Vascular endothelial responses to altered shear stress: pathologic implications for atherosclerosis.

Atherosclerosis preferentially develops at branches and curvatures of the arterial tree, where blood flow is disturbed from a laminar pattern, and wall shear stress is non-uniform and has an irregular distribution. Vascular endothelial cells (ECs), which form an interface between the flowing blood and the vessel wall, are exposed to blood flow-induced shear stress. There is increasing evidence suggesting that laminar blood flow and sustained high shear stress modulate the expression of EC genes and proteins that function to protect against atherosclerosis; in contrast, disturbed blood flow and the associated low and reciprocating shear stress upregulate proatherosclerotic genes and proteins that promote development of atherosclerosis. Understanding of the effects of shear stress on ECs will provide mechanistic insights into its role in the pathogenesis of atherosclerosis. The aim of this review article is to summarize current findings on the effects of shear stress on ECs, in terms of their signal transduction, gene expression, structure, and function. These endothelial cellular responses have important relevance to understanding the pathophysiological effects of altered shear stress associated with atherosclerosis and thrombosis and their complications.

Mechanisms of induction of endothelial cell E-selectin expression by smooth muscle cells and its inhibition by shear stress.

E-selectin is a major adhesion molecule expressed by endothelial cells (ECs), which are exposed to shear stress and neighboring smooth muscle cells (SMCs). We investigated the mechanisms underlying the modulation of EC E-selectin expression by SMCs and shear stress. SMC coculture induced rapid and sustained increases in expression of E-selectin and phosphorylation of interleukin-1 (IL-1) receptor-associated kinase glycoprotein-130, as well as the downstream mitogen-activated protein kinases (MAPKs) and Akt. By using specific inhibitors, dominant-negative mutants, and small interfering RNA, we demonstrated that activations of c-Jun-NH(2)-terminal kinase (JNK) and p38 of the MAPK pathways are critical for the coculture-induced E-selectin expression. Gel shifting and chromatin immunoprecipitation assays showed that SMC coculture increased the nuclear factor-kappaB (NF-kappaB)-promoter binding activity in ECs; inhibition of NF-kappaB activation by p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced E-selectin promoter activity. Protein arrays and blocking assays using neutralizing antibodies demonstrated that IL-1beta and IL-6 produced by EC/SMC cocultures are major contributors to the coculture induction of EC signaling and E-selectin expression. Preshearing of ECs at 12 dynes/cm(2) inhibited the coculture-induced EC signaling and E-selectin expression. Our findings have elucidated the molecular mechanisms underlying the SMC induction of EC E-selectin expression and the shear stress protection against this SMC induction.

Directional shear flow and Rho activation prevent the endothelial cell apoptosis induced by micropatterned anisotropic geometry.

To study the roles of anisotropic cell morphology and directionality of mechanical force in apoptosis, the spreading of human umbilical vein endothelial cells (HUVECs) was constrained by growing on micropatterned (MP) strips of fibronectin (FN, 20 microg/cm2) with widths of 15, 30, and 60 microm on silicone membrane. Cells on 30- and 60-microm strips, like cells on a nonpatterned (NP) surface coated with FN, showed clear actin stress fibers with anchoring spots of phosphorylated focal adhesion kinase (p-FAK) and no significant apoptosis. On 15-microm strips, cells had few stress fibers, no p-FAK, and significant apoptosis. After seeding for 12 h, the cells were subjected to pulsatile shear stress (12+/-4 dyn/cm2) parallel or perpendicular to MP strips, or kept under static condition. Parallel flow caused cell elongation with enhanced stress fibers and p-FAK, and a reduction in apoptosis, but perpendicular flow did not. The Rho inhibitory C3 exoenzyme abolished the effects of parallel flow. RhoV14, the constitutively active Rho, enhanced stress fibers and p-FAK, and prevented apoptosis of HUVECs on 15-microm strips under static condition. RhoV14 also reduced cell apoptosis under both parallel and perpendicular flows. Our results indicate that cell apoptosis can be modulated by changes in ECM micropatterning, anisotropic cell morphology, and mechanical forces. These extracellular microenvironment factors affect cell survival through alterations in Rho GTPase activity, stress fiber organization, and FAK phosphorylation.

Regulation of stretch-induced JNK activation by stress fiber orientation.

Cyclic mechanical stretch associated with pulsatile blood pressure can modulate cytoskeletal remodeling and intracellular signaling in vascular endothelial cells. The aim of this study was to evaluate the role of stretch-induced actin stress fiber orientation in intracellular signaling involving the activation of c-jun N-terminal kinase (JNK) in bovine aortic endothelial cells. A stretch device was designed with the capability of applying cyclic uniaxial and equibiaxial stretches to cultured endothelial cells, as well as changing the direction of cyclic uniaxial stretch. In response to 10% cyclic equibiaxial stretch, which did not result in stress fiber orientation, JNK activation was elevated for up to 6 h. In response to 10% cyclic uniaxial stretch, JNK activity was only transiently elevated, followed by a return to basal level as the actin stress fibers became oriented perpendicular to the direction of stretch. After the stress fibers had aligned perpendicularly and the JNK activity had subsided, a 90-degree change in the direction of cyclic uniaxial stretch reactivated JNK, and this activation again subsided as stress fibers became re-oriented perpendicular to the new direction of stretch. Disrupting actin filaments with cytochalasin D blocked the stress fiber orientation in response to cyclic uniaxial stretch and it also caused the uniaxial stretch-induced JNK activation to become sustained. These results suggest that stress fiber orientation perpendicular to the direction of stretch provides a mechanism for both structural and biochemical adaptation to cyclic mechanical stretch.

Integrins regulate VE-cadherin and catenins: dependence of this regulation on Src, but not on Ras.

Adhesions of cells to extracellular matrix and adjacent cells are mediated by integrins and VE-cadherin, respectively. Although these adhesion processes play crucial roles in vascular cell migration and angiogenesis, it remains unclear as to how they are coordinated to regulate cellular functions. We report here that integrin engagement by treating bovine endothelial aortic cell monolayers with beads coated with fibronectin (Fn) led to disruption of the VE-cadherin-containing adherens junctions. This disruption was accompanied by increases of tyrosine phosphorylation of beta-catenin, gamma-catenin, and p120ctn, as well as the dissociation of alpha-catenin and gamma-catenin from VE-cadherin. We applied a membrane-targeted Src reporter based on the fluorescence resonance energy transfer technique to visualize the dynamic Src activation at subcellular levels in live cells. The integrin engagement induced by Fn-coated beads caused the activation of Src around the beads and at adherens junctions, which are subsequently disrupted. The inhibition of Src with PP1 blocked the effects of integrin engagement on adherens junctions. Although Ras can also modulate adherens junctions, the resulting patterns of phosphorylation and association of junction proteins were distinct from those induced by integrin engagement. The inhibition of Ras by RasN17 did not rescue the disruption of adherens junctions induced by integrin engagement or by Src activation. Integrin engagement by Fn-coated beads also induced a significant alteration of cortical actin filaments at adherens junctions. The results indicate that integrin engagement disrupts VE-cadherin-containing adherens junctions via the activation of Src, but not Ras, possibly as a result of modulation of the actin network.

Synergistic roles of platelet-derived growth factor-BB and interleukin-1beta in phenotypic modulation of human aortic smooth muscle cells.

The phenotype of smooth muscle cells (SMCs) plays an important role in vascular function in health and disease. We investigated the mechanism of modulation of SMC phenotype (from contractile to synthetic) induced by the synergistic action of a growth factor (platelet-derived growth factor, PDGF-BB) and a cytokine (interleukin, IL-1beta). Human aortic SMCs grown on polymerized collagen showed high expression levels of contractile markers (smooth muscle alpha-actin, myosin heavy chain, and calponin). These levels were not significantly affected by PDGF-BB and IL-1beta individually, but decreased markedly after the combined usage of PDGF-BB and IL-1beta. PDGF/IL-1beta costimulation also induced a sustained phosphorylation of Akt and p70 ribosomal S6 kinase (p70S6K). The effects of PDGF/IL-1beta costimulation on contractile marker expression and Akt and p70S6K phosphorylation were blocked by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 and by adenovirus expressing a dominant-negative Akt, and they were mimicked by constitutively active Akt. PDGF-BB/IL-1beta induced a sustained phosphorylation of PDGF receptor (PDGFR)-beta and its association with IL-1 receptor (IL-1R1). Such activation and association of receptors were blocked by a PDGFR-beta neutralizing antibody (AF385), an IL-1R1 antagonist (IL-1ra), as well as a specific inhibitor of PDGFR-beta phosphorylation (AG1295); these agents also eliminated the PDGF-BB/IL-1beta-induced signaling and phenotypic modulation. PDGF-BB/IL-1beta inhibited the polymerized collagen-induced serum response factor DNA binding activity in the nucleus, and this effect was mediated by the PDGFR-beta/IL-1R1 association and phosphatidylinositol 3-kinase/Akt/p70S6K pathway. Our findings provide insights into the mechanism of SMC phenotypic modulation from contractile to synthetic, e.g., in atherosclerosis.

Roles of MAP kinases in the regulation of bone matrix gene expressions in human osteoblasts by oscillatory fluid flow.

We investigated the effects of oscillatory flow in regulating the gene expressions of type I collagen (COL1, the main component of human bone tissues) and osteopontin (OPN, the key gene for calcium deposition) in human osteoblast-like (MG-63) cells, and the roles of mitogen-activated protein kinases (MAPKs) in this regulation. The cells were subjected to oscillatory flow (0.5 +/- 4 dyn/cm(2)) or kept under static condition for various time periods (15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 16 h). Oscillatory flow caused significant up-regulations of both COL1 and OPN gene expressions over the 16 h of study, and a transient activation of MAPKs was starting at 15 min and declining to basal level in 2 h. The flow-induction of COL1 was blocked by an ERK inhibitor (PD98059) and reduced by a JNK inhibitor (SP600125), whereas that of OPN was abolished by PD98059. Analysis of the cis-elements in the COL1 and OPN promoters suggests the involvement of transacting factors Elk-1 and AP-1 in the transcription regulation. The ERK inhibitor (PD98059) blocked Elk-1 phosphorylation, as well as COL1 and OPN gene expression. The JNK inhibitor (SP600125) abolished c-jun phosphorylation and COL1 expression. These results suggest that the flow-induction of OPN was mediated through the ERK-Elk1-OPN pathway, and that COL1 was regulated by both the ERK-Elk1-COL1 and JNK-c-JUN-COL1 pathway.

Phosphatidylinositol 3-kinase/Akt pathway is involved in transforming growth factor-beta1-induced phenotypic modulation of 10T1/2 cells to smooth muscle cells.

Transforming growth factor-beta1 (TGF-beta1) is known to induce phenotypic modulation of mesenchymal cells to SMCs. However, the intracellular signals regulating induction of the SMC phenotype of mesenchymal cells have not been fully clarified. In the present study, we examined the role of the mitogen-activated protein kinase (MAPK) superfamily and phosphatidylinositol 3-kinase (PI3K)/Akt in the TGF-beta1-mediated phenotypic modulation of 10T1/2 mesenchymal cells to SMCs characterized by the expression of SMC-specific markers, including smooth muscle alpha-actin (SMalpha-actin), myosin heavy chain (SM-MHC), and protein 22-alpha (SM22alpha). The results showed the following: (1) TGF-beta1 induced SMalpha-actin and SM-MHC expressions in 10T1/2 cells in a time-dependent manner. (2) TGF-beta1 induced biphasic increases in extracellular signal-regulated kinase (ERK), p38 MAPK, c-Jun-NH2-terminal kinase (JNK), and Akt phosphorylation. (3) The inhibitor for PI3K/Akt (i.e., LY294002), but not those for MAPKs (i.e., SB203580, PD98059, and SP600125), attenuated the TGF-beta1-induced SMalpha-actin and SM-MHC expressions in 10T1/2 cells; in addition, transfection of 10T1/2 cells with the Akt-specific small interfering RNA (siRNA) significantly reduced their SMalpha-actin and SM-MHC expressions. (4) LY294002 and the Akt-specific siRNA inhibited the TGF-beta1-induced SM22alpha gene expression and promoter activity, suggesting that the TGF-beta1-induced gene expression was mediated by PI3K/Akt at the transcriptional level. (5) LY294002 inhibited the TGF-beta1-induced gene expression and DNA binding activity of serum response factor (SRF). These results indicate that TGF-beta1 is capable of inducing the SMC phenotype of 10T1/2 cells and that this induction is mediated through the PI3K/Akt signaling pathway.

Neutrophils, lymphocytes, and monocytes exhibit diverse behaviors in transendothelial and subendothelial migrations under coculture with smooth muscle cells in disturbed flow.

Atherosclerosis develops at regions of the arterial tree exposed to disturbed flow. The early stage of atherogenesis involves the adhesion of leukocytes (white blood cells [WBCs]) to and their transmigration across endothelial cells (ECs), which are located in close proximity to smooth muscle cells (SMCs). We investigated the effects of EC/SMC coculture and disturbed flow on the adhesion and transmigration of 3 types of WBCs (neutrophils, peripheral blood lymphocytes [PBLs], and monocytes) using our vertical-step flow (VSF) chamber, in which ECs were cocultured with SMCs in collagen gels. Such coculture significantly increased the adhesion and transmigration of neutrophils, PBLs, and monocytes under VSF, particularly in the reattachment area, where the rolling velocity of WBCs and their transmigration time were decreased, as compared with the other areas. Neutrophils, PBLs, and monocytes showed different subendothelial migration patterns under VSF. Their movements were more random and shorter in distance in the reattachment area. Coculture of ECs and SMCs induced their expressions of adhesion molecules and chemokines, which contributed to the increased WBC adhesion and transmigration. Our findings provide insights into the mechanisms of WBC interaction with the vessel wall (composed of ECs and SMCs) under the complex flow environments found in regions of prevalence for atherogenesis.

Cooperative effects of Rho and mechanical stretch on stress fiber organization.

The small GTPase Rho regulates the formation of actin stress fibers in adherent cells through activation of its effector proteins Rho-kinase and mDia. We found in bovine aortic endothelial cells that inhibitions of Rho, Rho-kinase, and mDia (with C3, Y27632, and F1F2Delta1, respectively) suppressed stress fiber formation, but fibers appeared after 10% cyclic uniaxial stretch (1-Hz frequency). In contrast to the predominately perpendicular alignment of stress fibers to the stretch direction in normal cells, the stress fibers in cells with Rho pathway inhibition became oriented parallel to the stretch direction. In cells with normal Rho activity, the extent of perpendicular orientation of stress fibers depended on the magnitude of stretch. Expressing active RhoV14 plasmid in these cells enhanced the stretch-induced stress fiber orientation by an extent equivalent to an additional approximately 3% stretch. This augmentation of the stretch-induced perpendicular orientation by RhoV14 was blocked by Y27632 and by F1F2Delta1. Thus, the activity of the Rho pathway plays a critical role in determining both the direction and extent of stretch-induced stress fiber orientation in bovine aortic endothelial cells. Our results demonstrate that the stretch-induced stress fiber orientation is a function of the interplay between Rho pathway activity and the magnitude of stretching.

Shear stress regulates gene expression in vascular endothelial cells in response to tumor necrosis factor-alpha: a study of the transcription profile with complementary DNA microarray.

We investigate the role of shear stress in regulating the gene expression in endothelial cells (ECs) in response to tumor necrosis factor-alpha (TNF-alpha). ECs were kept in static condition or pre-exposed to a high level (HSS, 20 dynes/cm2) or a low level of shear stress (LSS, 0.5 dynes/cm2) for 24 h, and TNF-alpha was added under static condition for 4 h. In static ECs, DNA microarray showed that TNF-alpha caused a significant increase in expression of 102 genes and a significant decrease in expression of 12 genes. Pre-shearing of ECs decreased the TNF-alpha-responsiveness of many pro-inflammatory, pro-coagulant, proliferative, and pro-apoptotic genes, whereas it increased the responsiveness of some antioxidant, anti-coagulant, and anti-apoptotic genes. LSS showed less regulatory effects than HSS on EC gene expression in response to TNF-alpha. The microarray data were confirmed by reverse-transcription polymerase chain reaction for 64 selected genes. Pre-shearing of ECs at HSS significantly inhibited the TNF-alpha-induced p65 and p50 mRNA expressions and nuclear factor-kappaB (NF-kappaB)-DNA binding activity. Inhibition of NF-kappaB activity with the p65-antisense or lactacystin under static condition blocked the expression of most of the genes that are TNF-alpha-inducible and shear stress-down-regulated. Our findings suggest that laminar shear stress serves protective functions against atherogenesis.

Visualizing the mechanical activation of Src.

The mechanical environment crucially influences many cell functions. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin-cytoskeleton interaction, which is essential for the transduction of mechanical stimuli. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean +/- s.e.m.) of 18.1 +/- 1.7 nm s(-1). This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.

Shear stress inhibits smooth muscle cell-induced inflammatory gene expression in endothelial cells: role of NF-kappaB.

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/cm2) 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-kappaB (NF-kappaB) activation by the p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced EC expression of proinflammatory genes, indicating that the NF-kappaB 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-kappaB recruitment to selected target promoters. Shear stress inhibited the SMC coculture-induced NF-kappaB activation in ECs and monocytic THP-1 cell adhesion to ECs. CONCLUSIONS: Our findings suggest that shear stress plays an inhibitory role in the proinflammatory gene expression in ECs located in close proximity to SMCs.

Effects of flow patterns on the localization and expression of VE-cadherin at vascular endothelial cell junctions: in vivo and in vitro investigations.

Atherosclerosis occurs preferentially at vascular curvature and branch sites where the vessel walls are exposed to fluctuating shear stress and have high endothelial permeability. Endothelial permeability is modulated by intercellular adhesion molecules such as VE-cadherin. This study was designed to elucidate the effects of different flow patterns on the localization and expression of VE-cadherin in endothelial cells (ECs) both in vivo and in vitro. VE-cadherin staining at EC borders was much stronger in the descending thoracic aorta and abdominal aorta, where the pulsatile flow has a strong net forward component than in the aortic arch and the poststenotic dilatation site beyond an experimental constriction, where the flow near the wall is complex and reciprocating with little net flow. With the use of flow chambers the effects of pulsatile flow (12 +/- 4 dyn/cm2 at 1 Hz) and reciprocating flow (0.5 +/- 4 dyn/cm2 at 1 Hz) on VE-cadherin organization in endothelial monolayers were studied in vitro. VE-cadherin staining was continuous along cell borders in static controls. Following 6 h of either pulsatile or reciprocating flow, the VE-cadherin staining at cell borders became intermittent. When the pulsatile flow was extended to 24, 48 or 72 h the staining around the cell borders became continuous again, but the staining was still intermittent when the reciprocating flow was similarly extended. Exposure to pulsatile or reciprocating flow for 6 and 24 h neither change the expression level of VE-cadherin nor its distribution between membrane and cytosol fractions as determined by Western blot and compared with static controls. These findings suggest that the cell junction remodeling induced by different flow patterns may result from a redistribution of VE-cadherin within the cell membrane. Both the in vivo and in vitro data indicate that pulsatile and reciprocating flow patterns have different effects on cell junction remodeling. The lack of junction reorganization in regions of reciprocating flow in vivo and in vitro may provide a mechanistic basis for the high permeability and the preferential localization of atherosclerosis in regions of the arterial stress with complex flow patterns and fluctuating shear stress.

Rho mediates the shear-enhancement of endothelial cell migration and traction force generation.

The migration of vascular endothelial cells in vivo occurs in a fluid dynamic environment due to blood flow, but the role of hemodynamic forces in cell migration is not yet completely understood. Here we investigated the effect of shear stress, the frictional drag of blood flowing over the cell surface, on the migration speed of individual endothelial cells on fibronectin-coated surfaces, as well as the biochemical and biophysical bases underlying this shear effect. Under static conditions, cell migration speed had a bell-shaped relationship with fibronectin concentration. Shear stress significantly increased the migration speed at all fibronectin concentrations tested and shifted the bell-shaped curve upwards. Shear stress also induced the activation of Rho GTPase and increased the traction force exerted by endothelial cells on the underlying substrate, both at the leading edge and the rear, suggesting that shear stress enhances both the frontal forward-pulling force and tail retraction. The inhibition of a Rho-associated kinase, p160ROCK, decreased the traction force and migration speed under both static and shear conditions and eliminated the shear-enhancement of migration speed. Our results indicate that shear stress enhances the migration speed of endothelial cells by modulating the biophysical force of tractions through the biochemical pathway of Rho-p160ROCK.

Shear stress increases ICAM-1 and decreases VCAM-1 and E-selectin expressions induced by tumor necrosis factor-[alpha] in endothelial cells.

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)-alpha. Shear stress increased the TNF-alpha-induced expression of intercellular adhesion molecule-1 (ICAM-1) at both mRNA and surface protein levels, but decreased the TNF-alpha-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-alpha-induced ICAM-1 mRNA expression vanished. The recovery of the TNF-alpha-induced VCAM-1 and E-selectin mRNA expressions following preshearing, however, required a static incubation time of >6 hours (complete recovery at 24 hours). Pre- and postshearing caused a reduction in the nuclear factor-kappaB-DNA binding activity induced by TNF-alpha in the EC nucleus. CONCLUSIONS: Our findings suggest that shear stress plays differential roles in modulating the TNF-alpha-induced expressions of ICAM-1 versus VCAM-1 and E-selectin genes in ECs.

Analysis of the effect of disturbed flow on monocytic adhesion to endothelial cells.

The preferential adhesion of monocytes to vascular endothelial cells (ECs) at regions near branches and curvatures of the arterial tree, where flow is disturbed, suggests that hemodynamic conditions play significant roles in monocyte adhesion. The present study aims to elucidate the effects of disturbed flow on monocyte adhesion to ECs and the adhesive properties of ECs. We applied, for the first time, the micron-resolution particle image velocimetry (microPIV) technique to analyze the characteristics of the disturbed flow produced in our vertical-step flow (VSF) chamber. The results demonstrated the existence of a higher near-wall concentration and a longer residence time of the monocytic analog THP-1 cells near the step and the reattachment point. THP-1 cells showed prominent adhesion to ECs pretreated with TNFalpha in the regions near the step and the reattachment point, but they showed virtually no adhesion to un-stimulated ECs. Pre-incubation of the TNFalpha-treated ECs with antibodies against intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin inhibited the THP-1 adhesion; the maximal inhibition was observed with a combination of these antibodies. Pre-exposure of ECs to disturbed flow in VSF for 24 h led to significant increases in their surface expressions of ICAM-1 and E-selectin, but not VCAM-1, and in the adhesion of THP-1 cells. Our findings demonstrate the importance of complex flow environment in modulating the adhesive properties of vascular endothelium and consequently monocyte adhesion in regions of prevalence of atherosclerotic lesions.

Signal transduction in matrix contraction and the migration of vascular smooth muscle cells in three-dimensional matrix.

The interaction of vascular smooth muscle cells (SMCs) and extracellular matrix plays important roles in vascular remodeling. We investigated the signaling pathways involved in SMC-induced matrix contraction and SMC migration in three-dimensional (3D) collagen matrix. Matrix contraction is inhibited by the disruption of actin filaments but not microtubules. Therefore, we investigated the roles of signaling pathways related to actin filaments in matrix contraction. SMC-induced matrix contraction was markedly blocked (-80%) by inhibiting the Rho-p160ROCK pathway and myosin light chain kinase, and was decreased to a lesser extent (30-40%) by a negative mutant of Rac and inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) or p38 mitogen-activated protein kinase (MAPK), but it was not affected by the inhibition of Ras and Cdc42-Wiskott-Aldrich syndrome protein (WASP) pathways. Inhibition of extracellular-signal-regulated kinase (ERK) decreased SMC-induced matrix contraction by only 15%. The migration speed and persistence of SMCs in the 3D matrix were decreased by the inhibition of p160ROCK, PI 3-kinase, p38 MAPK or WASP to different extents, and p160ROCK inhibitor had the strongest inhibitory effect. Our results suggest that the SMC-induced matrix contraction and the migration of SMCs in 3D matrix share some signaling pathways leading to force generation at cell-matrix adhesions and that various signaling pathways have different relative importance in the regulations of these processes in SMCs.

Shear stress inhibits adhesion molecule expression in vascular endothelial cells induced by coculture with smooth muscle cells.

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 with the flow direction. Under static conditions, coculture with SMCs induced EC gene expression of intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin, while attenuating EC gene expression of endothelial nitric oxide synthase (eNOS). Shear stress significantly inhibited SMC-induced adhesion molecule gene expression. These EC responses under static and shear conditions were not observed in the absence of close communication between ECs and SMCs, and they were also not observed when ECs were cocultured with fibroblasts instead of SMCs. Our findings indicate that under static conditions, coculture with SMCs induces ICAM-1, VCAM-1, and E-selectin gene expression in ECs. These coculture effects are inhibited by shear stress and require specific interaction between ECs and SMCs in close contact.

Differential regulation of Rho GTPases by beta1 and beta3 integrins: the role of an extracellular domain of integrin in intracellular signaling.

Integrins mediate cell adhesion and signal transduction at focal adhesions. Here we investigate the roles of integrin beta subunits in the regulation of actin cytoskeletal structure and the activities of Rho and Rac. The overexpression of beta3 integrin in Chinese hamster ovary cells enhances Rho activity and stress fiber formation, whereas the overexpression of beta1 integrin increases Rac activity and lamellipodia formation. The overexpression of a mutant beta1-3-1 integrin, in which the extracellular I-domain-like sequence of beta1 integrin has been replaced with the corresponding sequence of beta3 integrin, also enhances Rho activity and the formation of stress fibers. Our results demonstrate that beta1 and beta3 integrins differentially regulate the activities of Rho family GTPases and that the extracellular domains of integrin beta subunits play a critical role in transducing the extracellular ligand-binding information into specific intracellular signaling events.