Role of integrins in cellular responses to mechanical stress and adhesion.

Mechanical stresses are important environmental cues for both normal cellular functions and pathophysiological changes in conditions such as cardiac hypertrophy and atherosclerosis. There is increasing evidence that mechanotransduction processes in response to mechanical stresses share many common features with processes in cell adhesion, such as an increase in tyrosine phosphorylation of proteins in the focal adhesion sites. Recent findings suggest that integrins may function as mechanotransducers in cells.

Shear stress induction of the tissue factor gene.

Using flow channel, we report that the application of a laminar shear stress induced a transient increase of tissue factor (TF) procoagulant activity in human umbilical vein endothelial cells (HUVEC), which was accompanied by a rapid and transient induction of the TF mRNA in the HUVEC. Functional analysis of the 2.2 kb TF 5' promoter indicated that a GC-rich region containing three copies each of the EGR-1 and Sp1 sites was required for induction. Mutation of the Sp1 sites, but not the EGR-1 sites, attenuated the response of TF promoter to shear stress. Thus, Sp1 is a newly defined shear stress responsive element. Electrophoretic mobility shift assays showed there was no increase in binding of nuclear extracts from sheared cells to an Sp1 consensus site. In contrast, immunoblotting of these nuclear extracts with antibody against transcription factor Sp1 demonstrated that shear stress increased the phosphorylation of Sp1. We also showed that shear stress, like the phosphatase inhibitor okadaic acid, increased the transcriptional activity of Sp1. These findings suggest that the shear stress induction of TF gene expression is mediated through an increased Sp1 transcriptional activity with a concomitant hyperphosphorylation of Sp1.

Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress.

Shear stress, the tangential component of hemodynamic forces, plays an important role in endothelial remodeling. In this study, we investigated the role of Rho family GTPases Cdc42 and Rho in shear stress-induced signal transduction and cytoskeleton reorganization. Our results showed that shear stress induced the translocation of Cdc42 and Rho from cytosol to membrane. Although both Cdc42 and Rho were involved in the shear stress-induced transcription factor AP-1 acting on the 12-O-tetradecanoyl-13-phorbol-acetate-responsive element (TRE), only Cdc42 was sufficient to activate AP-1/TRE. Dominant-negative mutants of Cdc42 and Rho, as well as recombinant C3 exoenzyme, attenuated the shear stress activation of c-Jun NH2-terminal kinases (JNKs), suggesting that Cdc42 and Rho regulate the shear stress induction of AP-1/TRE activity through JNKs. Shear stress-induced cell alignment and stress fiber formation were inhibited by the dominant-negative mutants of Rho and p160ROCK, but not by the dominant-negative mutant of Cdc42, indicating that the Rho-p160ROCK pathway regulates the cytoskeletal reorganization in response to shear stress.

The Ras-JNK pathway is involved in shear-induced gene expression.

Hemodynamic forces play a key role in inducing atherosclerosis-implicated gene expression in vascular endothelial cells. To elucidate the signal transduction pathway leading to such gene expression, we studied the effects of fluid shearing on the activities of upstream signaling molecules. Fluid shearing (shear stress, 12 dynes/cm2 [1 dyne = 10(-5)N]) induced a transient and rapid activation of p21ras and preferentially activated c-Jun NH2 terminal kinases (JNK1 and JNK2) over extracellular signal-regulated kinases (ERK-1 and ERK-2). Cotransfection of RasN17, a dominant negative mutant of Ha-Ras, attenuated the shear-activated JNK and luciferase reporters driven by 12-O-tetradecanoylphorbol-13-acetate-responsive elements. JNK(K-R) and MEKK(K-M), the respective catalytically inactive mutants of JNK1 and MEKK, also partially inhibited the shear-induced luciferase reporters. In contrast, Raf301, ERK(K71R), and ERK(K52R), the dominant negative mutants of Raf-1, ERK-1, and ERK-2, respectively, had little effect on the activities of these reporters. The activation of JNK was also correlated with increased c-Jun transcriptional activity, which was attenuated by a negative mutant of Son of sevenless. Thus, mechanical stimulation exerted by fluid shearing activates primarily the Ras-MEKK-JNK pathway in inducing endothelial gene expression.

LDL-activated p38 in endothelial cells is mediated by Ras.

Endothelial dysfunction is a major atherogenic proinflammatory event. LDL causes the activation and phenotypic changes of cultured vascular endothelial cells (ECs). We previously reported that LDL activates c-Jun and AP-1 in ECs. In this study, we demonstrated that p38-ATF-2 is activated by LDL in human ECs and that this activation is mediated by Ras. When ECs are incubated with LDL in pathophysiological concentrations, the p38-mediated ATF-2 phosphorylation and ATF-2 transactivation are increased in a time- and dose-dependent manner. To elucidate the upstream mechanism in LDL-activated p38 in ECs, we demonstrate that LDL increases Ras translocation from the cytoplasm to the cellular membrane, with concurrent increases in Ras binding activity to GST-Raf-1. Overexpression of RasN17, a dominant negative mutant of Ras, attenuates the LDL-induced increases in (1) phosphorylation of ATF-2, (2) phosphorylation of c-Jun, (3) AP-1 binding, and (4) AP-1-driven luciferase activity. To study the effect of p38 in the regulation of an LDL targeting gene, we show that a specific p38 inhibitor attenuates LDL-induced E-selectin at the mRNA level. Thus, LDL activates both p38 and JNK signaling pathways through Ras activation, and furthermore, these events may play an important role in LDL-induced endothelial activation.

DNA microarray analysis of gene expression in endothelial cells in response to 24-h shear stress.

The recently developed DNA microarray technology provides a powerful and efficient tool to rapidly compare the differential expression of a large number of genes. Using the DNA microarray approach, we investigated gene expression profiles in cultured human aortic endothelial cells (HAECs) in response to 24 h of laminar shear stress at 12 dyn/cm(2). This relatively long-term shearing of cultured HAECs led to the modulation of the expression of a number of genes. Several genes related to inflammation and EC proliferation were downregulated, suggesting that 24-h shearing may keep ECs in a relatively noninflammatory and nonproliferative state compared with static cells. Some genes were significantly upregulated by the 24-h shear stress; these includes genes involved in EC survival and angiogenesis (Tie2 and Flk-1) and vascular remodeling (matrix metalloproteinase 1). These results provide information on the profile of gene expression in shear-adapted ECs, which is the case for the native ECs in the straight part of the aorta in vivo.

Biomechanical regulation of matrix metalloproteinase-9 in cultured chondrocytes.

Abnormal mechanical loading of joints may induce degeneration of articular cartilage. Shear stress is one mode of mechanical loading that may regulate chondrocyte metabolism. We investigated the mechanism by which shear stress induces the gene encoding matrix metalloproteinase-9, a mediator of the progressive degradation of articular cartilage in osteoarthritis. In vitro experiments using passaged rabbit chondrocytes in monolayer culture subjected to a shear stress of 16 dyn/cm2 (1.6 Pa) in a flow channel showed increased expression of the matrix metalloproteinase-9 gene. The induction of matrix metalloproteinase-9 appeared to depend on a region in the 5' promoter of the gene that contains a 12-0-tetradecanoylphorbol 13-acetate-responsive element. Transfection experiments using a construct containing a luciferase reporter driven by a 12-0-tetradecanoylphorbol 13-acetate-responsive element indicated that shear stress activated a 12-0-tetradecanoylphorbol 13-acetate-responsive element-mediated transcription in chondrocytes. Similar experiments showed that shear stress induced a matrix metalloproteinase-9 promoter construct (matrix metalloproteinase-9-luciferase). Shear stress activated c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38. Transfection of matrix metalloproteinase-9-luciferase together with the dominant negative mutant of c-Jun NH2-terminal kinase, but not with that of extracellular signal-regulated kinase or p38, attenuated the shear-induced matrix metalloproteinase-9 promoter activity. In addition, transfection of constructs encoding dominant negative mutants of Ras, Rac, and Cdc42 attenuated the induction of c-Jun transcriptional activity by shear stress. Thus. shear stimulation of chondrocytes stimulates Ras, Rac, and Cdc42, which subsequently activate c-Jun NH2-terminal kinase to induce a 12-0-tetradecanoylphorbol 13-acetate-responsive element-mediated expression of matrix metalloproteinase-9.

Mechanical compression modulates proliferation of transplanted chondrocytes.

The presence of an appropriate number of reparative cells in an articular cartilage defect is probably necessary for consistent and successful repair. Following the transplantation of chondrocytes into a defect, cell proliferation may modulate local defect cellularity. Transplanted cells can be compressed during cartilage repair as a result of joint-loading or press-fitting a graft into a cartilage defect. The objective of this study was to characterize the proliferative response of chondrocytes after attachment to cartilage and application of static compressive stress between cartilaginous surfaces in an ex vivo model. The chondrocytes were isolated from adult bovine cartilage, cultured in high-density monolayer, resuspended, and then transplanted onto the surface of devitalized cartilage at a density of 250,000 cells/cm2. The total DNA content of transplanted cell layers increased steadily to a plateau by 5 days and represented a 4-fold increase in cell number during incubation in medium including serum and ascorbate. Over the culture period, the level of DNA synthesis ([3H]thymidine incorporation), on a per cell basis, decreased steadily (88% between days 0 and 6). The application of 24 hours of static compressive stress (0.06-0.4 MPa) to the adherent cells at 1 and 4 days after transplantation inhibited overall DNA synthesis by 70-approximately 87% compared with unloaded controls. After release from load, cell proliferation generally remained at low levels. The marked proliferation of chondrocytes when attached to cartilage without applied load and the inhibition of this proliferation by relatively low-amplitude static compressive stress may be relevant to the occasional overgrowth of tissue in some chondrocyte transplantation procedures. The dosimetry of these effects suggests that the in vivo mechanical environment may have a marked effect on proliferation of transplanted chondrocytes.

Multiple cis-elements mediate shear stress-induced gene expression.

Fluid shear stress activates the expression of immediate early (IE) genes in vascular endothelial cells. The transcriptional regulation can be mediated through the shear stress-sensitive cis-acting elements at the 5' promoter regions of various IE genes such as the monocyte chemotactic protein-1 (MCP-1) gene. We linked wild-type and mutated MCP-1 promoters to the reporter gene luciferase and used such constructs to investigate the role of the phorbol ester TPA responsive element (TRE) in the shear-induced MCP-1 gene expression in vascular endothelial cells. Functional analysis showed that TGACTCC (a divergent TRE) located at nt -54 to -60 is necessary for shear-inducibility in bovine aortic endothelial cells (BAEC). The induction of the wild-type MCP-1 promoter construct by shear stress was attenuated by pretreating the cells with 1 microM dexamethasone or 1 microM retinoic acid 12 h before the shear stress experiments. The induction by shear stress reduced from 13-fold in the untreated cells to 7- and 3-folds in the dexamethasone- and retinoic acid-treated cells, respectively. These results demonstrate that the glucocorticoid receptor and retinoic acid receptor may interfere with the shear stress-activated AP-1/TRE. The reporter activity of HIV(LTR), which is a plasmid construct of the long terminal repeats of the human immunodeficiency virus and contains a kappa B enhancer element, was also activated by shear stress. The results of our investigations indicate that the shear stress-induced IE gene expression can be mediated through multiple cis-elements.

Mechanotransduction in endothelial responses to shear stress: review of work in Dr. Chien's laboratory.

Shear stresses play an important role in vascular biology in health and disease. While disturbed flows with low shear stresses in the bends and bifurcations of the arterial tree are atherogenic, laminar flows with high shear stresses in the straight part of the vessel is atheroresistant. Thus, elucidation of the mechanotransduction mechanism in vascular endothelial cells in response to shear stress has become an important research topic among bioengineers and vascular biologists. Here is a summary of studies performed in Dr. Shu Chien's laboratory on shear stress-induced signal transduction and gene expression during the period from 1992-1999. These studies, together with efforts from other research groups, demonstrate that integrins, which are transmembrane molecules that interact with both extracellular matrices and intracellular cytoskeleton and kinases in the focal adhesions, are important in mechanotransduction. This hypothesis is mainly supported by the similarity between cellular and molecular events elicited by shear stress and those activated during the integrin-mediated cell attachment to extracellular matrices. Evidence is also provided to show that the dynamic and specific interaction between integrin and extracellular matrices is essential for mechanotransduction.

The cis-acting phorbol ester "12-O-tetradecanoylphorbol 13-acetate"-responsive element is involved in shear stress-induced monocyte chemotactic protein 1 gene expression.

Vascular endothelial cells, serving as a barrier between vessel and blood, are exposed to shear stress in the body. Although endothelial responses to shear stress are important in physiological adaption to the hemodynamic environments, they can also contribute to pathological conditions--e.g., in atherosclerosis and reperfusion injury. We have previously shown that shear stress mediates a biphasic response of monocyte chemotactic protein 1 (MCP-1) gene expression in vascular endothelial cells and that the regulation is at the transcriptional level. These observations led us to functionally analyze the 550-bp promoter region of the MCP-1-encoding gene to define the cis element responding to shear stress. The shear stress/luciferase assay on the deletion constructs revealed that a 38-bp segment (-53 to -90 bp relative to the transcription initiation site) containing two divergent phorbol ester "12-O-tetradecanoylphorbol 13-acetate" (TPA)-responsive elements (TRE) is critical for shear inducibility. Site-specific mutations on these two sites further demonstrated that the proximal one (TGACTCC) but not the distal one (TCACTCA) was shear-responsive. Shear inducibility was lost after the mutation or deletion of the proximal site. This molecular mechanism of shear inducibility of the MCP-1 gene was functional in both the epithelial-like HeLa cells and bovine aortic endothelial cells (BAEC). In a construct with four copies of the TRE consensus sequences TGACTACA followed by the rat prolactin minimal promoter and luciferase gene, shear stress induced the reporter activities by 35-fold and 7-fold in HeLa cells and BAEC, respectively. The application of shear stress on BAEC also induced a rapid and transient phosphorylation of mitogen-activated protein kinases. Pretreatment of BAEC with TPA attenuated the shear-induced mitogen-activated protein kinase phosphorylation, suggesting that shear stress and TPA share a similar signal transduction pathway in activating cells. The present study provides a molecular basis for the transient induction of MCP-1 gene by shear stress.

Nitric oxide regulates monocyte chemotactic protein-1.

BACKGROUND: Monocyte chemotactic protein-1 (MCP-1) is a 76-amino-acid chemokine thought to be the major chemotactic factor for monocytes. We and others have demonstrated that NO inhibits monocyte-endothelial cell interactions and atherogenesis. We hypothesize that the antiatherogenic effect of NO may be due in part to its inhibition of MCP-1 expression. METHODS AND RESULTS: Smooth muscle cells (SMCs) were isolated from normal rabbit aortas by the explant method. Cells were then exposed to LPS (10 microg/mL), native LDL, or oxidized LDL (30 microg/mL) for 6 hours. The expression of MCP-1 in SMCs and chemotactic activity in the conditioned medium were induced by lipopolysaccharide (LPS) or by oxidized LDL but not native LDL. The induction of MCP-1 by cytokines or oxidized lipoproteins was associated with an increased generation of superoxide anion by the SMCs and increased activity of the transcriptional protein nuclear factor-kappaB (NFkappaB). The induced expression of MCP-1 and activation of NFkappaB were reduced by previous exposure of the SMCs to the NO donor DETA-NONOate (100 micromol/L) (P<.05). To determine whether NO exerted its effect at a transcriptional level, SMCs and COS cells were transfected with a 400-bp fragment of the MCP-1 promoter. Promoter activity was enhanced by oxidized LDL, and LPS was inhibited by DETA-NO. Nuclear run-on assays confirmed that the effect of NO occurred at a transcriptional level. To investigate the role of endogenous NO in the regulation of MCP-1 in vivo, New Zealand White rabbits were fed normal chow, normal chow plus nitro-L-arginine (LNA), high-cholesterol diet (Chol), or high-cholesterol diet supplemented with L-arginine (Arg). After 2 weeks, thoracic aortas were harvested and total RNA was isolated. Northern analysis using full-length MCP-1 cDNA demonstrated increased expression in Chol and LNA aortas; this expression was decreased in aortas from Arg animals. CONCLUSIONS: These studies indicate that the antiatherogenic effect of NO may be mediated in part by its inhibition of MCP-1 expression.

Sustained JNK activation induces endothelial apoptosis: studies with colchicine and shear stress.

The disruption of microtubules by treating bovine aortic endothelial cells with 10(-7)-10(-5) M colchicine caused apoptosis, as evidenced by DNA laddering and TdT-mediated dUTP nick end labeling fluorescence staining. Colchicine treatment also induced a sustained activation of c-Jun NH(2)-terminal kinase (JNK) that lasted for >/=12 h. The blockade of JNK activity by using the negative interfering mutant JNK(K-R) markedly decreased the apoptosis induced by colchicine. Exposure of bovine aortic endothelial cells to laminar shear stress (12 dyn/cm(2)) caused a transient (<2 h) activation of JNK, and there was no induction of apoptosis. The sustained activation of JNK may play a significant role in the apoptosis induced by colchicine.

A strain device imposing dynamic and uniform equi-biaxial strain to cultured cells.

The objective of this study is to design a new apparatus to allow the control of the magnitude and frequency of dynamic stretch applied uniformly to cells cultured on a silicon elastic membrane. The apparatus is designed to produce equi-biaxial dynamic stretches with area changes ranging from 0% to 55% and frequencies ranging from 0 to 2 Hz. Homogeneous finite strain analysis using triangles of markers was performed to compute the symmetric two-dimensional Lagrangian strain tensor on the membrane. Measurements of strain in both static and dynamic conditions showed that the shear component of the strain tensor (Erc) was near zero, and that there was no significant difference between radial (Err) and circumferential (Ecc) components, indicating the attainment of equi-biaxial strain. Bovine aortic endothelial cells were transiently transfected with a chimeric construct in which the luciferase reporter is driven by TPA-responsive elements (TRE). The transfected cells cultured on the membrane were stretched. The luciferase activity increased significantly only when the cells were stretched by 15% or more in area. Cells in different locations of the membrane showed similar induction of luciferase activities, confirming that strain is uniform and equi-biaxial across the membrane.

Effects of active and negative mutants of Ras on rat arterial neointima formation.

BACKGROUND: Ras protein is a key signal transducer in the cause of cell proliferation. We studied the effects of active and negative mutants of the Ras gene on arterial neointimal formation in rats, with the aim of elucidating the molecular mechanisms regulating restenosis following percutaneous transluminal coronary angioplasty. MATERIALS AND METHODS: AdRasV12 and AdRasN17, the recombinant adenoviruses containing a constitutively active mutant and a dominant negative mutant of Ras, respectively, were used to determine whether Ras is necessary and sufficient to modulate the smooth muscle cell proliferation and neointima formation. Following balloon injury, rat common carotid arteries were treated in their distal half with AdRasV12, AdRasN17, or AdLacZ, with the proximal half used as uninfected control. RESULTS: In rat arteries subjected to balloon injury, either uninfected or treated with AdLacZ, there were pronounced SMC proliferation and neointima formation. These changes were markedly augmented by AdRasV12 and reduced by AdRasN17. CONCLUSION: Ras is necessary and sufficient for SMC proliferation and neointima formation and may play a critical role in restenosis following balloon angioplasty.

Mechanotransduction in response to shear stress. Roles of receptor tyrosine kinases, integrins, and Shc.

Shear stress, the tangential component of hemodynamic forces, activates many signal transduction pathways in vascular endothelial cells. The conversion of mechanical stimulation into chemical signals is still unclear. We report here that shear stress (12 dynes/cm2) induced a rapid and transient tyrosine phosphorylation of Flk-1 and its concomitant association with the adaptor protein Shc; these are accompanied by a concurrent clustering of Flk-1, as demonstrated by confocal microscopy. Our results also show that shear stress induced an association of alphavbeta3 and beta1 integrins with Shc, and an attendant association of Shc with Grb2. These associations are sustained, in contrast to the transient Flk-1. Shc association in response to shear stress and the transient association between alphavbeta3 integrin and Shc caused by cell attachment to substratum. Shc-SH2, an expression plasmid encoding the SH2 domain of Shc, attenuated shear stress activation of extracellular signal-regulated kinases and c-Jun N-terminal kinases, and the gene transcription mediated by the activator protein-1/12-O-tetradecanoylphorbol-13-acetate-responsive element complex. Our results indicate that receptor tyrosine kinases and integrins can serve as mechanosensors to transduce mechanical stimuli into chemical signals via their association with Shc.

Fluid shear stress activation of IkappaB kinase is integrin-dependent.

Vascular endothelial cells (ECs), forming a boundary between the circulating blood and the vessel wall, are constantly subjected to fluid shear stress due to blood flow. The aim of this study was to determine the role of the recently identified IkappaB kinases (IKKs) in shear stress activation of NF-kappaB and to elucidate the upstream signaling mechanism that mediates IKK activation. Our results demonstrate that IKKs in ECs are activated by shear stress in a rapid and transient manner. This IKK activation is followed by IkappaB degradation and NF-kappaB translocation into the nucleus. Transfection of plasmids encoding catalytic inactive mutants of IKKs, i.e. hemagglutinin (HA)-IKKalpha(K44M) and HA-IKKbeta(K44A), inhibits shear stress-induced NF-kappaB translocation. In addition, constructs encoding antisense IKKs, i.e. HA-IKKalpha(AS) and HA-IKKbeta(AS), attenuate shear stress induction of a promoter driven by the kappaB enhancer element. Preincubation of the EC monolayer with a monoclonal anti-alphavbeta3 integrin antibody (clone LM609) attenuates shear stress induction of IKK. Inhibition of tyrosine kinases by genistein causes a similar down-regulating effect. These results suggest that the integrin-mediated signaling pathway regulates NF-kappaB through IKKs in ECs in response to shear stress.

Shear stress activates p60src-Ras-MAPK signaling pathways in vascular endothelial cells.

The aim of this study was to elucidate the upstream signaling mechanism that mediates the fluid shear stress activation of mitogen-activated protein kinases (MAPKs), including c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinases (ERKs), in vascular endothelial cells (ECs). Our results indicate that p60src is rapidly activated by fluid shear stress in bovine aortic endothelial cells (BAECs). Shear stress induction of the hemagglutinin (HA) epitope-tagged HA-JNK1 and the Myc epitope-tagged Myc-ERK2 was significantly attenuated by v-src(K295R) and c-src(K295R), the kinase-defective mutants ofv-src and c-src, respectively. HA-JNK1 and Myc-ERK2 were activated by c-src(F527), a constitutively activated form of p60src, and the activation was abolished by RasN17, a dominant-negative mutant of p2lras. In contrast, although HA-JNK1 and Myc-ERK2 were also activated by RasL61, an activated form of p21ras, the activation was not affected by v-src(K295R). These results indicate that p60src is upstream to the Ras-JNK and Ras-ERK pathways in response to shear stress. The shear stress inductions of the promoters of monocyte chemotactic protein-1 (MCP-1) and c-fos, driven by TPA-responsive element (TRE) and serum-responsive element (SRE), respectively, were attenuated by v-src(K295R). This attenuation is associated with decreased transcriptional activities of c-Jun and Elk-1, the transcription factors targeting TRE and SRE, respectively. Thus, p60src plays a critical role in the shear stress activation of MAPK pathways and induction of Activating Protein-1 (AP- 1)/TRE and Elk-1/SRE-mediated transcription in ECs.

Reactive oxygen species are involved in shear stress-induced intercellular adhesion molecule-1 expression in endothelial cells.

Vascular endothelial cells (ECs) are constantly subjected to flow-induced shear stress. Although the effects of shear stress on ECs are well known, the intracellular signal mechanisms remain largely unclear. Reactive oxygen species (ROS) have recently been suggested to act as intracellular second messengers. The potential role of ROS in shear-induced gene expression was examined in the present study by subjecting ECs to a shear force using a parallel-plate flow chamber system. ECs under shear flow increased their intracellular ROS as indicated by superoxide production. This superoxide production was maintained at an elevated level as shear flow remained. Sheared ECs, similar to TNF(alpha)-, PMA-, or H2O2-treated cells, increased their intercellular adhesion molecule-1 (ICAM-1) mRNA levels in a time-dependent manner. Pretreatment of ECs with an antioxidant, N-acetyl-cysteine (NAC) or catalase, inhibited this shear-induced or oxidant-induced ICAM-1 expression. ROS that were involved in the shear-induced ICAM-1 gene expression were further substantiated by functional analysis using a chimera containing the ICAM-1 promoter region (-850 bp) and the reporter gene luciferase. Shear-induced promoter activities were attenuated by pretreating sheared ECs with NAC and catalase. Flow cytometric analysis and monocytic adhesion assay confirmed the inhibitory effect of NAC and catalase on the shear-induced ICAM-1 expression on ECs. These results clearly demonstrate that shear flow to ECs can induce intracellular ROS generation that may result in an increase of ICAM-1 mRNA levels via transcriptional events. Our findings thus support the importance of intracellular ROS in modulating hemodynamically induced endothelial responses.