Fluid Shear Stress on Endothelial Cell Behavior

Arterial hemodynamic shear stress and blood vessel stiffening both significantly influence the arterial endothelial cell (EC) phenotype and atherosclerosis progression, and both have been shown to signal through cell-matrix adhesions. However, the cooperative effects of fluid shear stress and matrix stiffness on ECs remain unknown. To investigate these cooperative effects, we cultured bovine aortic ECs on hydrogels matching the elasticity of the intima of compliant, young, or stiff, aging arteries. The cells were then exposed to laminar fluid shear stress of 12 dyn/cm². Cells grown on more compliant matrices displayed increased elongation and tighter EC-cell junctions. Notably, cells cultured on more compliant substrates also showed decreased RhoA activation under laminar shear stress. Additionally, endothelial nitric oxide synthase and extracellular signal-regulated kinase phosphorylation in response to fluid shear stress occurred more rapidly in ECs cultured on more compliant substrates, and nitric oxide production was enhanced. Together, our results demonstrate that a signaling cross talk between stiffness and fluid shear stress exists within the vascular microenvironment, and, importantly, matrices mimicking young and healthy blood vessels can promote and augment the atheroprotective signals induced by fluid shear stress. These data suggest that targeting intimal stiffening and/or the EC response to intima stiffening clinically may improve vascular health.