Vascular smooth muscle cell glycocalyx modulates shear-induced proliferation, migration, and NO production responses

The endothelial cell glycocalyx, a structure coating the luminal surface of the vascular endothelium, and its related mechanotransduction have been studied by many over the last decade. However, the role of vascular smooth muscle cells (SMCs) glycocalyx in cell mechanotransduction has triggered little attention. This study addressed the role of heparan sulfate proteoglycans (HSPGs), a major component of the glycocalyx, in the shear-induced proliferation, migration, and nitric oxide (NO) production of the rat aortic smooth muscle cells (RASMCs). A parallel plate flow chamber and a peristaltic pump were employed to expose RASMC monolayers to a physiological level of shear stress (12 dyn/cm(2)). Heparinase III (Hep.III) was applied to selectively degrade heparan sulfate on the SMC surface. Cell proliferation, migration, and NO production rates were determined and compared among the following four groups of cells: 1) untreated with no flow, 2) Hep.III treatment with no flow, 3) untreated with flow of 12 dyn/cm(2) exposure, and 4) Hep.III treatment with flow of 12 dyn/cm(2) exposure. It was observed that flow-induced shear stress significantly suppressed SMC proliferation and migration, whereas cells preferred to aligning along the direction of flow and NO production were enhanced substantially. However, those responses were not found in the cells with Hep.III treatment. Under flow condition, the heparinase III-treated cells remained randomly oriented and proliferated as if there were no flow presence. Disruption of HSPG also enhanced wound closure and inhibited shear-induced NO production significantly. This study suggests that HSPG may play a pivotal role in mechanotransduction of SMCs.     

Smooth muscle cells contract in response to fluid flow via a Ca2+-independent signaling mechanism.

Smooth muscle cells (SMC) are exposed to fluid shear stress because of transmural (interstitial) flow across the arterial wall. This shear stress may play a role in the myogenic response and flow-mediated vasomotion. We, therefore, examined the effects of fluid flow on contraction of rat aortic SMC. SMC that had been serum-starved to induce a contractile phenotype were plated on quartz slides and exposed to controlled shear stress levels in a flow chamber. The area of the cells was quantified, and reduction in the cell area was reported as contraction. At 25 dyn/cm(2), significant area reduction was apparent 3 min after the onset of flow and exceeded 30% at 30 min. At 1 dyn/cm(2), significant contraction was not observed at 30 min. The threshold for significant shear-induced contraction appeared to be 11 dyn/cm(2). The signal transduction mechanism was studied at 25 dyn/cm(2). Intracellular calcium was imaged by using the calcium-sensitive fluorescent dye fura 2-AM. There was no detectable change in intracellular calcium during 10 min of exposure to shear stress, even though the cells displayed a significant calcium response to thapsigargin, calcium ionophore, and KCl. Further studies using pathway inhibitors provided evidence that the most important signal transduction pathway mediating calcium-independent contraction in response to fluid flow is the Rho-kinase pathway, although there was a suggestion that protein kinase C plays a secondary role.     

Shear stress inhibits smooth muscle cell migration via nitric oxide-mediated downregulation of matrix metalloproteinase-2 activity

Vascular smooth muscle cell (SMC) migration is a hallmark of intimal hyperplasia (IH), the progression of which is affected by hemodynamic conditions at the diseased site. The realization that SMCs are exposed to blood flow in both denuded vessels (direct blood flow) and intact vessels (interstitial blood flow) motivated this study of the effects of fluid flow shear stress (SS) on SMC migration. Rat aortic SMCs were seeded onto Matrigel-coated cell culture inserts, and their migratory activity toward PDGF-BB when exposed to SS in a rotating disk apparatus was quantified. Four hours of either 10 or 20 dyn/cm2 SS significantly inhibited SMC migration to the bottom side of the insert. This inhibition was associated with downregulation of SMC matrix metalloproteinase (MMP)-2 activation. Four hours of 10 dyn/cm2 SS also drastically increased SMC production of NO. A NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester; 100 microM) abolished the shear-induced increase in SMC NO production as well as the inhibition of migration and MMP-2 activity. A NO donor (S-nitroso-N-acetyl-penicillamine; 500 microM) suppressed SMC migration via the reduction of both total and active MMP-2 levels. Addition of 10 microM MMP-2 inhibitor I to inserts significantly reduced SMC migration. Western blots showed no effect of 4 h of 20 dyn/cm2 SS on SMC production of PDGF-AA, another chemical known to suppress SMC migration. Thus it appears that SS acts to suppress SMC migration by upregulating the cellular production of NO, which in turn inhibits MMP-2 activity.     

DNA microarray study on gene expression profiles in co-cultured endothelial and smooth muscle cells in response to 4- and 24-h shear stress

Shear stress, a major hemodynamic force acting on the vessel wall, plays an important role in physiological processes such as cell growth, differentiation, remodelling, metabolism, morphology, and gene expression. We investigated the effect of shear stress on gene expression profiles in co-cultured vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Human aortic ECs were cultured as a confluent monolayer on top of confluent human aortic SMCs, and the EC side of the co-culture was exposed to a laminar shear stress of 12 dyn/cm² for 4 or 24 h. After shearing, the ECs and SMCs were separated and RNA was extracted from the cells. The RNA samples were labelled and hybridized with cDNA array slides that contained 8694 genes. Statistical analysis showed that shear stress caused the differential expression (p ≤ 0.05) of a total of 1151 genes in ECs and SMCs. In the co-cultured ECs, shear stress caused the up-regulation of 403 genes and down-regulation of 470. In the co-cultured SMCs, shear stress caused the up-regulation of 152 genes and down-regulation of 126 genes. These results provide new information on the gene expression profile and its potential functional consequences in co-cultured ECs and SMCs exposed to a physiological level of laminar shear stress. Although the effects of shear stress on gene expression in monocultured and co-cultured EC are generally similar, the response of some genes to shear stress is opposite between these two types of culture (e.g., ICAM-1 is up-regulated in monoculture and down-regulated in co-culture), which strongly indicates that EC–SMC interactions affect EC responses to shear stress.     

The Adaptive Remodeling of Endothelial Glycocalyx in Response to Fluid Shear Stress

The endothelial glycocalyx is vital for mechanotransduction and endothelial barrier integrity. We previously demonstrated the early changes in glycocalyx organization during the initial 30 min of shear exposure. In the present study, we tested the hypothesis that long-term shear stress induces further remodeling of the glycocalyx resulting in a robust layer, and explored the responses of membrane rafts and the actin cytoskeleton. After exposure to shear stress for 24 h, the glycocalyx components heparan sulfate, chondroitin sulfate, glypican-1 and syndecan-1, were enhanced on the apical surface, with nearly uniform spatial distributions close to baseline levels that differed greatly from the 30 min distributions. Heparan sulfate and glypican-1 still clustered near the cell boundaries after 24 h of shear, but caveolin-1/caveolae and actin were enhanced and concentrated across the apical aspects of the cell. Our findings also suggest the GM₁-labelled membrane rafts were associated with caveolae and glypican-1/heparan sulfate and varied in concert with these components. We conclude that remodeling of the glycocalyx to long-term shear stress is associated with the changes in membrane rafts and the actin cytoskeleton. This study reveals a space- and time- dependent reorganization of the glycocalyx that may underlie alterations in mechanotransduction mechanisms over the time course of shear exposure.     

Heparan sulfate proteoglycan mediates shear stress-induced endothelial gene expression in mouse embryonic stem cell-derived endothelial cells

It has been shown that shear stress plays a critical role in promoting endothelial cell (EC) differentiation from embryonic stem cell (ESC)-derived ECs. However, the underlying mechanisms mediating shear stress effects in this process have yet to be investigated. It has been reported that the glycocalyx component heparan sulfate proteoglycan (HSPG) mediates shear stress mechanotransduction in mature EC. In this study, we investigated whether cell surface HSPG plays a role in shear stress modulation of EC phenotype. ESC-derived EC were subjected to shear stress (5 dyn/cm(2)) for 8 h with or without heparinase III (Hep III) that digests heparan sulfate. Immunostaining showed that ESC-derived EC surfaces contain abundant HSPG, which could be cleaved by Hep III. We observed that shear stress significantly increased the expression of vascular EC-specific marker genes (vWF, VE-cadherin, PECAM-1). The effect of shear stress on expression of tight junction protein genes (ZO-1, OCLD, CLD5) was also evaluated. Shear stress increased the expression of ZO-1 and CLD5, while it did not alter the expression of OCLD. Shear stress increased expression of vasodilatory genes (eNOS, COX-2), while it decreased the expression of the vasoconstrictive gene ET1. After reduction of HSPG with Hep III, the shear stress-induced expression of vWF, VE-cadherin, ZO-1, eNOS, and COX-2, were abolished, suggesting that shear stress-induced expression of these genes depends on HSPG. These findings indicate for the first time that HSPG is a mechanosensor mediating shear stress-induced EC differentiation from ESC-derived EC cells.     

Fluid shear stress stimulates incorporation of hyaluronan into endothelial cell glycocalyx

Vascular endothelial cells are shielded from direct exposure to flowing blood by the endothelial glycocalyx, a highly hydrated mesh of glycoproteins, sulfated proteoglycans, and associated glycosaminoglycans (GAGs). Recent data indicate that the incorporation of the unsulfated GAG hyaluronan into the endothelial glycocalyx is essential to maintain its permeability barrier properties, and we hypothesized that fluid shear stress is an important stimulus for endothelial hyaluronan synthesis. To evaluate the effect of shear stress on glycocalyx synthesis and the shedding of its GAGs into the supernatant, cultured human umbilical vein endothelial cells (i.e., the stable cell line EC-RF24) were exposed to 10 dyn/cm2 nonpulsatile shear stress for 24 h, and the incorporation of [3H]glucosamine and Na2[35S]O4 into GAGs was determined. Furthermore, the amount of hyaluronan in the glycocalyx and in the supernatant was determined by ELISA. Shear stress did not affect the incorporation of 35S but significantly increased the amount of glucosamine-containing GAGs incorporated in the endothelial glycocalyx [168 (SD 17)% of static levels, P < 0.01] and shedded into the supernatant [231 (SD 41)% of static levels, P < 0.01]. Correspondingly with this finding, shear stress increased the amount of hyaluronan in the glycocalyx [from 26 (SD 24) x 10(-4) to 46 (SD 29) x 10(-4) ng/cell, static vs. shear stress, P < 0.05] and in the supernatant [from 28 (SD 11) x 10(-4) to 55 (SD 16) x 10(-4) ng x cell(-1) x h(-1), static vs. shear stress, P < 0.05]. The increase in the amount of hyaluronan incorporated in the glycocalyx was confirmed by a threefold higher level of hyaluronan binding protein within the glycocalyx of shear stress-stimulated endothelial cells. In conclusion, fluid shear stress stimulates incorporation of hyaluronan in the glycocalyx, which may contribute to its vasculoprotective effects against proinflammatory and pro-atherosclerotic stimuli.     

Shear Stress Modulation of Smooth Muscle Cell Marker Genes in 2-D and 3-D Depends on Mechanotransduction by Heparan Sulfate Proteoglycans and ERK1/2

Background

During vascular injury, vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts (FBs/MFBs) are exposed to altered luminal blood flow or transmural interstitial flow. We investigate the effects of these two types of fluid flows on the phenotypes of SMCs and MFBs and the underlying mechanotransduction mechanisms.

Methodology/Principal Findings

Exposure to 8 dyn/cm² laminar flow shear stress (2-dimensional, 2-D) for 15 h significantly reduced expression of α-smooth muscle actin (α-SMA), smooth muscle protein 22 (SM22), SM myosin heavy chain (SM-MHC), smoothelin, and calponin. Cells suspended in collagen gels were exposed to interstitial flow (1 cmH₂O, ∼0.05 dyn/cm², 3-D), and after 6 h of exposure, expression of SM-MHC, smoothelin, and calponin were significantly reduced, while expression of α-SMA and SM22 were markedly enhanced. PD98059 (an ERK1/2 inhibitor) and heparinase III (an enzyme to cleave heparan sulfate) significantly blocked the effects of laminar flow on gene expression, and also reversed the effects of interstitial flow on SM-MHC, smoothelin, and calponin, but enhanced interstitial flow-induced expression of α-SMA and SM22. SMCs and MFBs have similar responses to fluid flow. Silencing ERK1/2 completely blocked the effects of both laminar flow and interstitial flow on SMC marker gene expression. Western blotting showed that both types of flows induced ERK1/2 activation that was inhibited by disruption of heparan sulfate proteoglycans (HSPGs).

Conclusions/Significance

The results suggest that HSPG-mediated ERK1/2 activation is an important mechanotransduction pathway modulating SMC marker gene expression when SMCs and MFBs are exposed to flow. Fluid flow may be involved in vascular remodeling and lesion formation by affecting phenotypes of vascular wall cells. This study has implications in understanding the flow-related mechanobiology in vascular lesion formation, tumor cell invasion, and stem cell differentiation.     

Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells

Platelet-derived growth factor (PDGF), a smooth muscle cell (SMC) mitogen, and heparin-like glycosaminoglycans, known inhibitors of SMC growth and migration, were found to regulate thrombospondin synthesis and matrix deposition by cultured rat aortic SMC. The synthesis and distribution of thrombospondin was examined in growth-arrested SMCs, in PDGF-stimulated SMCs, and in heparin-treated SMCs using metabolic labeling and immunofluorescence techniques. Thrombospondin synthesis in response to purified PDGF occurred within 1 h after addition of growth factor to growth-arrested SMCs, peaked at 2 h, and returned to baseline levels by 5 h. The induction of synthesis of thrombospondin by PDGF was dose dependent, with a maximal effect observed at 2.5 ng/ml. Actinomycin D (2 micrograms/ml) inhibited thrombospondin induction by PDGF, suggesting a requirement for new RNA synthesis. In the presence of heparin and related polyanions, the incorporation of thrombospondin into the SMC extracellular matrix was markedly reduced. This effect was dose dependent with a maximal effect observed at a heparin concentration of 1 microgram/ml. Heparin did not affect the ability of SMCs to synthesize thrombospondin in response to PDGF. We interpret these data to suggest a role for thrombospondin in the SMC proliferative response to PDGF and in the regulation of SMC growth and migration by glycosaminoglycans.     

Dynamic contact forces on leukocyte microvilli and their penetration of the endothelial glycocalyx

We develop a theoretical model to examine the combined effect of gravity and microvillus length heterogeneity on tip contact force (F(m)(z)) during free rolling in vitro, including the initiation of L-, P-, and E-selectin tethers and the threshold behavior at low shear. F (m)(z) grows nonlinearly with shear. At shear stress of 1 dyn/cm(2), F(m)(z) is one to two orders of magnitude greater than the 0.1 pN force for gravitational settling without flow. At shear stresses > 0.2 dyn/cm(2) only the longest microvilli contact the substrate; hence at the shear threshold (0.4 dyn/cm(2) for L-selectin), only 5% of microvilli can initiate tethering interaction. The characteristic time for tip contact is surprisingly short, typically 0.1-1 ms. This model is then applied in vivo to explore the free-rolling interaction of leukocyte microvilli with endothelial glycocalyx and the necessary conditions for glycocalyx penetration to initiate cell rolling. The model predicts that for arteriolar capillaries even the longest microvilli cannot initiate rolling, except in regions of low shear or flow reversal. In postcapillary venules, where shear stress is approximately 2 dyn/cm(2), tethering interactions are highly likely, provided that there are some relatively long microvilli. Once tethering is initiated, rolling tends to ensue because F(m)(z) and contact duration will both increase substantially to facilitate glycocalyx penetration by the shorter microvilli.     

A programmable, computer-controlled cone-plate viscometer for the application of pulsatile shear stress to platelet suspensions

Described is a special purpose cone-plate viscometer that is capable of acceleration or deceleration through a step change in speed in less than 0.7s. The speed of the rotating cone is controlled by a microcomputer which can be programmed to generate speed vs time ramp functions of variable slope. Prior calibration of motor power required to shear Newtonian fluids of known viscosity at various speeds provides the basis for determination of apparent suspension viscosity and enables the viscometer automatically to compensate for changing sample viscosity during shear. The viscometer was used to carry out a series of preliminary studies in which platelet-rich plasma (PRP) was subjected to continuous and pulsatile shear stress at 37 degrees C. Shear-induced platelet aggregation (SIPAG) was significantly greater in response to pulsatile versus continuous shearing except at the lowest applied stress (10 dyn/cm2). Increases ranged from about 40 percent at a stress amplitude of 25 dyn/cm2 to nearly 55 percent at dyn/cm2. This increasing trend with stress amplitude might be interpreted as a positive correlation between SIPAG and the loading rate. Dense granule release, as indicated by serotonin release, was dependent on both stress amplitude and number of pulses even at the higher stress where SIPAG was independent of pulse number.     

Pseudopod projection and cell spreading of passive leukocytes in response to fluid shear stress

Recent evidence suggests that circulating leukocytes respond to physiological levels of fluid shear stress. This study was designed to examine the shear stress response of individual leukocytes adhering passively to a glass surface. Human leukocytes were exposed to a step fluid shear stress with amplitude between 0.2 and 4 dyn/cm(2) and duration between 1 and 20 min. The response of the cells was determined in the form of projected cell area measurements by high-resolution observation before, during, and after fluid shear application. All cells selected initially had a round morphology. After application of fluid shear many cells projected pseudopodia and spread on the glass surface. The number of leukocytes responding with pseudopod projection and the extent of cell spreading increased with increasing amplitude and duration of fluid shear stress. Pseudopod projection after exposure to a step fluid shear occurs following a delay that is insensitive to the shear stress amplitude and duration. Leukocytes that did not project pseudopodia and spread in response to low shear stress could be shown to respond to a second shear step of higher amplitude. The spreading response requires an intact actin network and activated myosin molecules. Depleting the cell glycocalyx with protease treatment enhances the spreading response in sheared leukocytes. These results indicate that passive leukocytes respond to fluid shear stress with active pseudopod projection and cell spreading. This behavior may contribute to cell spreading on endothelium and other cells as well as to transendothelial migration of leukocytes in the microcirculation.     

Recruitment of endothelial caveolae into mechanotransduction pathways by flow conditioning in vitro

The luminal surface of rat lung microvascular endothelial cells in situ is sensitive to changing hemodynamic parameters. Acute mechanosignaling events initiated in response to flow changes in perfused lung microvessels are localized within specialized invaginated microdomains called caveolae. Here we report that chronic exposure to shear stress alters caveolin expression and distribution, increases caveolae density, and leads to enhanced mechanosensitivity to subsequent changes in hemodynamic forces within cultured endothelial cells. Flow-preconditioned cells expressed a fivefold increase in caveolin (and other caveolar-residing proteins) at the luminal surface compared with no-flow controls. The density of morphologically identifiable caveolae was enhanced sixfold at the luminal cell surface of flow-conditioned cells. Laminar shear stress applied to static endothelial cultures (flow step of 5 dyn/cm2), enhanced the tyrosine phosphorylation of luminal surface proteins by 1.7-fold, including caveolin-1 by 1.3-fold, increased Ser1179 phosphorylation of endothelial nitric oxide synthase (eNOS) by 2.6-fold, and induced a 1.4-fold activation of mitogen-activated protein kinases (ERK1/2) over no-flow controls. The same shear step applied to endothelial cells preconditioned under 10 dyn/cm2 of laminar shear stress for 6 h and induced a sevenfold increase of total phosphotyrosine signal at the luminal endothelial cell surface enhanced caveolin-1 tyrosine phosphorylation 5.8-fold and eNOS phosphorylation by 3.3-fold over static control values. In addition, phosphorylated caveolin-1 and eNOS proteins were preferentially localized to caveolar microdomains. In contrast, ERK1/2 activation was not detected in conditioned cells after acute shear challenge. These data suggest that cultured endothelial cells respond to a sustained flow environment by directing caveolae to the cell surface where they serve to mediate, at least in part, mechanotransduction responses.     

Pattern formation of vascular smooth muscle cells subject to nonuniform fluid shear stress: mediation by gradient of cell density

Smooth muscle cells (SMCs) are organized in various patterns in blood vessels. Whereas straight blood vessels mainly contain circumferentially aligned SMCs, curved blood vessels are composed of axially aligned SMCs in regions with vortex blood flow. The vortex flow-dependent feature of SMC alignment suggests a role for nonuniform fluid shear stress in regulating the pattern formation of SMCs. Here, we demonstrate that, in experimental models with vascular polymer implants designed for the observation of neointima formation and SMC migration under defined fluid shear stress, nonuniform shear stress possibly plays a role in regulating the direction of SMC migration and alignment in the neointima of the vascular implant. It was found that fluid shear stress inhibited cell growth, and the presence of nonuniform shear stress influenced the distribution of total cell density and induced the formation of cell density gradients, which in turn directed SMC migration and alignment. In contrast, uniform fluid shear stress in a control model influenced neither the distribution of total cell density nor the direction of SMC migration and alignment. In both the uniform and nonuniform shear models, the gradient of total cell density was consistent with the alignment of SMCs. These observations suggest that nonuniform shear stress may regulate the pattern formation of SMCs, possibly via mediating the gradient of cell density in the neointima of vascular polymer implants.     

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     

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.     

Heparan sulfate proteoglycans mediate interstitial flow mechanotransduction regulating MMP-13 expression and cell motility via FAK-ERK in 3D collagen

Background

Interstitial flow directly affects cells that reside in tissues and regulates tissue physiology and pathology by modulating important cellular processes including proliferation, differentiation, and migration. However, the structures that cells utilize to sense interstitial flow in a 3-dimensional (3D) environment have not yet been elucidated. Previously, we have shown that interstitial flow upregulates matrix metalloproteinase (MMP) expression in rat vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts via activation of an ERK1/2-c-Jun pathway, which in turn promotes cell migration in collagen. Herein, we focused on uncovering the flow-induced mechanotransduction mechanism in 3D.

Methodology/Principal Findings

Cleavage of rat vascular SMC surface glycocalyx heparan sulfate (HS) chains from proteoglycan (PG) core proteins by heparinase or disruption of HS biosynthesis by silencing N-deacetylase/N-sulfotransferase 1 (NDST1) suppressed interstitial flow-induced ERK1/2 activation, interstitial collagenase (MMP-13) expression, and SMC motility in 3D collagen. Inhibition or knockdown of focal adhesion kinase (FAK) also attenuated or blocked flow-induced ERK1/2 activation, MMP-13 expression, and cell motility. Interstitial flow induced FAK phosphorylation at Tyr925, and this activation was blocked when heparan sulfate proteoglycans (HSPGs) were disrupted. These data suggest that HSPGs mediate interstitial flow-induced mechanotransduction through FAK-ERK. In addition, we show that integrins are crucial for mechanotransduction through HSPGs as they mediate cell spreading and maintain cytoskeletal rigidity.

Conclusions/Significance

We propose a conceptual mechanotransduction model wherein cell surface glycocalyx HSPGs, in the presence of integrin-mediated cell-matrix adhesions and cytoskeleton organization, sense interstitial flow and activate the FAK-ERK signaling axis, leading to upregulation of MMP expression and cell motility in 3D. This is the first study to describe a flow-induced mechanotransduction mechanism via HSPG-mediated FAK activation in 3D. This study will be of interest in understanding the flow-related mechanobiology in vascular lesion formation, tissue morphogenesis, cancer cell metastasis, and stem cell differentiation in 3D, and also has implications in tissue engineering.     

Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.     

High Glucose Attenuates Shear-Induced Changes in Endothelial Hydraulic Conductivity by Degrading the Glycocalyx

Diabetes mellitus is a risk factor for cardiovascular disease; however, the mechanisms through which diabetes impairs homeostasis of the vasculature have not been completely elucidated. The endothelium interacts with circulating blood through the surface glycocalyx layer, which serves as a mechanosensor/transducer of fluid shear forces leading to biomolecular responses. Atherosclerosis localizes typically in regions of low or disturbed shear stress, but in diabetics, the distribution is more diffuse, suggesting that there is a fundamental difference in the way cells sense shear forces. In the present study, we examined the effect of hyperglycemia on mechanotranduction in bovine aortic endothelial cells (BAEC). After six days in high glucose media, we observed a decrease in heparan sulfate content coincident with a significant attenuation of the shear-induced hydraulic conductivity response, lower activation of eNOS after exposure to shear, and reduced cell alignment with shear stress. These studies are consistent with a diabetes-induced change to the glycocalyx altering endothelial response to shear stress that could affect the distribution of atherosclerotic plaques.     

CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced endothelial cell migration

The migration of endothelial cells (ECs) plays critical roles in vascular physiology and pathology. The receptors CXCR1 and CXCR2, known as G protein-coupled receptors which are essential for migratory response of ECs toward the shear stress-dependent CXCL8 (interleukin-8), are potential mechano-sensors for mechanotransduction of the hemodynamic forces. In present study, the mRNA and protein expression of CXCR1 and CXCR2 in EA.hy926 cells was detected by RT-PCR and Western blot analysis under three conditions of laminar shear stress (5.56, 10.02 and 15.27 dyn/cm(2)) respectively. Using a scratched-wound assay, the effects of CXCR1 and CXCR2 were assessed by the percentage of wound closure while CXCR1 and CXCR2 were functional blocked by the CXCL8 receptor antibodies. The results showed that the mRNA and protein expression of CXCR1 and CXCR2 was both upregulated by 5.56 dyn/cm(2) laminar shear stress, but was both downregulated by 15.27 dyn/cm(2). The wound closure was inhibited significantly while cells were treated with those antibodies in all the conditions. It was suggested that CXCR1 and CXCR2 are involved in mediating the laminar shear stress-induced EC migration. Taken together, these findings indicated that CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced EC migration. Understanding this expanded mechanism of laminar shear stress-induced cell migration will provide novel molecular targets for therapeutic intervention in cancer and cardiovascular diseases.