Shear stress enhances glutathione peroxidase expression in endothelial cells

Hemodynamic forces have profound effects on vasculature. Laminar shear stress upregulates superoxide dismutase (SOD) expression in endothelial cells. SOD converts superoxide anion to H(2)O(2), which, however, promotes atherosclerosis. Therefore, defense against H(2)O(2) may be crucial in reducing oxidative stress. Since glutathione peroxidase (GPx-1) reduces H(2)O(2) to H(2)O, the regulation of GPx-1 expression by mechanical stress was examined. Cultured bovine aortic endothelial cells (BAECs) were subjected to laminar shear stress and stretch force. Shear stress upregulated GPx-1 mRNA expression in a time- and force-dependent manner in BAECs, whereas stretch force was without effect. Furthermore, shear stress increased GPx activity. L-NAME, an inhibitor of nitric oxide synthase, did not affect shear stress-induced GPx-1 mRNA expression. The ability of laminar shear stress to induce GPx-1 expression in endothelial cells may be an important mechanism whereby shear stress protects vascular cells against oxidative stress.     

Shear stress enhances prostacyclin release from endocardial endothelial cells

The effect of shear stress on the release of prostacyclin (PGI2) from cultured endocardial endothelial cells (EECs) was investigated. EECs were harvested from the right ventricle (RV) and the left ventricle (LV) of porcine heart. Confluent EECs were incubated under various degrees of shear stress (0.2, 1, 4 and 6 dyne/cm2) and PGI2 release from each cell was measured. PGI2 release from LV-EECs and RV-EECs was enhanced by the elevation of shear stress in a shear-dependent manner with a rapid increase at the onset of flow; however, there was no significant difference in PGI2 production between RV-EECs and LV-EECs. production of PGI2 was significantly inhibited from cells exposed to 8-(dimetilamino) octyl 3,4,5-trymethoxybenzoate hydrochloride (10 and 100 microM: an inhibitor of intracellular calcium mobilization) or cyclopiazonic acid (10 microM: an endoplasmic reticulum Ca2+-ATPase inhibitor). These results indicate that shear stress enhances PGI2 release from cultured EECs and that mechanotransduction of shear stress depends on calcium mobilization in EECs.     

Shear stress mediates tyrosylprotein sulfotransferase isoform shift in human endothelial cells

In this study, we examined expression of tyrosylprotein sulfotransferase (TPST) isoforms TPST1 and TPST2 in primary cultures of human umbilical vein endothelial cells. For the first time coexpression of both isoforms is shown in primary human cells. Application of physiological levels of shear stress regulates expression of TPST isoforms in a time- and dose-dependent manner. Sustained application of arterial laminar shear stress causes downregulation of TPST1 mRNA and protein expression, while TPST2 is upregulated. This TPST isoform shift is mediated by different signaling pathways. Shear stress-dependent downregulation of TPST1 involves tyrosine kinase, while upregulation of TPST2 is mediated by a protein kinase C-dependent pathway [corrected].     

Shear stress activates Tie2 receptor tyrosine kinase in human endothelial cells

The receptor tyrosine kinase (RTK) Tie2 is expressed predominantly on endothelial cells. Tie2 is critical for vasculogenesis during development and could be important for maintaining endothelial cell survival and integrity in adult blood vessels. Although most RTKs are activated by shear stress in the absence of ligand activation, the effect of shear stress on Tie2 is unknown. Therefore, we examined the effect of shear stress on Tie2 phosphorylation in primary cultured endothelial cells. Interestingly, shear stress (20 dyne/cm(2)) produced a rapid, marked, and sustained Tie2 phosphorylation, while it produced a rapid but slight and transient phosphorylation of insulin receptor and VEGF receptor 2 (Flk1). In addition, Tie2 phosphorylation in response to shear stress was velocity-dependent, while phosphorylation of insulin receptor and Flk1 was not. Shear stress also produced Akt phosphorylation in a time-, velocity-, and PI 3-kinase-dependent manner. Accordingly, shear stress suppressed serum deprivation-induced endothelial cell apoptosis. Taken together, our results indicated that activation of Tie2/PI 3-kinase/Akt in response to shear stress could be an important signaling cascade for maintaining endothelial survival and integrity in blood vessels.     

Shear stress induces a time- and position-dependent increase in endothelial cell membrane fluidity

Blood flow-associatedshear stress may modulate cellular processes through its action on theplasma membrane. We quantified the spatial and temporal aspects of theeffects of shear stress () on the lipid fluidity of1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC₁₆(13)]-stained plasma membranesof bovine aortic endothelial cells in a flow chamber. A confocalmicroscope was used to determine the DiI diffusion coefficient(D) by fluorescence recovery after photobleaching on cellsunder static conditions, after a step- of 10 or 20 dyn/cm², and after the cessation of . The methodallowed the measurements of D on the upstream and downstreamsides of the cell taken midway between the respective cell borders andthe nucleus. In <10 s after a step- of 10 dyn/cm²,D showed an upstream increase and a downstream decrease, and both changes disappeared rapidly. There was a secondary, larger increase in upstream D, which reached a peak at 7 min and decreased thereafter, despite the maintenance of .D returned to near control values within 5 s aftercessation of . Downstream D showed little secondarychanges throughout the 10-min shearing, as well as after its cessation.Further investigations into the early phase, with simultaneousmeasurements of upstream and downstream D, confirmed that astep- of 10 dyn/cm² elicited a rapid (5-s) but transientincrease in upstream D and a concurrent decrease indownstream D, yielding a significant difference between thetwo sites. A step- of 20 dyn/cm² caused D toincrease at both sites at 5 s, but by 30 s and 1 min theupstream D became significantly higher than the downstream D. These results demonstrate shear-induced changes inmembrane fluidity that are time dependent and spatially heterogeneous. These changes in membrane fluidity may have important implications inshear-induced membrane protein modulation.

     

Shear stress-induced detachment of human polymorphonuclear leukocytes from endothelial cell monolayers

We employed a static-incubation assay to determine the intensity of wall shear stress (tau) needed to detach human polymorphonuclear leukocytes (HPMNs) from human umbilical vein endothelial cell (HUVE) monolayers. Confluent monolayers of HUVE were placed in a parallel-plate flow chamber which was mounted on the stage of an inverted tissue culture microscope, attached to a perfusion system and maintained at 37 degrees C. All events in the selected fields were recorded using videomicroscopy. HPMNs were co-incubated for 15 minutes with the HUVE monolayers under control conditions or in the presence of 10(-7) M formyl-methionyl-leucyl-phenylalanine (FMLP). Following this static incubation, a series of five individual flows, each 1 minute in duration, were driven through the flow channel, exposing the cells to 1.0, 2.0, 3.8, 7.6 and 14.8 dyn/cm2 wall shear stresses. Under control conditions, the percentage of HPMNs remaining attached to the HUVE monolayers following exposure to each shear stress was 61, 38, 25, 12 and 5, respectively. In the FMLP-treated condition, the percentage of HPMNs remaining attached to the monolayers was significantly greater than control at all five levels of tau. Thus, under control conditions, adherent HPMNs can be detached from endothelial cell monolayers in vitro with levels of shear stress normally found in the microcirculation (18). In the presence of FMLP, the level of shear stress needed to overcome the adhesions is increased significantly.     

Shear stress during early embryonic stem cell differentiation promotes hematopoietic and endothelial phenotypes

Pluripotent embryonic stem cells (ESCs) are a potential source for cell‐based tissue engineering and regenerative medicine applications, but their translation into clinical use will require efficient and robust methods for promoting differentiation. Fluid shear stress, which can be readily incorporated into scalable bioreactors, may be one solution for promoting endothelial and hematopoietic phenotypes from ESCs. Here we applied laminar shear stress to differentiating ESCs using a 2D adherent parallel plate configuration to systematically investigate the effects of several mechanical parameters. Treatment similarly promoted endothelial and hematopoietic differentiation for shear stress magnitudes ranging from 1.5 to 15 dyne/cm² and for cells seeded on collagen‐, fibronectin‐ or laminin‐coated surfaces. Extension of the treatment duration consistently induced an endothelial response, but application at later stages of differentiation was less effective at promoting hematopoietic phenotypes. Furthermore, inhibition of the FLK1 protein (a VEGF receptor) neutralized the effects of shear stress, implicating the membrane protein as a critical mediator of both endothelial and hematopoietic differentiation by applied shear. Using a systematic approach, studies such as these help elucidate the mechanisms involved in force‐mediated stem cell differentiation and inform scalable bioprocesses for cellular therapies. Biotechnol. Bioeng. 2013; 110: 1231–1242. © 2012 Wiley Periodicals, Inc.     

Shear stress regulates endothelial cell autophagy via redox regulation and Sirt1 expression

Disturbed cell autophagy is found in various cardiovascular disease conditions. Biomechanical stimuli induced by laminar blood flow have important protective actions against the development of various vascular diseases. However, the impacts and underlying mechanisms of shear stress on the autophagic process in vascular endothelial cells (ECs) are not entirely understood. Here we investigated the impacts of shear stress on autophagy in human vascular ECs. We found that shear stress induced by laminar flow, but not that by oscillatory or low-magnitude flow, promoted autophagy. Time-course analysis and flow cessation experiments confirmed that this effect was not a transient adaptive stress response but appeared to be a sustained physiological action. Flow had no effect on the mammalian target of rapamycin-ULK pathway, whereas it significantly upregulated Sirt1 expression. Inhibition of Sirt1 blunted shear stress-induced autophagy. Overexpression of wild-type Sirt1, but not the deacetylase-dead mutant, was sufficient to induce autophagy in ECs. Using both of gain- and loss-of-function experiments, we showed that Sirt1-dependent activation of FoxO1 was critical in mediating shear stress-induced autophagy. Shear stress also induced deacetylation of Atg5 and Atg7. Moreover, shear stress-induced Sirt1 expression and autophagy were redox dependent, whereas Sirt1 might act as a redox-sensitive transducer mediating reactive oxygen species-elicited autophagy. Functionally, we demonstrated that flow-conditioned cells are more resistant to oxidant-induced cell injury, and this cytoprotective effect was abolished after inhibition of autophagy. In summary, these results suggest that Sirt1-mediated autophagy in ECs may be a novel mechanism by which laminar flow produces its vascular-protective actions.Vascular endothelial cells (ECs) are fundamentally important in maintaining structural and functional homeostasis of blood vessels. Normal biological functions of ECs are highly sensitive to the biomechanical stimuli induced by blood flow, of which shear stress acting on the surface of EC has been recognized to be one of the most important vasoactive factors in EC.^{1, 2} A relatively high level of laminar shear stress is cytoprotective, whereas abnormal (low-magnitude or oscillatory) shear stress is a detrimental cellular stress to ECs.¹ Transduction of the mechanical signals involves multiple messenger molecules and signaling proteins, which collectively regulate important endothelial functions, such as gene expression, proliferation, migration, morphogenesis, permeability, thrombogenicity, and inflammation.²Autophagy (also known as macroautophagy) is an evolutionarily conserved cellular stress response.^{3, 4} Autophagy is a cellular self-digestion process, which is responsible for degradation of misfolded proteins and damaged organelles. Autophagic process is mainly mediated by the formation of autophagosome, a double-membrane vacuole structure containing engulfed cellular components. This process requires expression of a group of key genes involved in autophagy, including LC3A, beclin-1, Atg5, Atg7, and Atg12, for example.^{3, 5} Autophagosomes fuse with lysosomes, forming autolysosomes, where the cellular components are degraded by various hydrolases in an acidified environment.^{4, 5} In ECs, an autophagic response can be initiated by different stress stimuli.^{6, 7, 8} It is noted that the cellular outcome following autophagy induction in ECs varies depending on the nature of stimuli and specific experimental settings.^{6, 7, 9, 10} Moreover, there is evidence showing that autophagy may also be involved in modulating other EC functions such as angiogenesis and cellular senescence.^{11, 12} Therefore, understanding the regulatory mechanisms of autophagy in ECs will be important for discovery of strategies to protect normal endothelial functions. Recently, Guo et al. provided some evidence indicating that the autophagic process in EC might be affected by shear stress.¹³ This argument, however, was only based on observations of changed expression levels of LC3 and beclin-1; further experimental evidence is needed to confirm such an effect of shear stress on autophagy. More importantly, the mechanisms underlying this phenomenon are not understood. Different signaling pathways may be involved in modulating autophagy in ECs.^{14, 15, 16} For example, inhibition of the mTOR (mammalian target of rapamycin) pathway by rapamycin-induced endothelial autophagy and prevented energy stress-triggered cell damage.¹⁶ There is also evidence indicating a potential role of Sirt1.¹⁴ Moreover, accumulating evidence has suggested that reactive oxygen species (ROS) are closely implicated in modulating autophagic responses via complex interactions with other autophagy-related factors.¹⁵ Despite of these results, the signaling mechanisms of shear stress-regulated autophagy in EC remain to be defined. Hence, here we aim to delineate the impacts and underlying mechanisms of shear stress on autophagy in human vascular ECs.     

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.     

Shear stress augments the endothelial cell differentiation marker expression in late EPCs by upregulating integrins

Vascular endothelial cell injury has been implicated in the onset of atherosclerosis. A number of previous studies have demonstrated that endothelial progenitor cells (EPCs), in particular late EPCs, play important roles in endothelial maintenance and repair. Recent evidence has revealed shear stress as a key regulator for EPC differentiation. However, the detailed events that contribute to the shear stress-induced EPC differentiation, in particular the mechanisms of mechanotransduction, remain to be identified. The present study was undertaken to further confirm the effects of shear stress on the late EPC differentiation, and to investigate the role of integrins in this procedure. Shear stress was observed to increase the expression of endothelial cell differentiation markers, such as vWF and CD31, in late EPCs isolated from rat bone marrow. Shear stress moreover enhanced the mRNA expression of integrin subunits β(1) and β(3) in a time-dependent manner, and also upregulated specific integrins in late EPCs plated on substrates containing various extracellular matrix (ECM) proteins. In addition, the shear stress-induced vWF and CD31 expression were found to be related to the levels of integrin β(1) and β(3), and were inhibited in late EPCs treated with RGD peptide (Gly-Arg-Gly-Asp-Asn-Pro, GRGDNP) that blocks the binding of integrins to the extracellular matrix. Additionally, this increase was also attenuated by both anti-β(1) integrin and anti-β(3) integrin antibodies. The integrin subunits β(1) and β(3) thus play important roles in regulating the shear stress-induced endothelial cell differentiation marker expression in late EPCs. This may provide novel insights into the mechanisms of mechanotransduction in shear stress-mediated late EPC differentiation.     

Pycnogenol enhances endothelial cell antioxidant defenses

Summary

Reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), superoxide anion (O₂^?), and hydroxyl radical (OH^?) have been implicated in mediating various pathological events such as cancer, atherosclerosis, diabetes, ischemia, inflammatory diseases, and the aging process. The glutathione (GSH) redox cycle and antioxidant enzymes—superoxide dismutase (SOD) and catalase (CAT)—play an important role in scavenging ROS and preventing cell injury. Pycnogenol has been shown to protect endothelial cells against oxidant-induced injury. The present study determined the effects of pycnogenol on cellular metabolism of H₂O₂ and O₂^? and on glutathione-dependent and -independent antioxidant enzymes in bovine pulmonary artery endothelial cells (PAEC). Confluent monolayers of PAEC were incubated with pycnogenol, and oxidative stress was triggered by hypoxanthine and xanthine oxidase or H₂O₂. Pycnogenol caused a concentration-dependent enhancement of H₂O₂ and O₂^? clearance. It increased the intracellular GSH content and the activities of GSH peroxidase and GSH disulfide reductase. It also increased the activities of SOD and CAT. The results suggest that pycnogenol promotes a protective antioxidant state by upregulating important enzymatic and nonenzymatic oxidant scavenging systems.     

Effects of corticosteroid-induced apoptosis on airway epithelial wound closure in vitro

Damage to the airway epithelium is common in asthma. Corticosteroids induce apoptosis in and suppress proliferation of airway epithelial cells in culture. Whether apoptosis contributes to impaired epithelial cell repair after injury is not known. We examined whether corticosteroids would impair epithelial cell migration in an in vitro model of wound closure. Wounds (approximately 0.5-1.3 mm2) were created in cultured 1HAEo- human airway epithelial cell monolayers, after which cells were treated with up to 10 microM dexamethasone or budesonide for 24 h. Cultured cells were pretreated for 24 or 48 h with dexamethasone to observe the effect of long-term exposure on wound closure. After 12 h, the remaining wound area in monolayers pretreated for 48 h with 10 microM dexamethasone was 43+/-18% vs. 10+/-8% for untreated control monolayers. The addition of either corticosteroid immediately after injury did not slow closure significantly. After 12 h the remaining wound area in monolayers treated with 10 microM budesonide was 39+/-4% vs. 43+/-3% for untreated control monolayers. The proportion of apoptotic epithelial cells as measured by terminal deoxynucleotidyltransferase-mediated dUTP biotin nick end labeling both at and away from the wound edge was higher in monolayers treated with budesonide compared with controls. However, wound closure in the apoptosis-resistant 1HAEo-.Bcl-2+ cell line was not different after dexamethasone treatment. We demonstrate that corticosteroid treatment before mechanical wounding impairs airway epithelial cell migration. The addition of corticosteroids after injury does not slow migration, despite their ability to induce apoptosis in these cells.     

Nitric oxide enhances hydrogen peroxide-mediated endothelial permeability in vitro

The objective ofthis study was to evaluate the effects of nitric oxide (NO) onH₂O₂-mediatedendothelial permeability.H₂O₂ (0.1 mM) increased permeability at 90 min to 298% of baseline. Spermine NONOate (SNO), an NO donor, at 0.1 or 1 mM did not alter permeability. However, 0.1 mMH₂O₂ + 1 mM SNO increased permeability to 764%, twice that of 0.1 mMH₂O₂alone. These treatments were not directly toxic to endothelial cells.This NO effect was concentration dependent, inasmuch as 0.1 mM SNO didnot significantly change H₂O₂-mediatedpermeability. The NO-enhanced,H₂O₂-dependentpermeability required the simultaneous presence of NO andH₂O₂,inasmuch as preincubation with SNO for 30 min followed by 0.1 mMH₂O₂did not alter permeability. Staining of endothelial junctions showed widening of the intercellular space only in junctions of cells exposedtoH₂O₂(0.1 mM) + SNO (1 mM). Furthermore, NO did not affectH₂O₂metabolism by endothelial cells but significantly depletedintracellular glutathione. This reduction of cell glutathione producedby NO exposure recovered 15-30 min after removal of the NO donor.NO-enhanced permeability was completely blocked by methionine (1 mM), ascavenger of reactive oxygen species, and by the iron chelatordesferrioxamine (0.1 mM). These results suggest that NO may exacerbatethe effects ofH₂O₂-dependentincrease in endothelial monolayer permeability via the iron-catalyzedformation of reactive oxygen metabolites.

     

Laminar shear stress enhances endothelial cell survival through a NADPH oxidase 2-dependent mechanism

The beneficial effects of laminar shear stress (LSS) due to blood flow include inhibition of endothelial cell death, but the associated mechanism is not well understood. This issue was addressed in the present study. In a normal growth medium, the endothelial cell death rate was below 5%, but this value increased beyond 30% when the serum was depleted. However, when cells were exposed to LSS during the serum depletion period, cell viability recovered to the levels of the serum-provided cells. The pro-survival effect of LSS was not affected by l-arginine methyl ester, but it was abrogated by apocynin, indicating that NADPH oxidases (NOX) play key roles in the mechanism. The pro-survival effect of LSS was reduced by NOX2 siRNA, but not by NOX4 siRNA. LSS increased the expressions of p47^phox and p67^phox, the subunits of NOX2 complex. These observations suggest that LSS prevents apoptotic death of endothelial cells through a NOX2-dependent mechanism.     

Shear stress induces caveolin-1 translocation in cultured endothelial cells

Considering that vascular endothelial caveolae could be flow sensors converting mechanical stimuli into chemical signals transmitted into the cell, this work studied, in vitro, the change of caveolin-1 expression and distribution of cultured endothelial cells exposed to laminar flows. Experimental results showed that, in control cells, caveolin-1 were primarily localized on the cell surface, and presented some local concentrations. In cells exposed to laminar flows, caveolin-1 distribution showed a time-dependent variation. After 24 h of shear (1.0 Pa), the expression of caveolin-1 increased and a local caveolin-1 concentration was found, in most cells, at the upstream side of the cell body where the hydrostatic pressure and the spatial gradient of shear stress were at a maximum. As a comparison, tumor necrosis factor-a induced a decrease of caveolin-1 in the cells.     

Simulated hypogravity stimulates cell spreading and wound healing in cultured human vascular endothelial cells

It is well known that endothelial cells (EC) are highly sensitive to mechanical influences such as hemodynamic conditions or pulsatile stretch. However, it is still unknown, how endothelium responds to the changed gravity. The results of some studies suggest that cellular elements of vascular wall and, particularly, endothelium, may directly participate in development of physiological responces to microgravity. On our suggestion, this is extremely attractive since vascular endothelium is one of the main regulators of vascular tone (via its interaction with vascular smooth muscle cells) and, consequently, can play not last role in maintaining of normal cardiovascular system operation in microgravity. On the other hand, the endothelium itself may be regarded as a widely dispersed organ of approximately 1.5 kg in weight (in the adult human organism). Finally, endothelium is not just a passive barrier between vascular wall and circulating blood but synthesizes, metabolizes, and releases a substances which act on adjacent cell systems or distant cell structures. The main aims of this study were: 1) the development of experimental model, allowing to study functional parameters of human endothelial cells in hypogravity conditions in vitro; 2) the verification of endothelial sensitivity to gravitational micro-environment.     

Shear stress in the microvasculature: influence of red blood cell morphology and endothelial wall undulation

Biomechanics and Modeling in Mechanobiology - The effect of red blood cells and the undulation of the endothelium on the shear stress in the microvasculature is studied numerically using the...