Advances in endothelial shear stress proteomics

The vascular endothelium lining the luminal surface of all blood vessels is constantly exposed to shear stress exerted by the flowing blood. Blood flow with high laminar shear stress confers protection by activation of antiatherogenic, antithrombotic and anti-inflammatory proteins, whereas low or oscillatory shear stress may promote endothelial dysfunction, thereby contributing to cardiovascular disease. Despite the usefulness of proteomic techniques in medical research, however, there are relatively few reports on proteome analysis of cultured vascular endothelial cells employing conditions that mimic in vivo shear stress attributes. This review focuses on the proteome studies that have utilized cultured endothelial cells to identify molecular mediators of shear stress and the roles they play in the regulation of endothelial function, and their ensuing effect on vascular function in general. It provides an overview on current strategies in shear stress-related proteomics and the key proteins mediating its effects which have been characterized so far.     

Individual and combined effects of shear stress magnitude and spatial gradient on endothelial cell gene expression

The apparent tendency of atherosclerotic lesions to form in complex blood flow environments has led to many theories regarding the importance of hemodynamic forces in endothelium-mediated atherosusceptibility. The effects of shear stress magnitude and spatial shear stress gradient on endothelial cell gene expression in vitro were examined in this study. Converging-width flow channels were designed to impose physiological ranges of shear stress gradient and magnitude on porcine aortic endothelial cells, and real-time quantitative PCR was performed to evaluate their expression of five genes of interest. Although vascular cell adhesion molecule-1 expression was insensitive to either variable, each of the remaining genes exhibited a unique dependence on shear stress magnitude and gradient. Endothelial nitric oxide synthase showed a strong positive dependence on magnitude but was insensitive to gradient. The expression of c-jun was weakly correlated with magnitude and gradient, without an interaction effect. Monocyte chemoattractant protein-1 expression varied inversely with gradient and also depended on the interaction of gradient with magnitude. Intercellular adhesion molecule-1 expression also exhibited an interaction effect, and increased with shear magnitude. These results support the notion that vascular endothelial cells are able to sense shear gradient and magnitude independently.     

Influence of TNF-α and biomechanical stress on endothelial anti- and prothrombotic genes

Biomechanical stress modulates vascular tone, vascular remodelling and the spatial localisation of atherosclerotic plaques. Inflammatory cytokines, such as TNF-α, regulate expression of genes that impair the function of endothelial cells. This study investigates the combinatory effect of different biomechanical stresses and TNF-α on the expression of endothelial anti- and prothrombotic genes. Human umbilical vein endothelial cells were exposed to TNF-α and different levels of static/pulsatile tensile stress or shear stress. The response in endothelial cells to TNF-α was not modulated by tensile stress. However, shear stress was a more potent stimulus. Shear stress counteracted the cytokine-induced expression of VCAM-1, and the cytokine-suppressed expression of thrombomodulin and eNOS. Shear stress and TNF-α additively induced PAI-1, whereas shear stress blocked the cytokine effect on t-PA and u-PA. A flow profile characterized by high laminar shear stress seems to render the endothelial cell more resistant to inflammatory stress.     

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.     

KLF2-dependent, shear stress-induced expression of CD59: a novel cytoprotective mechanism against complement-mediated injury in the vasculature

Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress led us to explore its influence on endothelial cell expression of complement inhibitory proteins CD59 and decay-accelerating factor. Human umbilical vein and aortic endothelial cells were exposed to laminar shear stress (12 dynes/cm(2)) or disturbed flow (+/- 5 dynes/cm(2) at 1Hz) in a parallel plate flow chamber. Laminar shear induced a flow rate-dependent increase in steady-state CD59 mRNA, reaching 4-fold at 12 dynes/cm(2). Following 24-48 h of laminar shear stress, cell surface expression of CD59 was up-regulated by 100%, whereas decay-accelerating factor expression was unchanged. The increase in CD59 following laminar shear was functionally significant, reducing C9 deposition and complement-mediated lysis of flow-conditioned endothelial cells by 50%. Although CD59 induction was independent of PI3-K, ERK1/2 and nitric oxide, an RNA interference approach demonstrated dependence upon an ERK5/KLF2 signaling pathway. In contrast to laminar shear stress, disturbed flow failed to induce endothelial cell CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional laminar shear stress, when compared with atheroprone areas exposed to disturbed flow. We propose that up-regulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement-mediated injury and protects from atherogenesis in regions of laminar shear stress.     

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in ¹ and ²). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells ^1,3,4 and in vivo in larger animals and humans ^1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. ⁹ introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery ¹⁰. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics ¹¹ as confirmed by in vivo Magnetic Resonance Imaging (MRI) ¹²: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions ¹¹. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.     

Molecular and mechanical bases of focal lipid accumulation in arterial wall

Mechanical forces such as shear stress can modulate gene and protein expressions and hence cellular functions by activating membrane sensors and intracellular signaling. Using cultured endothelial cells, we have shown that laminar shear stress causes a transient increase in monocyte chemotactic protein-1 (MCP-1) expression, which involves the Ras-MAP kinase signaling pathway. We have demonstrated that integrins and the vascular endothelial growth factor receptor Flk-1 can sense shear stress, with integrins being upstream to Flk-1. Other possible membrane components involved in the sensing of shear stress include G-protein coupled receptors, intercellular junction proteins, membrane glycocalyx, and the lipid bilayer. Mechano-transduction involves the participation of a multitude of sensors, signaling molecules, and genes. Microarray analysis has demonstrated that shear stress can upregulate and downregulate different genes. Sustained shear stress downregulates atherogenic genes (e.g., MCP-1 and the genes that facilitate lipid accumulation) and upregulates growth-arrest genes. In contrast, disturbed flow observed at branch points and simulated in step-flow channels causes sustained activation of MCP-1 and the genes facilitating cell turnover and lipid accumulation. These findings provide a molecular basis for the explanation of the preferential localization of atherosclerotic lesions at regions of disturbed flow, such as the arterial branch points. The combination of mechanics and biology (from molecules-cells to organs-systems) can help to elucidate the physiological processes of mechano-chemical transduction and improving the methods of the management of important clinical conditions such as coronary artery disease.     

Shear Stress and Atherosclerosis

Hemodynamic shear stress, the frictional force acting on vascular endothelial cells, is crucial for endothelial homeostasis under normal physiological conditions. When discussing blood flow effects on various forms of endothelial (dys)function, one considers two flow patterns: steady laminar flow and disturbed flow because endothelial cells respond differently to these flow types both in vivo and in vitro. Laminar flow which exerts steady laminar shear stress is atheroprotective while disturbed flow creates an atheroprone environment. Emerging evidence has provided new insights into the cellular mechanisms of flow-dependent regulation of vascular function that leads to cardiovascular events such as atherosclerosis, atherothrombosis, and myocardial infarction. In order to study effects of shear stress and different types of flow, various models have been used. In this review, we will summarize our current views on how disturbed flow-mediated signaling pathways are involved in the development of atherosclerosis.     

Role of shear stress direction in endothelial mechanotransduction

Fluid shear stress due to blood flow can modulate functions of endothelial cells (ECs) in blood vessels by activating mechano-sensors, signaling pathways, and gene and protein expressions. Laminar shear stress with a definite forward direction causes transient activations of many genes that are atherogenic, followed by their down-regulation; laminar shear stress also up-regulates genes that inhibit EC growth. In contrast, disturbed flow patterns with little forward direction cause sustained activations of these atherogenic genes and enhancements of EC mitosis and apoptosis. In straight parts of the arterial tree, laminar shear stress with a definite forward direction has anti-atherogenic effects. At branch points, the complex flow patterns with little net direction are atherogenic. Thus, the direction of shear stress has important physiological and pathophysiological effects on vascular ECs.     

Proliferation, differentiation, and tube formation by endothelial progenitor cells in response to shear stress.

Endothelial progenitor cells (EPCs), circulating in peripheral blood, migrate toward target tissue, differentiate, and contribute to the formation of new vessels. In this study, we report that shear stress generated by blood flow or tissue fluid flow can accelerate the proliferation, differentiation, and capillary-like tube formation of EPCs. When EPCs cultured from human peripheral blood were subjected to laminar shear stress, the cells elongated and oriented their long axes in the direction of flow. The cell density of the EPCs exposed to shear stress was higher, and a larger percentage of these cells were in the G2-M phase of the cell cycle, compared with EPCs cultured under static conditions. Shear stress markedly increased the EPC expression of two vascular endothelial growth factor receptors, kinase insert domain-containing receptor and fms-like tyrosine kinase-1, and an intercellular adhesion molecule, vascular endothelial-cadherin, at both the protein and mRNA levels. Assays for tube formation in the collagen gels showed that the shear-stressed EPCs formed tubelike structures and developed an extensive tubular network significantly faster than the static controls. These findings suggest that EPCs are sensitive to shear stress and that their vasculogenic activities may be modulated by shear stress.     

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.     

Hypo- and Hyperglycemia Impair Endothelial Cell Actin Alignment and Nitric Oxide Synthase Activation in Response to Shear Stress

Uncontrolled blood glucose in people with diabetes correlates with endothelial cell dysfunction, which contributes to accelerated atherosclerosis and subsequent myocardial infarction, stroke, and peripheral vascular disease. In vitro, both low and high glucose induce endothelial cell dysfunction; however the effect of altered glucose on endothelial cell fluid flow response has not been studied. This is critical to understanding diabetic cardiovascular disease, since endothelial cell cytoskeletal alignment and nitric oxide release in response to shear stress from flowing blood are atheroprotective. In this study, porcine aortic endothelial cells were cultured in 1, 5.55, and 33 mM D-glucose medium (low, normal, and high glucose) and exposed to 20 dynes/cm² shear stress for up to 24 hours in a parallel plate flow chamber. Actin alignment and endothelial nitric oxide synthase phosphorylation increased with shear stress for cells in normal glucose, but not cells in low and high glucose. Both low and high glucose elevated protein kinase C (PKC) levels; however PKC blockade only restored actin alignment in high glucose cells. Cells in low glucose instead released vascular endothelial growth factor (VEGF), which translocated β-catenin away from the cell membrane and disabled the mechanosensory complex. Blocking VEGF in low glucose restored cell actin alignment in response to shear stress. These data suggest that low and high glucose alter endothelial cell alignment and nitric oxide production in response to shear stress through different mechanisms.     

HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells

Reendothelialization involves endothelial progenitor cell (EPC) homing, proliferation, and differentiation, which may be influenced by fluid shear stress and local flow pattern. This study aims to elucidate the role of laminar flow on embryonic stem (ES) cell differentiation and the underlying mechanism. We demonstrated that laminar flow enhanced ES cell-derived progenitor cell proliferation and differentiation into endothelial cells (ECs). Laminar flow stabilized and activated histone deacetylase 3 (HDAC3) through the Flk-1-PI3K-Akt pathway, which in turn deacetylated p53, leading to p21 activation. A similar signal pathway was detected in vascular endothelial growth factor-induced EC differentiation. HDAC3 and p21 were detected in blood vessels during embryogenesis. Local transfer of ES cell-derived EPC incorporated into injured femoral artery and reduced neointima formation in a mouse model. These data suggest that shear stress is a key regulator for stem cell differentiation into EC, especially in EPC differentiation, which can be used for vascular repair, and that the Flk-1-PI3K-Akt-HDAC3-p53-p21 pathway is crucial in such a process.     

Endothelial cell morphometry of atherosclerotic lesions and flow profiles at aortic bifurcations in cholesterol fed rabbits.

Observations on shapes of endothelial cells both in sudanophilic and nonsudanophilic regions at bifurcations of the brachiocephalic (BC) and left subclavian (SA) arteries in hyperlipidemic rabbits were performed under a SEM. The stagnation point of flow and leading edges of flow dividers were nonsudanophilic and covered by round and long fusiform endothelial cells, respectively. The hips of flow dividers of both branchings, proven to be relatively low shear stress regions, by movement of microspheres in steady flow, were sudanophilic and covered by ellipsoidal cells. Similar studies were carried out in normolipidemic rabbits. It might be concluded that lipid deposition in hyperlipidemic rabbits occurs in relatively low shear stress regions, where endothelial cells are functionally activated, rather than in laminar high shear stress regions at the flow divider.     

Molecular basis of rheological modulation of endothelial functions: importance of stress direction

Vascular endothelial cells (EC) play significant roles in regulating circulatory functions. Shear stress and stretch can modulate EC functions by activating mechano-sensors, signaling pathways, and gene and protein expressions. Laminar shear stress with a significant forward direction causes transient activations of monocyte chemotactic protein-1 (MCP-1), sterol response element binding protein (SREBP), and proliferative genes. Sustained laminar shear stress down-regulates these genes and up-regulates genes that inhibit EC growth. In EC subjected to complex flow patterns with little forward direction, activations of MCP-1, SREBP, and proliferation genes become sustained, and mitosis and apoptosis are enhanced. Cyclic uniaxial stretch causes actin stress fibers to orient perpendicular to stretch direction, with a consequent reduction of intracellular stress, transient JNK activation, and protection of EC against apoptosis. Cyclic biaxial stretch without a preferred direction has opposite effects. In the straight part of arterial tree, laminar shear stress with a net forward direction and uniaxial strain in the circumferential direction have anti-atherogenic effects. At vascular branch points, the complex shear flow and mechanical strain with little net direction are atherogenic. Therefore, the direction of stress has important influences on the biorheological effects of flow and deformation on vascular endothelium in health and disease.     

Endothelial function and coronary artery disease

The endothelium produces a number of vasodilator and vasoconstrictor substances that not only regulate vasomotor tone, but also the recruitment and activity of inflammatory cells and the propensity towards thrombosis. Endothelial vasomotor function is a convenient way to assess these other functions, and is related to the long-term risk of cardiovascular disease. Lipids (particularly low density lipoprotein cholesterol) and oxidant stress play a major role in impairing these functions, by reducing the bioavailability of nitric oxide and activating pro-inflammatory signalling pathways such as nuclear factor kappa B. Biomechanical forces on the endothelium, including low shear stress from disturbed blood flow, also activate the endothelium increasing vasomotor dysfunction and promoting inflammation by upregulating pro-atherogenic genes. In contrast, normal laminar shear stress promotes the expression of genes that may protect against atherosclerosis. The sub-cellular structure of endothelial cells includes caveolae that play an integral part in regulating the activity of endothelial nitric oxide synthase. Low density lipoprotein cholesterol and oxidant stress impair caveolae structure and function and adversely affect endothelial function. Lipid-independent pathways of endothelial cell activation are increasingly recognized, and may provide new therapeutic targets. Endothelial vasoconstrictors, such as endothelin, antagonize endothelium-derived vasodilators and contribute to endothelial dysfunction. Some but not all studies have linked certain genetic polymorphisms of the nitric oxide synthase enzyme to vascular disease and impaired endothelial function. Such genetic heterogeneity may nonetheless offer new insights into the variability of endothelial function.     

Low shear stress regulates monocyte adhesion to oxidized lipid-induced endothelial cells via an IkappaBalpha dependent pathway

In regions of a vessel that experience low shear stress and reversing flow patterns, early features in the pathogenesis of atherosclerosis include the accumulation of oxidized LDL (OxLDL) and adhesion of monocytes to endothelial cells (EC). Here we investigated the hypothesis that low shear stress (2 dyn/cm2) and OxLDL are synergistic for enhanced expression of vascular cell adhesion molecule (VCAM-1) and human aortic endothelial cell (HAEC)-monocyte adhesion. This study shows low shear stress can significantly reduce IkappaBalpha levels, activate NF-kappaB, increase the expression of VCAM-1 in HAEC and binding of monocytes. OxLDL itself cannot significantly increase the expression of VCAM-1 in HAEC and binding of monocytes, but through activation of NF-kappaB and degradation of IkappaBalpha induced by low shear stress it can significantly enhance VCAM-1 expression and monocyte adhesion, over that in unmodified LDL or control. These results suggest that low shear stress can regulate monocyte adhesion to oxidized lipid-induced endothelial cells via an IkappaBalpha-dependent pathway, and that low shear stress together with OxLDL may likely play an important role in atherogenesis.