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.     

Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.     

HBP induces the expression of monocyte chemoattractant protein-1 via the FAK/PI3K/AKT and p38 MAPK/NF-κB pathways in vascular endothelial cells

Inflammation is characterized by early influx of polymorphonuclear neutrophils (PMNs), followed by a second wave of monocyte recruitment. PMNs mediate monocyte recruitment via their release of heparin binding protein (HBP), which activates CCR2 (CC-chemokine receptor 2) on monocytes. However, the pathways for such signal transmission remain unknown. Accumulating evidences have highlighted the importance of leukocyte-endothelial cell interactions in the initiation of inflammation. In this study, an interesting finding is that HBP enhances the secretion of monocyte chemotactic protein 1(MCP-1), ligand of CCR2, from a third party, the endothelial cells (ECs). HBP-induced increase in MCP-1 production was demonstrated at the protein, mRNA and secretion levels. Exposure of ECs to HBP elicited rapid phosphorylation of FAK/PI3K/AKT and p38 MAPK/NF-κB signaling. MCP-1 levels were attenuated during the response to HBP stimulation by pretreatment with a FAK inhibitor (or siRNA), a PI3K inhibitor, an AKT inhibitor, a p38 inhibitor (or siRNA) and two NF-κB inhibitors. Additionally, pretreatment with inhibitors to FAK, PI3K and AKT led to a decrease in HBP-induced phosphorylation of p38/NF-κB axis. These results showed that HBP induced MCP-1 expression via a sequential activation of the FAK/PI3K/AKT pathway and p38 MAPK/NF-κB axis. Interestingly, the patterns of HBP regulation of the expression of the adhesion molecular VCAM-1 were similar to those seen in MCP-1 after pretreatment with inhibitors (or not). These findings may help to determine key pharmacological points of intervention, thus slowing the progress of inflammatory-mediated responses in certain diseases where inflammation is detrimental to the host.     

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

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

Globular adiponectin induces adhesion molecule expression through the sphingosine kinase pathway in vascular endothelial cells

The signaling pathways that couple adiponectin receptors to functional, particularly inflammatory, responses have remained elusive. We report here that globular adiponectin induces endothelial cell activation, as measured by the expression of adhesion proteins such as vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin and MCP-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with globular adiponectin resulted in NF-kappaB activation and increased mRNA levels of VCAM-1, ICAM-1, E-selectin and MCP-1. Sphingosine 1-phosphate (S1P), but not ceramide or sphingosine, was a potent stimulator of adhesion protein expression. As S1P is generated from sphingosine by SKase, we treated cells with siRNA for SKase to silence the effects of S1P in the endothelial cells. Treatment with SKase siRNA inhibited globular adiponectin-induced NF-kappaB activation and markedly decreased the globular adiponectin-induced mRNA levels of adhesion protein. Thus, we demonstrated that the SKase pathway, through the generation of S1P, is critically involved in mediating globular adiponectin-induced endothelial cell activation.     

Laminar shear stress up-regulates peroxiredoxins (PRX) in endothelial cells: PRX 1 as a mechanosensitive antioxidant

Shear stress plays a significant role in endothelial cell biology and atherosclerosis development. Previous work by our group has shown that fluid flow stimulates important functional changes in cells through protein expression regulation. Peroxiredoxins (PRX) are a family of antioxidant enzymes but have yet to be investigated in response to shear stress. Studies have shown that oscillatory shear stress (OS) increases reactive oxygen species (ROS) levels in endothelial cells, whereas laminar shear stress (LS) blocks this response. We hypothesized that PRX are responsible for the anti-oxidative effect of LS. To test this hypothesis, bovine aortic endothelial cells (BAEC) were subjected to LS (15 dyn/cm(2)), OS (+/-5 dyn/cm(2), 1 Hz), or static conditions for 24 h. Using Western blot and immunofluorescence staining, all six isoforms of PRX were identified in BAEC. When compared with OS and static, exposure to chronic LS up-regulated PRX 1 levels intracellularly. LS also increased expression of PRX 5 relative to static controls, but not OS. PRX exhibited broad subcellular localization, with distribution in the cytoplasm, Golgi, mitochondria, and intermediate filaments. In addition, PRX 1 knock down, using specific small interference RNA, attenuated LS-dependent reactive oxygen species reduction in BAEC. However, PRX 5 depletion did not. Together, these results suggest that PRX 1 is a novel mechanosensitive antioxidant, playing an important role in shear-dependent regulation of endothelial biology and atherosclerosis.     

Shear stress attenuates endothelin and endothelin-converting enzyme expression through oxidative stress

Shear stress is known to dilate blood vessels and exert an antiproliferative effect on vascular walls. These effects have partly been ascribed to shear stress-induced regulation of the secretion of endothelium-derived vasoactive substances. In this study, to elucidate the role of shear stress in endothelin production by endothelial cells, we examined the effect of physiological shear stress on the mRNA expression of endothelin-converting enzyme-1 (ECE-1) as well as endothelin-1 (ET-1) in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs), using a parallel plate-type flow chamber. ECE-1 mRNA expression was significantly down-regulated by shear stress in an intensity- and time-dependent manner within the physiological range (1.5 to 15 dyn/cm(2)). ET-1 mRNA expression decreased together with ECE-1 mRNA expression. Shear stress at 15 dyn/cm(2) for 30 min induced a significant increase in the intracellular peroxide concentration, and the down-regulation of ECE-1 and ET-1 mRNA expression by shear stress was attenuated almost completely on treatment with N-acetyl cysteine (NAC), an antioxidant (20 mM). Furthermore, when H(2)O(2) (0.5 to 2 mM) was added to BAECs in static culture, the ECE-1 as well as ET-1 mRNA expression was attenuated in proportion to the concentration of H(2)O(2). It is suggested that endothelial cells sense shear stress as oxidative stress and transduce signal for the regulation of the gene expression of ECE as well as ET to attenuate vascular tone and inhibit the proliferation of vascular smooth muscle cells.     

Sphingosine kinase 1 expression is regulated by signaling through PI3K, AKT2, and mTOR in human coronary artery smooth muscle cells

Sphingosine kinase 1 (SphK1) is a lipid kinase implicated in mitogenic signaling pathways in vascular smooth muscle cells. We demonstrate that human coronary artery smooth muscle (HCASM) cells require SphK1 for growth and that SphK1 mRNA and protein levels are elevated in PDGF stimulated HCASM cells. To determine the mechanism of PDGF-induced SphK1 expression, we used pharmacological inhibitors of the PI3K/AKT/mTOR signaling pathway. Wortmannin, SH-5, and rapamycin significantly blocked PDGF-stimulated induction of SphK1 mRNA and protein expression, indicating a regulatory role of the PI3K/AKT/mTOR pathway in SphK1 expression. To determine which isoform of AKT regulates SphK1 mRNA and protein levels, siRNAs specific for AKT1, AKT2, and AKT3 were used. We show that AKT2 siRNA significantly blocked PDGF-stimulated increases in SphK1 mRNA and protein expression levels as well as SphK1 enzymatic activity levels. In contrast, AKT1 or AKT3 siRNA did not have an effect. Together, these results demonstrate that the PI3K/AKT/mTOR signaling pathway is involved in regulation of SphK1, with AKT2 playing a key role in PDGF-induced SphK1 expression in HCASM cells.     

Shear stress-dependent expression of apoptosis-regulating genes in endothelial cells

Laminar shear stress exerts potent anti-apoptotic effects. Therefore, we analyzed the influence of laminar shear stress on the expression of apoptosis-regulating genes in human umbilical vein endothelial cells (HUVEC). Application of high levels of laminar shear stress (15 and 30 dyn/cm(2)) decreased the susceptibility of HUVEC to undergo apoptosis, whereas low shear stress (1 dyn/cm(2)) had no effect. These diminished signs of apoptosis were accompanied by a decreased mRNA expression of apoptosis-inducing Fas receptor. Furthermore, mRNA and protein expression of anti-apoptotic, soluble Fas isoform FasExo6Del and anti-apoptotic Bcl-x(L) were induced. Surprisingly, high shear stress also elevated mRNA and protein expression of pro-apoptotic Bak. The shear stress-induced up-regulation of Bcl-x(L) and Bak mRNA can be abrogated by inhibition of the endothelial NO synthase. We propose that altered expression of Bcl-x(L) and the Fas system is involved in the protective effect of laminar shear stress against apoptosis in human endothelial cells.     

Sphingosine kinase 1 expression is regulated by signaling through PI3K, AKT2, and mTOR in human coronary artery smooth muscle cells

Sphingosine kinase 1 (SphK1) is a lipid kinase implicated in mitogenic signaling pathways in vascular smooth muscle cells. We demonstrate that human coronary artery smooth muscle (HCASM) cells require SphK1 for growth and that SphK1 mRNA and protein levels are elevated in PDGF stimulated HCASM cells. To determine the mechanism of PDGF-induced SphK1 expression, we used pharmacological inhibitors of the PI3K/AKT/mTOR signaling pathway. Wortmannin, SH-5, and rapamycin significantly blocked PDGF-stimulated induction of SphK1 mRNA and protein expression, indicating a regulatory role of the PI3K/AKT/mTOR pathway in SphK1 expression. To determine which isoform of AKT regulates SphK1 mRNA and protein levels, siRNAs specific for AKT1, AKT2, and AKT3 were used. We show that AKT2 siRNA significantly blocked PDGF-stimulated increases in SphK1 mRNA and protein expression levels as well as SphK1 enzymatic activity levels. In contrast, AKT1 or AKT3 siRNA did not have an effect. Together, these results demonstrate that the PI3K/AKT/mTOR signaling pathway is involved in regulation of SphK1, with AKT2 playing a key role in PDGF-induced SphK1 expression in HCASM cells.     

NF-kappaB, but not p38 MAP kinase, is required for TNF-alpha-induced expression of cell adhesion molecules in endothelial cells

In response to inflammation stimuli, tumor necrosis factor-alpha (TNF-alpha) induces expression of cell adhesion molecules (CAMs) in endothelial cells (ECs). Studies have suggested that the nuclear factor-kappaB (NF-kappaB) and the p38 MAP kinase (p38) signaling pathways play central roles in this process, but conflicting results have been reported. The objective of this study is to determine the relative contributions of the two pathways to the effect of TNF-alpha. Our initial data indicated that blockade of p38 activity by chemical inhibitor SB203580 (SB) at 10 microM moderately inhibited TNF-alpha-induced expression of three types of CAMs; ICAM-1, VCAM-1 and E-selectin, indicating that p38 may be involved in the process. However, subsequent analysis revealed that neither 1 microM SB that could completely inhibit p38 nor specific knockdown of p38alpha and p38beta with small interference RNA (siRNA) had an apparent effect, indicating that p38 activity is not essential for TNF-alpha-induced CAMs. The most definitive evidence to support this conclusion was from the experiments using cells differentiated from p38alpha knockout embryonic stem cells. We could show that deletion of p38alpha gene did not affect TNF-alpha-induced ICAM-1 and VCAM-1 expression when compared with wild-type cells. We further demonstrated that inhibition of NF-kappaB completely blocked TNF-alpha-induced expression of ICAM-1, VCAM-1 and E-selectin. Taken together, our results clearly demonstrate that NF-kappaB, but not p38, is critical for TNF-alpha-induced CAM expression. The inhibition of SB at 10 microM on TNF-alpha-induced ICAM-1, VCAM-1 and E-selectin is likely due to the nonspecific effect of SB.     

Inhibition of c-Jun N-terminal kinase attenuates low shear stress-induced atherogenesis in apolipoprotein E-deficient mice

Atherosclerosis begins as local inflammation of arterial walls at sites of disturbed flow, such as vessel curvatures and bifurcations with low shear stress. c-Jun NH?-terminal kinase (JNK) is a major regulator of flow-dependent gene expression in endothelial cells in atherosclerosis. However, little is known about the in vivo role of JNK in low shear stress in atherosclerosis. We aimed to observe the effect of JNK on low shear stress-induced atherogenesis in apolipoprotein E-deficient (ApoE(-/-)) mice and investigate the potential mechanism in human umbilical vein endothelial cells (HUVECs). We divided 84 male ApoE(-/-) mice into two groups for treatment with normal saline (NS) (n = 42) and JNK inhibitor SP600125 (JNK-I) (n = 42). Perivascular shear stress modifiers were placed around the right carotid arteries, and plaque formation was studied at low shear stress regions. The left carotid arteries without modifiers represented undisturbed shear stress as a control. The NS group showed atherosclerotic lesions in arterial regions with low shear stress, whereas the JNK-I group showed almost no atherosclerotic lesions. Corresponding to the expression of proatherogenic vascular cell adhesion molecule 1 (VCAM-1), phospho-JNK (p-JNK) level was higher in low shear stress regions with NS than with JNK-I inhibitor. In HUVECs under low shear stress, siRNA knockdown and SP600125 inhibition of JNK attenuated nuclear factor (NF)-κB activity and VCAM-1 expression. Furthermore, siRNA knockdown of platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31) reduced p-JNK and VCAM-1 levels after low shear stress stimulation. JNK may play a critical role in low shear stress-induced atherogenesis by a PECAM-1-dependent mechanosensory pathway and modulating NF-κB activity and VCAM-1 expression.     

Plaque-prone hemodynamics impair endothelial function in pig carotid arteries

Hemodynamic forces play an active role in vascular pathologies, particularly in relation to the localization of atherosclerotic lesions. It has been established that low shear stress combined with cyclic reversal of flow direction (oscillatory shear stress) affects the endothelial cells and may lead to an initiation of plaque development. The aim of the study was to analyze the effect of hemodynamic conditions in arterial segments perfused in vitro in the absence of other stimuli. Left common porcine carotid segments were mounted into an ex vivo arterial support system and perfused for 3 days under unidirectional high and low shear stress (6 +/- 3 and 0.3 +/- 0.1 dyn/cm(2)) and oscillatory shear stress (0.3 +/- 3 dyn/cm(2)). Bradykinin-induced vasorelaxation was drastically decreased in arteries exposed to oscillatory shear stress compared with unidirectional shear stress. Impaired nitric oxide-mediated vasodilation was correlated to changes in both endothelial nitric oxide synthase (eNOS) gene expression and activation in response to bradykinin treatment. This study determined the flow-mediated effects on native tissue perfused with physiologically relevant flows and supports the hypothesis that oscillatory shear stress is a determinant factor in early stages of atherosclerosis. Indeed, oscillatory shear stress induces an endothelial dysfunction, whereas unidirectional shear stress preserves the function of endothelial cells. Endothelial dysfunction is directly mediated by a downregulation of eNOS gene expression and activation; consequently, a decrease of nitric oxide production and/or bioavailability occurs.     

Fluid shear stress suppresses interleukin 8 production by vascular endothelial cells.

The effects of shear stress on interleukin 8 (IL-8) production by human umbilical vein endothelial cells (HUVEC) were studied by subjecting the HUVEC to a steady flow laminar shear stress of up to 0.7 N/m(2) in a parallel plate flow chamber. Shear stress decreased IL-8 mRNA expression in a dose and time-dependent fashion. High glucose concentrations increased IL-8 mRNA levels in a MAPK-p38-dependent manner, which was suppressed by shear stress. Measurement of IL-8 protein in HUVEC culture media by ELISA demonstrated that IL-8 secretion was also increased by high glucose and suppressed by shear stress. These results suggest that the anti-atherogenic effect of shear stress arises partly from the suppression of the production of IL-8 which has been shown to trigger the adhesion of monocytes to a vascular endothelium and also acts as a mitogen and chemoattractant for vascular smooth muscle cells.