Depletion of plasma membrane cholesterol dampens hydrostatic pressure and shear stress-induced mechanotransduction pathways in osteoblast cultures

The preferential association of cholesterol and sphingolipids within plasma membranes forms organized compartments termed lipid rafts. Addition of caveolin proteins to this lipid milieu induces the formation of specialized invaginated plasma membrane structures called caveolae. Both lipid rafts and caveolae are purported to function in vesicular transport and cell signaling. We and others have shown that disassembly of rafts and caveolae through depletion of plasma membrane cholesterol mitigates mechanotransduction processes in endothelial cells. Because osteoblasts are subjected to fluid-mechanical forces, we hypothesize that cholesterol-rich plasma membrane microdomains also serve the mechanotransduction process in this cell type. Cultured human fetal osteoblasts were subjected to either sustained hydrostatic pressure or laminar shear stress using a pressure column or parallel-plate apparatus, respectively. We found that sustained hydrostatic pressure induced protein tyrosine phosphorylation, activation of extracellular signal-regulated kinase (ERK)1/2, and enhanced expression of c-fos in both time- and magnitude-dependent manners. Similar responses were observed in cells subjected to laminar shear stress. Both sustained hydrostatic pressure- and shear stress-induced signaling were significantly reduced in osteoblasts pre-exposed to either filipin or methyl--cyclodextrin. These mechanotransduction responses were restored on reconstitution of lipid rafts and caveolae, which suggests that cholesterol-rich plasma membrane microdomains participate in the mechanotransduction process in osteoblasts. In addition, mechanical force-induced phosphoproteins were localized within caveolin-containing membranes. These data support the concept that lipid rafts and caveolae serve a general function as cell surface mechanotransduction sites within the plasma membrane. lipid rafts; caveolae; extracellular signal-regulated kinase     

Fluid shear stress‐induced JNK activity leads to actin remodeling for cell alignment

Fluid shear stress (FSS) exerted on endothelial cell (EC) surfaces induces actin cytoskeleton remodeling through mechanotransduction. This study was designed to determine whether FSS activates Jun N‐terminal kinase (JNK), to examine the spatial and temporal distribution of active JNK relative to the actin cytoskeleton in ECs exposed to different FSS conditions, and to evaluate the effects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm² FSS induced higher activity levels of JNK than the lower 2 and 4 dyn/cm² flow conditions. At the higher FSS treatments, JNK activity increased with increasing exposure time, peaking 30 min after flow onset with an eightfold activity increase compared to cells in static culture. FSS‐induced phospho‐JNK co‐localized with actin filaments at cell peripheries, as well as with stress fibers. Pharmacologically blocking JNK activity altered FSS‐induced actin structure and distribution as a response to FSS. Our results indicate that FSS‐induced actin remodeling occurs in three phases, and that JNK plays a role in at least one, suggesting that this kinase activity is involved in mechanotransduction from the apical surface to the actin cytoskeleton in ECs. J. Cell. Physiol. 226: 110–121, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae

The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH(2) terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 mug/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.     

Endothelial actin cytoskeleton remodeling during mechanostimulation with fluid shear stress

Fluid shear stress stimulation induces endothelial cells to elongate and align in the direction of applied flow. Using the complementary techniques of photoactivation of fluorescence and fluorescence recovery after photobleaching, we have characterized endothelial actin cytoskeleton dynamics during the alignment process in response to steady laminar fluid flow and have correlated these results to motility. Alignment requires 24 h of exposure to fluid flow, but the cells respond within minutes to flow and diminish their movement by 50%. Although movement slows, the actin filament turnover rate increases threefold and the percentage of total actin in the polymerized state decreases by 34%, accelerating actin filament remodeling in individual cells within a confluent endothelial monolayer subjected to flow to levels used by dispersed nonconfluent cells under static conditions for rapid movement. Temporally, the rapid decrease in filamentous actin shortly after flow stimulation is preceded by an increase in actin filament turnover, revealing that the earliest phase of the actin cytoskeletal response to shear stress is net cytoskeletal depolymerization. However, unlike static cells, in which cell motility correlates positively with the rate of filament turnover and negatively with the amount polymerized actin, the decoupling of enhanced motility from enhanced actin dynamics after shear stress stimulation supports the notion that actin remodeling under these conditions favors cytoskeletal remodeling for shape change over locomotion. Hours later, motility returned to pre-shear stress levels but actin remodeling remained highly dynamic in many cells after alignment, suggesting continual cell shape optimization. We conclude that shear stress initiates a cytoplasmic actin-remodeling response that is used for endothelial cell shape change instead of bulk cell translocation. atherosclerosis; cytoskeletal dynamics; endothelial cells; mechanotransduction     

Endothelial glycocalyx as a critical signalling platform integrating the extracellular haemodynamic forces and chemical signalling

The glycocalyx covers the human mammalian cells and plays important roles in stroke, inflammation and atherosclerosis. It has also been shown to be involved in endothelial mechanotransduction of shear stress. Shear stress induces the remodelling of the major component of the glycocalyx including glypican‐1, a cell membrane heparan sulphate proteoglycan. Other factors, such as sphingosine‐1‐phosphate (S1P), protect the glycocalyx against syndecan‐1 ectodomain shedding and induce the synthesis of heparan sulphate. In this study, we reviewed the role of shear stress and S1P in glycocalyx remodelling and revealed that the glycocalyx is a critical signalling platform, integrating the extracellular haemodynamic forces and chemical signalling, such as S1P, for determining the fate of endothelial cells and vascular diseases. This review integrated our current understanding of the structure and function of the glycocalyx and provided new insight into the role of the glycocalyx that might be helpful for investigating the underlying biological mechanisms in certain human diseases, such as atherosclerosis.     

Membrane tension regulates syndecan-1 expression through actin remodelling

BackgroundThe endothelial glycocalyx, located at the interface of vascular lumen, is a carbohydrate-rich complex that controls vascular functions such as solute permeation and mechanotransduction. It anchors to the cell membrane through core proteins, e.g. syndecan-1, which couple to the actin cytoskeleton. Membrane tension plays an important role in the reorganisation of membrane-bound proteins, however, little is known on the effect of the membrane tension on the various components of the glycocalyx.MethodsHypo-osmotic stress is used to investigate the effect of the membrane tension on syndecan-1 expression.ResultsFollowing 20 min exposure to hypo-osmotic medium, the expression of syndecan-1 in the endothelial glycocalyx layer is reduced to 84.7 ± 3.6% (255 mOsm) and 64.7 ± 2.1% (167 mOsm). This reduction, however, is transient and partial recovery is observed at the end of 2 h exposure to the hypo-osmotic medium. The transient reduction of syndecan-1 is associated with depolymerisation of the actin cytoskeleton. Further examination of the effect of actin manipulation reveals that actin depolymerisation by cytochalasin D results in sustained syndecan-1 reduction. In contrast, stabilising actin using jasplakinolide abolishes the transient reduction of syndecan-1completely.ConclusionsWe demonstrate, for the first time, that membrane tension plays an important role in the regulation of syndecan-1 expression and this effect is mediated by the reorganisation of the actin cytoskeleton.General significanceFindings in this study suggest a new venue of research on the protective role of the glycocalyx in vascular pathophysiology and diseases.     

Mapping the dynamics of shear stress-induced structural changes in endothelial cells

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.     

Structural alteration of the endothelial glycocalyx: contribution of the actin cytoskeleton

The endothelial glycocalyx is a carbohydrate–protein layer that lines the luminal surface of the endothelium. It anchors to the cell membrane via its core proteins that share extended link to the actin cytoskeleton. It is widely accepted that those protein domains and the attached carbohydrates are susceptible to pathological changes. It is unclear, however, to what extent the actin cytoskeleton contributes to the glycocalyx stability. In this study, we investigate the role of the actin cytoskeleton in the maintenance of the glycocalyx under static and laminar flow conditions in vitro. Our results show that in the static culture medium neither rapid actin depolymerisation nor prolonged actin disturbance leads to glycocalyx disruption from the apical surface of human umbilical vein endothelial cells. However, when endothelial cells are exposed to laminar flow for 24 h, the glycocalyx is seen to shift to the downstream peripheral region of the cell surface. The mean fluorescence intensity decreases to \(91.9 \pm 2.5\%\) of the control. When actin depolymerisation is introduced, the intensity decreases significantly to \(54.7 \pm 1.3\%\), indicating a severe disruption of the glycocalyx. Similar changes are observed in human aortic endothelial cells, where the intensity of the glycocalyx is reduced to \(72.8 \pm 1.6\%\) of the control. Collectively, we demonstrate that the actin cytoskeleton contributes to structural stability of the glycocalyx under shear stress. Our results can be used to develop new strategies to prevent shedding of the glycocalyx in cardiovascular diseases.     

Endostatin associates with lipid rafts and induces reorganization of the actin cytoskeleton via down-regulation of RhoA activity

Endostatin, the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Observations that endostatin inhibits endothelial cell migration and induces disassembly of the actin cytoskeleton provide putative cellular mechanisms for this effect. To understand the mechanisms of endostatin-induced intracellular signaling, we analyzed the association of recombinant endostatin with endothelial cell lipid rafts and the roles of its heparin- and integrin-binding properties in this interaction. We observed that a fraction of cell surface-bound endostatin partitioned in low density membrane raft fractions together with caveolin-1. Heparinase treatment of cells prevented the recruitment of endostatin to the lipid rafts but did not affect the association of endostatin with the non-raft fraction, whereas preincubation of endostatin with soluble alpha5beta1 integrin prevented the association of endostatin with the endothelial cell membrane. Endostatin treatment induced recruitment of alpha5beta1 integrin into the raft fraction via a heparan sulfate proteoglycan-dependent mechanism. Subsequently, through alpha5beta1 integrin, heparan sulfate, and lipid raft-mediated interactions, endostatin induced Src-dependent activation of p190RhoGAP with concomitant decrease in RhoA activity and disassembly of actin stress fibers and focal adhesions. These observations provide a cell biological mechanism, which plausibly explains the anti-angiogenic mechanisms of endostatin in vivo.     

Vascular smooth muscle cell glycocalyx influences shear stress-mediated contractile response.

This study addressed the influence of the rate of shear stress application on aortic smooth muscle cell (SMC) contraction and the role of specific glycosaminoglycans in this mechanotransduction. Rat aortic SMCs were exposed to either a step increase in shear stress (0 to 25 dyn/cm(2)) or a ramp increase in shear stress (0 to 25 dyn/cm(2) over 5 min) in a parallel plate flow chamber, and cell contraction was characterized by cell area reduction. SMCs contracted at levels similar to those reported previously and equally in response to both a step and ramp increase in shear stress. When the cells were pretreated with heparinase III or chondroitinase ABC to remove the glycosaminoglycans heparan sulfate and chondroitin sulfate, respectively, from the glycocalyx, the contraction response to increases in shear stress was significantly inhibited. These studies indicate that specific components of the SMC glycocalyx play an important role in the mechanotransduction of shear stress into a contractile response and that the rate of application of shear stress does not affect the SMC contraction.     

Insulin-induced endothelial cell cortical actin filament remodeling: a requirement for trans-endothelial insulin transport

Insulin's trans-endothelial transport (TET) is critical for its metabolic action on muscle and involves trafficking of insulin bound to its receptor (or at high insulin concentrations, the IGF-I receptor) via caveolae. However, whether caveolae-mediated insulin TET involves actin cytoskeleton organization is unknown. Here we address whether insulin regulates actin filament organization in bovine aortic endothelial cells (bAEC) and whether this affects insulin uptake and TET. We found that insulin induced extensive cortical actin filament remodeling within 5 min. This remodeling was inhibited not only by disruption of actin microfilament organization but also by inhibition of phosphatidylinositol 3-kinase (PI3K) or by disruption of lipid rafts using respective specific inhibitors. Knockdown of either caveolin-1 or Akt using specific small interfering RNA also eliminated the insulin-induced cortical actin filament remodeling. Blocking either actin microfilament organization or PI3K pathway signaling inhibited both insulin uptake and TET. Disruption of actin microfilament organization also reduced the caveolin-1, insulin receptor, and IGF-I receptor located at the plasma membrane. Exposing bAEC for 6 h to either TNFα or IL-6 blocked insulin-induced cortical actin remodeling. Extended exposure (24 h) also inhibited actin expression at both mRNA and protein levels. We conclude that insulin-induced cortical actin filament remodeling in bAEC is required for insulin's TET in a PI3K/Akt and plasma membrane lipid rafts/caveolae-dependent fashion, and proinflammatory cytokines TNFα and IL-6 block this process.     

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.     

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.     

Interaction of membrane/lipid rafts with the cytoskeleton: Impact on signaling and function : Membrane/lipid rafts,mediators of cytoskeletal arrangement and cell signaling

The plasma membrane in eukaryotic cells contains microdomains that are enriched in certain glycosphingolipids, gangliosides, and sterols (such as cholesterol) to form membrane/lipid rafts (MLR). These regions exist as caveolae, morphologically observable flask-like invaginations, or as a less easily detectable planar form. MLR are scaffolds for many molecular entities, including signaling receptors and ion channels that communicate extracellular stimuli to the intracellular milieu. Much evidence indicates that this organization and/or the clustering of MLR into more active signaling platforms depends upon interactions with and dynamic rearrangement of the cytoskeleton. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to MLR and help regulate lateral diffusion of membrane proteins and lipids in response to extracellular events (e.g., receptor activation, shear stress, electrical conductance, and nutrient demand). MLR regulate cellular polarity, adherence to the extracellular matrix, signaling events (including ones that affect growth and migration), and are sites of cellular entry of certain pathogens, toxins and nanoparticles. The dynamic interaction between MLR and the underlying cytoskeleton thus regulates many facets of the function of eukaryotic cells and their adaptation to changing environments. Here, we review general features of MLR and caveolae and their role in several aspects of cellular function, including polarity of endothelial and epithelial cells, cell migration, mechanotransduction, lymphocyte activation, neuronal growth and signaling, and a variety of disease settings. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Glycocalyx modulates the motility and proliferative response of vascular endothelium to fluid shear stress

Flow-induced mechanotransduction in vascular endothelial cells has been studied over the years with a major focus on putative connections between disturbed flow and atherosclerosis. Recent studies have brought in a new perspective that the glycocalyx, a structure decorating the luminal surface of vascular endothelium, may play an important role in the mechanotransduction. This study reports that modifying the amount of the glycocalyx affects both short-term and long-term shear responses significantly. It is well established that after 24 h of laminar flow, endothelial cells align in the direction of flow and their proliferation is suppressed. We report here that by removing the glycocalyx by using the specific enzyme heparinase III, endothelial cells no longer align under flow after 24 h and they proliferate as if there were no flow present. In addition, confluent endothelial cells respond rapidly to flow by decreasing their migration speed by 40% and increasing the amount of vascular endothelial cadherin in the cell-cell junctions. These responses are not observed in the cells treated with heparinase III. Heparan sulfate proteoglycans (a major component of the glycocalyx) redistribute after 24 h of flow application from a uniform surface profile to a distinct peripheral pattern with most molecules detected above cell-cell junctions. We conclude that the presence of the glycocalyx is necessary for the endothelial cells to respond to fluid shear, and the glycocalyx itself is modulated by the flow. The redistribution of the glycocalyx also appears to serve as a cell-adaptive mechanism by reducing the shear gradients that the cell surface experiences.     

Participation of caveolae in beta1 integrin-mediated mechanotransduction

We previously reported that caveolin-1 is a key component in a beta1 integrin-dependent mechanotransduction pathway suggesting that caveolae organelles and integrins are functionally linked in their mechanotransduction properties. Here, we exposed BAEC monolayers to shear stress then isolated caveolae vesicles form the plasma membrane. While little beta1 integrin was detected in caveolae derived from cells kept in static culture, shear stress induced beta1 integrin transposition to the caveolae. To evaluate the significance of shear-induced beta1 integrin localization to caveolae, cells were pretreated with cholesterol sequestering compounds or caveolin-1 siRNA to disrupt caveolae structural domains. Cholesterol depletion attenuated integrin-dependent caveolin-1 phosphorylation, Src activation and Csk association with beta1 integrin. Reduction of both caveolin-1 protein and membrane cholesterol inhibited downstream shear-induced, integrin-dependent phosphorylation of myosin light chain. Taken together with our previous findings, the data supports the concept that beta1 integrin-mediated mechanotransduction is mediated by caveolae domains.     

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.     

Fluid Flow-induced Soluble Vascular Endothelial Growth Factor Isoforms Regulate Actin Adaptation in Osteoblasts

Although load-induced mechanical signals play a key role in bone formation and maintenance of bone mass and structure, the cellular mechanisms involved in the translation of these signals are still not well understood. Recent identification of a novel flow-induced mechanosignaling pathway involving VEGF in osteoblasts and the known VEGF regulation of actin reorganization in various cell types has led us to hypothesize that fluid shear stress-induced Vegf up-regulation underlies the actin cytoskeleton adaptation observed in osteoblasts during mechanotransduction. Our results show that MC3T3-E1 cells secrete significant VEGF in response to 5 h of pulsatile fluid shear stress (PFSS; 5 dynes/cm² at 1 Hz), whereas expression of VEGF receptors (VEGFR-1, VEGFR-2, or NRP1) is unaffected. These receptors, in particular VEGFR-2, participate in PFSS-induced VEGF release. Exposure to flow-conditioned medium or exogenous VEGF significantly induces stress fiber formation in osteoblasts that is comparable with PFSS-induced stress fiber formation, whereas VEGF knockdown abrogates this response to PFSS, thereby providing evidence that flow-induced VEGF release plays a role in actin polymerization. Using neutralizing antibodies against the receptors and VEGF isoforms, we found that soluble VEGFs, in particular VEGF₁₆₄, play a crucial role in transient stress fiber formation during osteoblast mechanotransduction, most likely through VEGFR-2 and NRP1. Based on these data we conclude that flow-induced VEGF release from osteoblasts regulates osteoblast actin adaptation during mechanotransduction and that VEGF paracrine signaling may provide potent cross-talk among bone cells and endothelial cells that is essential for fracture healing, bone remodeling, and osteogenesis.     

Shear stress-induced redistribution of the glycocalyx on endothelial cells in vitro

The glycocalyx is the inner most layer of the endothelium that is in direct contact with the circulating blood. Shear stress affects its synthesis and reorganization. This study focuses on changes in the spatial distribution of the glycocalyx caused by shear stimulation and its recovery following the removal of the shear stress. Sialic acid components of the glycocalyx on human umbilical vain endothelial cells are observed using confocal microscopy. The percentage area of the cell membrane covered by the glycocalyx, as well as the average fluorescence intensity ratio between the apical and edge areas of the cell is used to assess the spatial distribution of the glycocalyx on the cell membrane. Our results show that following 24 h shear stimulation, the glycocalyx relocates near the edge of endothelial cells (i.e., cell–cell junction regions). Following the removal of the shear stress, the glycocalyx redistributes and gradually appears in the apical region of the cell membrane. This redistribution is faster in the early hours ( $<$ 4 h) after shear stimulation than that in the later stage (e.g., between 8 and 24 h). We further investigate the recovery of the glycocalyx after its enzyme degradation under either static or shear flow conditions. Our results show that following 24 h recovery under shear flow, the glycocalyx reappears predominantly near the edge of endothelial cells. Static and shear flow conditions result in notable changes in the spatial recovery of the glycocalyx, but the difference is not statistically significant. We hypothesize that newly synthesized glycocalyx is not structurally well developed. Its weak interaction with flow results in less than significant redistribution, contrary to what has been observed for a well-developed glycocalyx layer.