Interleukin-6-induced JAK2/STAT3 signaling pathway in endothelial cells is suppressed by hemodynamic flow

Endothelial cells (ECs) are constantly exposed to shear stress, the action of which triggers signaling pathways and cellular responses. During inflammation, cytokines such as IL-6 increase in plasma. In this study, we examined the effects of steady flow on IL-6-induced endothelial responses. ECs exposed to IL-6 exhibited STAT3 activation via phosphorylation of Tyr705. However, when ECs were subjected to shear stress, shear force-dependent suppression of IL-6-induced STAT3 phosphorylation was observed. IL-6 treatment increased the phosphorylation of JAK2, an upstream activator of STAT3. Consistently, shear stress significantly reduced IL-6-induced JAK2 activation. Pretreatment of ECs with an inhibitor of MEK1 did not alter this suppression by shear stress, indicating that extracellular signal-regulated kinase (ERK1/2) was not involved. However, pretreatment of ECs with an endothelial nitric oxide synthase inhibitor (nitro-L-arginine methyl ester) attenuated this inhibitory effect of shear stress on STAT3 phosphorylation. Shear stress-treated ECs displayed decreased nuclear transmigration of STAT3 and reduced STAT3 binding to DNA. Intriguingly, ECs exposed to IL-6 entered the cell cycle, as evidenced by increasing G₂/M phase, and shear stress to these ECs significantly reduced IL-6-induced cell cycle progression. STAT3-mediated IL-6-induced cell cycle was confirmed by the inhibition of the cell cycle in ECs infected with adenovirus carrying the inactive mutant of STAT3. Our study clearly shows that shear stress exerts its inhibitory regulation by suppressing the IL-6-induced JAK2/STAT3 signaling pathway and thus inhibits IL-6-induced EC proliferation. This shear force-dependent inhibition of IL-6-induced JAK2/STAT3 activation provides new insights into the vasoprotective effects of steady flow on ECs against cytokine-induced responses. shear stress; nitric oxide; cell cycle     

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.     

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.     

Induction of lactadherin mediates the apoptosis of endothelial cells in response to advanced glycation end products and protective effects of grape seed procyanidin B2 and resveratrol

One of characteristics of diabetes mellitus (DM) is endothelial cell (EC) dysfunction and apoptosis which contributes to the development of vasculopathy. Advanced glycation end products (AGEs) continuously produced in the setting of DM play an important role in causing EC dysfunction and apoptosis. However, the underlying molecular mechanism remains largely elusive. Lactadherin, a secreted glycoprotein of milk-fat globule, is expressed by multiple cell types of arterial wall including ECs. Our previous proteomic studies showed that the expression of lactadherin was significantly increased in the aorta of diabetic rats as compared with control rats and treatment with grape seed procyanidin extracts significantly inhibited the lactadherin expression in diabetic rats. We hypothesized that lactadherin plays a critical role in AGEs-induced EC apoptosis; grape seed procyanidin B2 (GSPB2) and resveratrol protect against AGEs-induced EC apoptosis through lactadherin regulation. Our results showed that AGEs upregulated lactadherin expression and lactadherin RNA interference significantly attenuated AGEs-induced EC apoptosis. Overexpression of lactadherin increased EC apoptosis with up-regulation of Bax/Bcl-2 ratio, cytochrome c release, caspase-9 and caspase-3 activation suggesting the involvement of mitochondria apoptosis pathway. Mechanistically, overexpression of lactadherin reduced the phosphorylation of GSK3beta at baseline. Our study also revealed nine proteins interacting with lactadherin in HUVEC and study of these candidate proteins could unveil further underlying molecular mechanisms. In summary, our study identified lactadherin as a key player responsible for AGEs-induced EC apoptosis and antioxidants GSPB2 and resveratrol protect against AGEs-induced EC apoptosis by inhibiting lactadherin. Targeting lactadherin with antioxidant could be translated into clinical application in the fighting against DM complications.     

15-deoxy-delta 12,14-prostaglandin J2 and laminar fluid shear stress stabilize c-IAP1 in vascular endothelial cells

Laminar shear stress strongly inhibits vascular endothelial cell apoptosis by unknown mechanisms. We reported that shear stress stimulates endothelial cells to produce 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) by elevating the expression level of lipocalin-type prostaglandin D synthase. To investigate the role of 15d-PGJ2 produced in the vascular wall, we examined the effect of 15d-PGJ2 on endothelial cell apoptosis. We induced apoptosis in human umbilical vein endothelial cells (HUVECs) by growth factor deprivation. 15d-PGJ2 strongly inhibited DNA ladder formation, nuclear fragmentation, and caspase-3-like activity in HUVECs. To elucidate the mechanism by which 15d-PGJ2 inhibits endothelial cell apoptosis, we examined expression of the inhibitor of apoptosis proteins (IAP) cellular-IAP1 (c-IAP1), c-IAP2, x-linked IAP, and survivin in HUVECs. In parallel with the inhibition of apoptosis, 15d-PGJ2 elevated the expression level of c-IAP1 protein in a dose- and time-dependent manner without changing the mRNA level. Laminar shear stress also induced c-IAP1 expression. Chase experiments with the use of cycloheximide revealed that 15d-PGJ2 and shear stress both inhibited the proteolytic degradation of c-IAP1 protein. These results suggested that 15d-PGJ2 inhibits endothelial cell apoptosis through, at least in part, c-IAP1 protein stabilization. This mechanism might be involved in the antiapoptotic effect of laminar shear stress.     

Heparan sulfate proteoglycan mediates shear stress-induced endothelial gene expression in mouse embryonic stem cell-derived endothelial cells

It has been shown that shear stress plays a critical role in promoting endothelial cell (EC) differentiation from embryonic stem cell (ESC)-derived ECs. However, the underlying mechanisms mediating shear stress effects in this process have yet to be investigated. It has been reported that the glycocalyx component heparan sulfate proteoglycan (HSPG) mediates shear stress mechanotransduction in mature EC. In this study, we investigated whether cell surface HSPG plays a role in shear stress modulation of EC phenotype. ESC-derived EC were subjected to shear stress (5 dyn/cm(2)) for 8 h with or without heparinase III (Hep III) that digests heparan sulfate. Immunostaining showed that ESC-derived EC surfaces contain abundant HSPG, which could be cleaved by Hep III. We observed that shear stress significantly increased the expression of vascular EC-specific marker genes (vWF, VE-cadherin, PECAM-1). The effect of shear stress on expression of tight junction protein genes (ZO-1, OCLD, CLD5) was also evaluated. Shear stress increased the expression of ZO-1 and CLD5, while it did not alter the expression of OCLD. Shear stress increased expression of vasodilatory genes (eNOS, COX-2), while it decreased the expression of the vasoconstrictive gene ET1. After reduction of HSPG with Hep III, the shear stress-induced expression of vWF, VE-cadherin, ZO-1, eNOS, and COX-2, were abolished, suggesting that shear stress-induced expression of these genes depends on HSPG. These findings indicate for the first time that HSPG is a mechanosensor mediating shear stress-induced EC differentiation from ESC-derived EC cells.     

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.     

Effects of shear stress on endothelial cell haptotaxis on micropatterned surfaces

Endothelial cell (EC) migration plays a critical role in vascular remodeling. Here we investigated the interactions between haptotaxis (induced by extracellular matrix gradient) and mechanotaxis (induced by mechanical forces) during EC migration. A micropatterning technique was used to generate step changes of collagen surface density. Due to haptotaxis, ECs developed focal adhesions and migrated into the area with higher surface density of collagen. Different levels of fluid shear stress were applied on ECs in the direction perpendicular to collagen strips. Shear stress at 2 dyn/cm2 did not affect haptotaxis, while shear stress at 3 dyn/cm2 or higher was sufficient to drive the migration of most ECs in the flow direction and against haptotaxis. Immunostaining revealed the increase of focal adhesions and lamellipodial protrusion in the direction of flow. These results suggest that shear stress beyond a certain threshold can be a predominant factor to determine the direction of EC migration.     

Shear stress attenuates tumor necrosis factor-alpha-induced monocyte chemotactic protein-1 expressions in endothelial cells

The interplay between shear stress and cytokines in regulating vascular endothelial function remains largely unexplored. In the present study, the potential role of shear stress in regulating tumor necrosis factor-alpha (TNF-alpha)-induced gene expression in endothelial cells (ECs) was investigated. The TNF-alpha-induced monocyte chemotactic protein-1 (MCP-1) mRNA expressions were significantly attenuated in ECs subjected to a high level of shear stress (20 dynes/cm2) for 4 or 24 h prior to the addition of TNF-alpha in the presence of flow. Less inhibition of TNF-alpha-induced MCP-1 mRNA expression was found in ECs pre-exposed to a low level of shear stress (1.2 dynes/cm2) for 24 h as compared with the cells presheared (pre-exposed to shear stress) for 4 h. Simultaneous exposure of ECs to TNF-alpha and a high or low level of shear stress down-regulated TNF-alpha-induced MCP-1 gene expressions, suggesting that the post-flow condition modulates endothelial responses to cytokine stimulation. Individually or combined, an endothelial nitric oxide synthase (eNOS) inhibitor and a glutathione (GSH) biosynthesis inhibitor had no effect on this shear stress-mediated inhibition. Moreover, in ECs either presheared or remained in a static condition prior to stimulation by TNF-alpha while under shear flow, the ability of TNF-alpha to induce AP-1-DNA binding activity in the nucleus was reduced. Our findings suggest that shear stress plays a protective role in vascular homeostasis by inhibiting endothelial responses to cytokine stimulation.     

Rac1 mediates laminar shear stress-induced vascular endothelial cell migration

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. ECs are constantly subjected to shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular behaviors and functions. The aim of this study is to elucidate the effects of Rac1, which is the member of small G protein family, on EC migration under different laminar shear stress (5.56, 10.02, and 15.27 dyn/cm²). The cell migration distance under laminar shear stress increased significantly than that under the static culture condition. Especially, under relative high shear stress (15.27 dyn/cm²) there was a higher difference at 8 h (P < 0.01) and 2 h (P < 0.05) compared with static controls. RT-PCR results further showed increasing mRNA expression of Rac1 in ECs exposed to laminar shear stress than that exposed to static culture. Using plasmids encoding the wild-type (WT), an activated mutant (Q61L), and a dominant-negative mutant (T17N), plasmids encoding Rac1 were transfected into EA.hy 926 cells. The average net migration distance of Rac1Q61L group increased significantly, while Rac1T17N group decreased significantly in comparison with the static controls. These results indicated that Rac1 mediated shear stress-induced EC migration. Our findings conduce to elucidate the molecular mechanisms of EC migration induced by shear stress, which is expected to understand the pathophysiological basis of wound healing in health and diseases.     

Mitochondrial fission in endothelial cells after simulated ischemia/reperfusion: role of nitric oxide and reactive oxygen species

Ischemia (I)/reperfusion (RP)-induced endothelial cell (EC) injury is thought to be due to mitochondrial reactive oxygen species (mtROS) production. MtROS have been implicated in mitochondrial fission. We determined whether cultured EC exposure to simulated I/RP causes morphological changes in the mitochondrial network and the mechanisms behind those changes. Because shear stress results in nitric oxide (NO)-mediated endothelial mtROS generation, we simulated I/RP as hypoxia (H) followed by oxygenated flow over the ECs (shear stress of 10dyn/cm(2)). By exposing ECs to shear stress, H, H/reoxygenation (RO), or simulated I/RP and employing MitoTracker staining, we assessed the differential effects of changes in mechanical forces and/or O(2) levels on the mitochondrial network. Static or sheared ECs maintained their mitochondrial network. H- or H/RO-exposed ECs underwent changes, but mitochondrial fission was significantly less compared to that in ECs exposed to I/RP. I/RP-induced fission was partially inhibited by antioxidants, a NO synthase inhibitor, or an inhibitor of the fission protein dynamin-related protein 1 (Drp1) and was accompanied by Drp1 oligomerization and phosphorylation (Ser616). Hence, shear-induced NO, ROS (including mtROS), and Drp1 activation are responsible for mitochondrial fission in I/RP-exposed ECs, and excessive fission may be an underlying cause of EC dysfunction in postischemic hearts.     

Shear-induced reactive nitrogen species inhibit mitochondrial respiratory complex activities in cultured vascular endothelial cells

There is evidence that nitric oxide (NO), superoxide (O₂^–), and their associated reactive nitrogen species (RNS) produced by vascular endothelial cells (ECs) in response to hemodynamic forces play a role in cell signaling. NO is known to impair mitochondrial respiration. We sought to determine whether exposure of human umbilical vein ECs (HUVECs) to steady laminar shear stress and the resultant NO production modulate electron transport chain (ETC) enzymatic activities. The activities of respiratory complexes I, II/III, and IV were dependent on the presence of serum and growth factor supplement in the medium. EC exposure to steady laminar shear stress (10 dyn/cm²) resulted in a gradual inhibition of each of the complexes starting as early as 5 min from the flow onset and lasting up to 16 h. Ramp flow resulted in inhibition of the complexes similar to that of step flow. When ECs were sheared in the presence of the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 100 µM), the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; 100 µM), or the peroxynitrite (ONOO^–) scavenger uric acid (UA; 50 µM), the flow-inhibitory effect on mitochondrial complexes was attenuated. In particular, L-NAME and UA abolished the flow effect on complex IV. Increased tyrosine nitration was observed in the mitochondria of sheared ECs, and UA blocked the shear-induced nitrotyrosine staining. In summary, shear stress induces mitochondrial RNS formation that inhibits the electron flux of the ETC at multiple sites. This may be a critical mechanism by which shear stress modulates EC signaling and function. oxidative stress; mitochondria; endothelium     

A model for studying the effect of shear stress on interactions between vascular endothelial cells and smooth muscle cells

Vascular endothelial cells (ECs) are constantly subjected to blood flow-induced shear stress and the influences of neighboring smooth muscle cells (SMCs). In the present study, a coculture flow system was developed to study the effect of shear stress on EC-SMC interactions. ECs and SMCs were separated by a porous membrane with only the EC side subjected to the flow condition. When ECs were exposed to a shear stress of 12 dynes/cm2 for 24 h, the cocultured SMCs tended to orient perpendicularly to the flow direction. This perpendicular orientation of the cocultured SMCs to flow direction was not observed when ECs were exposed to a shear stress of 2 dynes/cm2. Under the static condition, long and parallel actin bundles were observed in the central regions of the cocultured SMCs, whereas the actin filaments localized mainly at the periphery of the cocultured ECs. After 24 h of flow application, the cocultured ECs displayed very long, well-organized, parallel actin stress fibers aligned with the flow direction in the central regions of the cells. Immunostaining of platelet endothelial cell adhesion molecule-1 confirmed the elongation and alignment of the cocultured ECs with the flow direction. Coculture with SMCs under static condition induced EC gene expressions of growth-related oncogene-alpha and monocyte chemotactic protein-1, and shear stress was found to abolish these SMC-induced gene expressions. Our results suggest that shear stress may serve as a down-regulator for the pathophysiologically relevant gene expression in ECs cocultured with SMCs.     

Hepatocyte growth factor inhibits apoptosis by the profibrotic factor angiotensin II via extracellular signal-regulated kinase 1/2 in endothelial cells and tissue explants

Hepatocyte growth factor (HGF), an endogenous tissue repair factor, attenuates apoptosis in many primary cell types, but the mechanism is not completely understood. Our laboratory demonstrated that angiotensin (Ang) II activates the intrinsic apoptotic pathway in primary endothelial cells (ECs) via reduction of the antiapoptotic protein Bcl-x(L). Ang II decreased Bcl-x(L) mRNA half-life by reducing its binding to nucleolin, a protein that normally binds a 3' AU-rich region and stabilizes Bcl-x(L) mRNA. We hypothesized HGF may block apoptosis induced by Ang II. We used primary EC and ex vivo cultures of rat lung tissue to investigate HGF inhibition of Ang II-induced apoptosis. Our data indicated HGF abrogated Ang II-induced apoptosis by inhibiting cytochrome c release, caspase-3 activation, and DNA fragmentation. RNA-immunoprecipitation experiments demonstrated that HGF stabilized Bcl-x(L) mRNA by increasing nucleolin binding to the 3'-untranslated region that was associated with cytoplasmic localization of nucleolin. Cytoplasmic localization of nucleolin and Bcl-x(L) mRNA stabilization required HGF activation of extracellular signal-regulated kinase (ERK)1/2, but not phosphatidylinositol 3-kinase. HGF also blocked Ang II-induced caspase-3 activation and lactate dehydrogenase release in tissue explants in an ERK-dependent manner.     

CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced endothelial cell migration

The migration of endothelial cells (ECs) plays critical roles in vascular physiology and pathology. The receptors CXCR1 and CXCR2, known as G protein-coupled receptors which are essential for migratory response of ECs toward the shear stress-dependent CXCL8 (interleukin-8), are potential mechano-sensors for mechanotransduction of the hemodynamic forces. In present study, the mRNA and protein expression of CXCR1 and CXCR2 in EA.hy926 cells was detected by RT-PCR and Western blot analysis under three conditions of laminar shear stress (5.56, 10.02 and 15.27 dyn/cm(2)) respectively. Using a scratched-wound assay, the effects of CXCR1 and CXCR2 were assessed by the percentage of wound closure while CXCR1 and CXCR2 were functional blocked by the CXCL8 receptor antibodies. The results showed that the mRNA and protein expression of CXCR1 and CXCR2 was both upregulated by 5.56 dyn/cm(2) laminar shear stress, but was both downregulated by 15.27 dyn/cm(2). The wound closure was inhibited significantly while cells were treated with those antibodies in all the conditions. It was suggested that CXCR1 and CXCR2 are involved in mediating the laminar shear stress-induced EC migration. Taken together, these findings indicated that CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced EC migration. Understanding this expanded mechanism of laminar shear stress-induced cell migration will provide novel molecular targets for therapeutic intervention in cancer and cardiovascular diseases.     

Hypoxic preconditioning protects microvascular endothelial cells against hypoxia/reoxygenation injury by attenuating endoplasmic reticulum stress

Endothelial cells (ECs) are directly exposed to hypoxia and contribute to injury during myocardial ischemia/reperfusion. Hypoxic preconditioning (HPC) protects ECs against hypoxia injury. This study aimed to explore whether HPC attenuates hypoxia/reoxygenation (H/R) injury by suppressing excessive endoplasmic reticulum stress (ERS) in cultured microvascular ECs (MVECs) from rat heart. MVECs injury was measured by lactate dehydrogenase (LDH) leakage, cytoskeleton destruction, and apoptosis. Expression of glucose regulating protein 78 (GRP78) and C/EBP homologous protein (CHOP), activation of caspase-12 (pro-apoptosis factors) and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) were detected by western blot analysis. HPC attenuated H/R-induced LDH leakage, cytoskeleton destruction, and cell apoptosis, as shown by flow cytometry, Bax/Bcl-2 ratio, caspase-3 activation and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling. HPC suppressed H/R-induced ERS, as shown by a decrease in expression of GRP78 and CHOP, and caspase-12 activation. HPC enhanced p38 MAPK phosphorylation but decreased that of protein kinase R-like ER kinase (PERK, upstream regulator of CHOP). SB202190 (an inhibitor of p38 MAPK) abolished HPC-induced cytoprotection, downregulation of GRP78 and CHOP, and activation of caspase-12, as well as PERK phosphorylation. HPC may protect MVECs against H/R injury by suppressing CHOP-dependent apoptosis through p38 MAPK mediated downregulation of PERK activation.