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.     

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.     

Shear stress enhances human endothelial cell wound closure in vitro

Repair of the endothelium occurs in the presence of continued blood flow, yet the mechanisms by which shear forces affect endothelial wound closure remain elusive. Therefore, we tested the hypothesis that shear stress enhances endothelial cell wound closure. Human umbilical vein endothelial cells (HUVEC) or human coronary artery endothelial cells (HCAEC) were cultured on type I collagen-coated coverslips. Cell monolayers were sheared for 18 h in a parallel-plate flow chamber at 12 dyn/cm(2) to attain cellular alignment and then wounded by scraping with a metal spatula. Subsequently, the monolayers were exposed to a laminar shear stress of 3, 12, or 20 dyn/cm(2) under shear-wound-shear (S-W-sH) or shear-wound-static (S-W-sT) conditions for 6 h. Wound closure was measured as a percentage of original wound width. Cell area, centroid-to-centroid distance, and cell velocity were also measured. HUVEC wounds in the S-W-sH group exposed to 3, 12, or 20 dyn/cm(2) closed to 21, 39, or 50%, respectively, compared with only 59% in the S-W-sT cells. Similarly, HCAEC wounds closed to 29, 49, or 33% (S-W-sH) compared with 58% in the S-W-sT cells. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate. These results suggest that physiological levels of shear stress enhance endothelial repair.     

Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase

Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10-20 dyn/cm(2)) in conditionally immortalized human GEnC. Laminar shear stress caused an orientation of GEnC and stress fibers parallel to the direction of flow and induced Akt and eNOS phosphorylation along with NO production. Inhibition of the phophatidylinositol (PI)3-kinase/Akt pathway attenuated laminar shear stress-induced eNOS phosphorylation and NO production. Laminar shear stress of 10 dyn/cm(2) had a dramatic effect on GEnC permeability, reversibly decreasing the electrical resistance across GEnC monolayers. Finally, the laminar shear stress-induced reduction in electrical resistance was attenuated by the NOS inhibitors l-N(G)-monomethyl arginine (l-NMMA) and l-N(G)-nitroarginine methyl ester (l-NAME) and also by inhibition of the PI3-kinase/Akt pathway. Hence we have shown for GEnC in vitro that acute permeability responses to laminar shear stress are dependent on NO, produced via activation of the PI3-kinase/Akt pathway and increased eNOS phosphorylation. These results suggest the importance of laminar shear stress and NO in regulating the contribution of GEnC to the permeability properties of the glomerular capillary wall.     

Resistance to induced apoptosis in the human neuroblastoma cell line SK-N-SH in relation to neuronal differentiation. Role of Bcl-2 protein family.

Much evidence suggests that apoptosis plays a crucial role in cell population homeostasis that depends on the expression of various genes implicated in the control of cell life and death. The sensitivity of human neuroblastoma cells SK-N-SH to undergo apoptosis induced by thapsigargin was examined. SK-N-SH were previously differentiated into neuronal cells by treatments with retinoic acid (RA), 4 beta-phorbol 12-myristate 13-acetate (PMA) which increases protein kinase C (PKC) activity, and staurosporine which decreases PKC activity. Neuronal differentiation was evaluated by gamma-enolase, microtubule associated protein 2 (MAP2) and synaptophysin immunocytochemistry. The sensitivity of the cells to thapsigargin-induced apoptosis was evaluated by cell viability and nuclear fragmentation (Hoechst 33258) and compared with pro-(Bcl-2, Bcl-x(L)) and anti-apoptotic (Bax, Bak) protein expression of the Bcl-2 family. Cells treated with RA and PMA were more resistant to apoptosis than controls. Conversely, the cells treated with staurosporine were more susceptible to apoptosis. In parallel with morphological modifications, the expression of inhibitors and activators of apoptosis was directly dependent upon the differentiating agent used. Bcl-2 expression was strongly increased by PMA and drastically decreased by staurosporine as was Bcl-x(L) expression. Bax and Bak expression were not significantly modified. These results demonstrate that drugs that modulate PKC activity may induce a modification of Bcl-2 expression as well as resistance to the apoptotic process. Furthermore, the expression of Bcl-2 was reduced by toxin B from Clostridium difficile and, to a lesser extent, by wortmannin suggesting a role of small G-protein RhoA and PtdIns3 kinase in the control of Bcl-2 expression. Our data demonstrate a relationship between the continuous activation of PKC, the expression of Bcl-2 protein family and the resistance of differentiated SK-N-SH to apoptosis.     

p14(ARF)-induced apoptosis in p53 protein-deficient cells is mediated by BH3-only protein-independent derepression of Bak protein through down-regulation of Mcl-1 and Bcl-xL proteins

The p14(ARF) tumor suppressor plays a central role in regulating cell cycle arrest and apoptosis. We reported previously that p14(ARF) is capable of triggering apoptosis in a p53-independent manner. However, the mechanism remained unclear. Here we demonstrate that the p53-independent activation of the mitochondrial apoptosis pathway by p14(ARF) is primarily mediated by the pro-apoptotic Bax-homolog Bak. Expression of p14(ARF) exclusively triggers a N-terminal conformational switch of Bak, but not Bax, which allows for mitochondrial permeability shift, release of cytochrome c, activation of caspases, and subsequent fragmentation of genomic DNA. Although forced expression of Bak markedly sensitizes toward p14(ARF)-induced apoptosis, re-expression of Bax has no effect. Vice versa, knockdown of Bak by RNA interference attenuates p14(ARF)-induced apoptosis, whereas down-regulation of Bax has no effect. Bak activation coincides with a prominent, caspase-independent deprivation of the endogenous Bak inhibitors Mcl-1 and Bcl-x(L). In turn, mitochondrial apoptosis is fully blocked by overexpression of either Mcl-1 or Bcl-x(L). Taken together, these data indicate that in the absence of functional p53 and Bax, p14(ARF) triggers mitochondrial apoptosis signaling by activating Bak, which is facilitated by down-regulating anti-apoptotic Mcl-1 and Bcl-x(L). Moreover, our data suggest that the simultaneous inhibition of two central endogenous Bak inhibitors, i.e. Mcl-1 and Bcl-x(L), may be sufficient to activate mitochondrial apoptosis in the absence of BH3-only protein regulation.     

KLF2-dependent, shear stress-induced expression of CD59: a novel cytoprotective mechanism against complement-mediated injury in the vasculature

Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress led us to explore its influence on endothelial cell expression of complement inhibitory proteins CD59 and decay-accelerating factor. Human umbilical vein and aortic endothelial cells were exposed to laminar shear stress (12 dynes/cm(2)) or disturbed flow (+/- 5 dynes/cm(2) at 1Hz) in a parallel plate flow chamber. Laminar shear induced a flow rate-dependent increase in steady-state CD59 mRNA, reaching 4-fold at 12 dynes/cm(2). Following 24-48 h of laminar shear stress, cell surface expression of CD59 was up-regulated by 100%, whereas decay-accelerating factor expression was unchanged. The increase in CD59 following laminar shear was functionally significant, reducing C9 deposition and complement-mediated lysis of flow-conditioned endothelial cells by 50%. Although CD59 induction was independent of PI3-K, ERK1/2 and nitric oxide, an RNA interference approach demonstrated dependence upon an ERK5/KLF2 signaling pathway. In contrast to laminar shear stress, disturbed flow failed to induce endothelial cell CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional laminar shear stress, when compared with atheroprone areas exposed to disturbed flow. We propose that up-regulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement-mediated injury and protects from atherogenesis in regions of laminar shear stress.     

Distinct mechanisms of cardiomyocyte apoptosis induced by doxorubicin and hypoxia converge on mitochondria and are inhibited by Bcl-xL

Hypoxia and doxorubicin can cause cardiotoxicity and loss of myocardial function. These effects are due, in part, to an induction of apoptosis. Herein we identify the apoptotic pathways activated in H9c2 cells in response to hypoxia (O(2)/N(2)/CO(2), 0.5:94.5:5) and doxorubicin (0.5 muM). Although the apoptosis induced was accompanied by induction of Fas and Fas ligand, the death receptor pathway was not critical for caspase activation by either stimulus. Hypoxia induced the expression of endoplasmic reticulum (ER) stress mediators and processed ER-resident pro-caspase-12 whereas doxorubicin did not induce an ER stress response. Most importantly, both stimuli converged on mitochondria to promote apoptosis. Accumulation of cytochrome c in the cytosol coincided with the processing of pro-caspase-9 and -3. Increasing the expression of the anti-apoptotic protein Bcl-x(L), either by dexamethasone or adenovirus-mediated transduction, protected H9c2 cells from doxorubicin- and hypoxia-induced apoptosis. Bcl-x(L) attenuated mitochondrial cytochrome crelease and reduced downstream pro-caspase processing and apoptosis. These data demonstrate that two distinct cardiomyocyte-damaging stimuli converge on mitochondria thus presenting this organelle as a potentially important therapeutic target for anti-apoptotic strategies for cardiovascular diseases.     

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.     

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.     

Temporal gradient in shear-induced signaling pathway: involvement of MAP kinase, c-fos, and connexin43

The effect of a temporal gradient in shear and steady shear on the activity of extracellular signal-regulated protein kinases 1 and 2 (ERK1/ERK2), c-fos, and connexin43 (Cx43) in human endothelial cells was investigated. Three laminar flow profiles (16 dyn/cm(2)), including impulse flow (shear stress abruptly applied for 3 s), ramp flow (shear stress smoothly transitioned at flow onset), and step flow (shear stress abruptly applied at flow onset) were utilized. Relative to static controls, impulse flow stimulated the phosphorylation of ERK1/ERK2 8.5- to 7.5-fold, respectively at 10 min, as well as the mRNA expression of c-fos 51-fold at 30 min, and Cx43 8-fold at 90 min. These high levels of mRNA expression were sustained for at least 4 h. In contrast, ramp flow was unable to significantly induce gene expression and even inhibited the activation of ERK1/ERK2. Step flow, which contains both a sharp temporal gradient in shear stress and a steady shear component, elicited only moderate and transient responses, indicating the distinct role of these fluid shear stimuli in endothelial signal transduction. The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 inhibited impulse flow-induced c-fos and Cx43 mRNA expression. Thus these findings implicate the involvement of ERK1/ERK2, c-fos, and Cx43 in the signaling pathway induced by the temporal gradient in shear.     

IL-8 directly enhanced endothelial cell survival,proliferation, and matrix metalloproteinases production and regulated angiogenesis

IL-8, a member of the chemokine family, has been shown to play an important role in tumor growth, angiogenesis, and metastasis. The objective of this study was to determine the mechanism of IL-8-mediated angiogenesis. We examined the direct role of IL-8 in angiogenesis by examining IL-8 receptor expression on endothelial cells and their proliferation, survival, and matrix metalloproteinases (MMPs) production. We demonstrate that HUVEC and human dermal microvascular endothelial cells constitutively express CXCR1 and CXCR2 mRNA and protein. Recombinant human IL-8 induced endothelial cell proliferation and capillary tube organization while neutralization of IL-8 by anti-IL-8 Ab blocks IL-8-mediated capillary tube organization. Incubation of endothelial cells with IL-8 inhibited endothelial cell apoptosis and enhanced antiapoptotic gene expression. Endothelial cells incubated with IL-8 had higher levels of Bcl-x(L):Bcl-x(S) and Bcl-2:Bax ratios. Furthermore, incubation of endothelial cells with IL-8 up-regulated MMP-2 and MMP-9 production and mRNA expression. Our data suggest that IL-8 directly enhanced endothelial cell proliferation, survival, and MMP expression in CXCR1- and CXCR2-expressing endothelial cells and regulated angiogenesis.     

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.