Objective-Vascular endothelial cells (ECs) are subjected to shear stress and cytokine stimulation. We studied the interplay between shear stress and cytokine in modulating the expression of adhesion molecule genes in ECs. Methods and Results-Shear stress (20 dynes/cm2 ) was applied to ECs prior to and/or following the addition of tumor necrosis factor (TNF)-␣. Shear stress increased the TNF-␣-induced expression of intercellular adhesion molecule-1 (ICAM-1) at both mRNA and surface protein levels, but decreased the TNF-␣-induced expression of vascular adhesion molecule-1 (VCAM-1) and E-selectin. Transfection studies using promoter reporter gene constructs of ICAM-1, VCAM-1, and E-selectin demonstrated that these shear stress modulations of gene expression occur at the transcriptional levels. After 24-hour preshearing followed by 1 hour of static incubation, the effect of preshearing on TNF-␣-induced ICAM-1 mRNA expression vanished. The recovery of the TNF-␣-induced VCAM-1 and E-selectin mRNA expressions following preshearing, however, required a static incubation time of Ͼ6 hours (complete recovery at 24 hours). Pre-and postshearing caused a reduction in the nuclear factor-B-DNA binding activity induced by TNF-␣ in the EC nucleus. V ascular endothelial cells (ECs) are constantly exposed to fluid shear stress, a tangential force generated by the velocity gradient in viscous fluid flow. The nature and magnitude of shear stress play a significant role in the homeostasis of the structure and function of the blood vessel. Recent evidence suggests that physiological levels of laminar shear stress modulate cellular signaling and EC function and are protective against atherogenesis. 1 In human carotid and coronary arteries, atherosclerotic plaques are found in the vicinity of arterial bifurcations and bends, where the local flow is disturbed. 2 In contrast, regions of artery that experience laminar non-oscillatory shear stress were protected from atherosclerosis. The cytokine tumor necrosis factor-␣ (TNF-␣) is an important mediator of the inflammatory processes that occur during the progression of atherosclerosis. 3 Produced by macrophages that infiltrate the lesion, cytokines such as TNF-␣ are known to induce the expression of many endothelial genes that contribute to the complex processes involved in atherogenesis. 3 Well know examples include the transcriptional regulation of various adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. 3 In contrast to laminar shear stress, cytokines are generally considered to be proatherogenic factors. Conclusions-OurDespite the intensive studies on the effects of fluid shear stress on ECs, the interplay of shear stress and cytokines in modulating EC gene expression and function has not fully been clarified. It has been shown that physiological levels of laminar shear stress inhibit the apoptosis of ECs induced by TNF-␣ and H 2 O 2 . 4,5 This inhibitory effect of shear stress is mediated by signalin...

Vascular endothelial cells (ECs), which exist in close proximity to vascular smooth muscle cells (SMCs), are constantly subjected to blood flow-induced shear stress. Although the effect of shear stress on endothelial biology has been extensively studied, the influence of SMCs on endothelial response to shear stress remains largely unexplored. We examined the potential role of SMCs in regulating the shear stress-induced gene expression in ECs, using a parallel-plate coculture flow system in which these 2 types of cells were separated by a porous membrane. In this coculture system, SMCs tended to orient perpendicularly to the flow direction, whereas the ECs were elongated and aligned IntroductionVascular endothelial cells (ECs), which provide an interface between the blood and the vessel wall, are constantly subjected to hemodynamic forces, including the shear stress imposed by blood flow. Shear stress affects leukocyte-EC interaction and the subsequent leukocyte extravasation into inflamed tissues. 1,2 The effects of fluid shear stress on endothelial biology and gene expression have been extensively studied. [3][4][5][6][7] In addition to its physical influence on leukocyte-EC adhesion, 8,9 shear stress can alter the adhesive properties of ECs by modulating the surface expression of adhesive proteins, for example, intercellular adhesion molecule-1 (ICAM-1), 10-15 vascular cell adhesion molecule-1 (VCAM-1), 10,11,13,14,16,17 and E-selectin. 13,18 Exposure of ECs to laminar flow increases the gene and protein expression of ICAM-1. [10][11][12][13][14][15] Our recent study demonstrated that this shear flow-induced increase in ICAM-1 expression is partially due to an elevation of the levels of intracellular reactive oxygen species (ROS) in ECs. 15 In contrast to ICAM-1, shear stress treatment of ECs has only a minor effect on the surface expression of VCAM-1, or may even cause a reduction. 10,11,13,14,16,17 E-selectin has been reported to be less responsive to laminar shear stress 13 and more responsive to oscillatory flow condition. 18 Most studies on how shear stress affects ECs have been performed by using a cultured EC monolayer as an experimental model. The vessel wall is composed of several types of cells, including ECs, smooth muscle cells (SMCs), and fibroblasts. There is increasing evidence that cell-to-cell interactions can control cellular growth, migration, differentiation, and function. 19 Physical contact between ECs and SMCs via myoendothelial bridges has been demonstrated in vivo 20,21 and may play an important role in cellular communication. Hence, in vitro models for studying ECs need to simulate the in vivo environment by coculturing ECs in close proximity to SMCs. Several coculture models have recently been developed. [22][23][24][25][26] Ziegler et al 22 designed a coculture model that includes SMCs, ECs, and a matrix of collagen gel. They demonstrated that ECs cocultured with SMCs aligned with the flow direction more rapidly than ECs grown on plastic. Redmond et al 23 developed a system in whic...

Atherosclerosis is commonly appreciated to represent a chronic inflammatory response of the vascular wall, and its complications cause high mortality in patients. Angioplasty with stent replacement is commonly performed in patients with atherosclerotic disease. However, the restenosis usually has a high incidence rate in angioplasty patients. Although the pathophysiological mechanisms underlying atherosclerosis and restenosis have been well established, new signaling molecules that control the progress of these pathologies have continuously been discovered. MicroRNAs (miRs) have recently emerged as a novel class of gene regulators that work via transcriptional degradation and translational inhibition or activation. Over 30% of genes in the cell can be directly regulated by miRs. Thus, miRs are recognized as crucial regulators in normal development, physiology and pathogenesis. AIterations of miR expression profiles have been revealed in diverse vascular diseases. A variety of functions of vascular cells, such as cell differentiation, contraction, migration, proliferation and inflammation that are involved in angiogenesis, neointimal formation and lipid metabolism underlying various vascular diseases, have been found to be regulated by miRs. This review summarizes current research progress and knowledge on the roles of miRs in regulating vascular cell function in atherosclerosis and restenosis. These discoveries are expected to present opportunities for clinical diagnostic and therapeutic approaches in vascular diseases resulting from atherosclerosis and restenosis.

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Vascular endothelial cells (ECs) and smooth muscle cells (VSMCs) are constantly exposed to haemodynamic forces, including blood flow-induced fluid shear stress and cyclic stretch from blood pressure. These forces modulate vascular cell gene expression and function and, therefore, influence vascular physiology and pathophysiology in health and disease. Epigenetics, including DNA methylation, histone modification/chromatin remodelling and RNA-based machinery, refers to the study of heritable changes in gene expression that occur without changes in the DNA sequence. The role of haemodynamic force-induced epigenetic modifications in the regulation of vascular gene expression and function has recently been elucidated. This review provides an introduction to the epigenetic concepts that relate to vascular physiology and pathophysiology. Through the studies of gene expression, cell proliferation, angiogenesis, migration and pathophysiological states, we present a conceptual framework for understanding how mechanical force-induced epigenetic modifications work to control vascular gene expression and function and, hence, the development of vascular disorders. This research contributes to our knowledge of how the mechanical environment impacts the chromatin state of ECs and VSMCs and the consequent cellular behaviours.

Rationale In atherosclerotic lesions, synthetic smooth muscle cells (sSMCs) induce aberrant microRNA (miR) profiles in endothelial cells (ECs) under flow stagnation. Increase in shear stress induces favorable miR modulation to mitigate sSMC-induced inflammation. Objective To address the role of miRs in sSMC-induced EC inflammation and its inhibition by shear stress. Methods and Results Co-culturing ECs with sSMCs under static condition causes initial increases of four anti-inflammatory miRs (146a/708/451/98) in ECs followed by decreases below basal levels at 7 days; the increases for miR-146a/708 peaked at 24 h and those for miR-451/98 lasted for only 6-12 h. Shear stress (12 dynes/cm2) to co-cultured ECs for 24 h augments these four miR expressions. In vivo, these four miRs are highly expressed in neointimal ECs in injured arteries under physiological levels of flow, but not expressed under flow stagnation. MiR-146a, -708, -451, and -98 target interleukin (IL)-1 receptor-associated kinase, inhibitor of nuclear factor-κB (NF-κB) kinase subunit-γ, IL-6 receptor, and conserved helix-loop-helix ubiquitous kinase, respectively, to inhibit NF-κB signaling, which exerts negative feedback control on the biogenesis of these miRs. NF-E2-related factor-2 (Nrf-2) is critical for shear-induction of miR-146a in co-cultured ECs. Silencing either Nrf-2 or miR-146a led to increased neointima formation of injured rat carotid artery under physiological levels of flow. Overexpressing miR-146a inhibits neointima formation of rat or mouse carotid artery induced by injury or flow cessation. Conclusions Nrf-2-mediated miR-146a expression is augmented by atheroprotective shear stress in ECs adjacent to sSMCs to inhibit neointima formation of injured arteries.

Objectives-Vascular endothelial cells (ECs) are influenced by shear stress and neighboring smooth muscle cells (SMCs).We investigated the inflammation-relevant gene expression in EC/SMC cocultures under static condition and in response to shear stress. Materials and Methods-Under static condition, DNA microarrays and reverse-transcription polymerase chain reaction identified 23 inflammation-relevant genes in ECs whose expression was significantly affected by coculture with SMCs, with 18 upregulated and 5 downregulated. Application of shear stress (12 dynes/cm 2 ) to the EC side of the coculture for 6 hours inhibited most of the proinflammatory gene expressions in ECs induced by coculture with SMCs. Inhibition of nuclear factor-B (NF-B) activation by the p65-antisense, lactacystin, and N-acetyl-cysteine blocked the cocultureinduced EC expression of proinflammatory genes, indicating that the NF-B binding sites in the promoters of these genes play a significant role in their expression as a result of coculture with SMCs. Chromatin immunoprecipitation assays demonstrated the in vivo regulation of NF-B recruitment to selected target promoters. Shear stress inhibited the SMC coculture-induced NF-B activation in ECs and monocytic THP-1 cell adhesion to ECs. The interactions between these cells play significant roles in the homeostasis of the structure and function of the blood vessel. As an interface between the blood and the vessel wall, ECs occupy a unique location directly exposed to blood flow-induced shear stress, which can influence interactions between ECs and SMCs. By incorporating the SMCs into a matrix of collagen gel, Ziegler et al 1 demonstrated that ECs cocultured with SMCs aligned with the flow direction more rapidly than the EC monocultures. Imberti et al 2 further demonstrated that the EC response to shear stress in this collagen coculture model was influenced by preconditioning the SMC-seeded collagen gel with cyclic strain. Redmond et al 3 have developed a system in which ECs and SMCs were cocultured on opposite sides of porous polypropylene capillary tubes. A series of research studies have been conducted using this coculture system, which represents a significant advance over homogeneous culture. 3-5 Nackman et al 6 and Rainger and Nash 7 constructed a system by combining a parallel plate flow chamber and a coculture module in which ECs and SMCs were grown on opposite sides of a 10-mthick permeable membrane containing 0.4-m pores. They demonstrated that the EC/SMC coculture affected SMC proliferation 6 and the EC response to tumor necrosis factor-␣ (TNF-␣). 7 Although these studies have contributed to the understanding of the effects of hemodynamic forces on the EC-SMC interactions and functional modulations, there is a lack of systematic analysis of the alteration of gene expression program of ECs and SMCs as a result of their coculture under static condition or in response to shear stress. Conclusions-OurIn the present study, we applied the high-throughput DNA microarray technology to investigate ...