PKC is required for activation of ROCK by RhoA in human endothelial cells

Rho/Rho-kinase (ROCK) complex formation is the only proposed mechanism for ROCK activation. Rho/ROCK and PKC can exhibit a convergence of cellular effects such as suppression of endothelial nitric oxide synthase (eNOS) expression. We, therefore, investigated the role of PKC in RhoA/ROCK complex formation and activation linked to eNOS expression in cultured human umbilical vein endothelial cells. We showed that expression of constitutively active RhoA (Rho63) or ROCK (CAT) suppressed eNOS gene expression. This effect of Rho63 but not that of CAT was abolished by phorbol ester-sensitive PKC depletion. Accordingly, depletion or inhibition of PKC prevented ROCK activation by Rho63 without affecting RhoA/ROCK complex formation. Similarly, suppression of eNOS expression and activation of ROCK, but not RhoA by thrombin were prevented by PKC inhibition or depletion. These results indicate that RhoA/ROCK complex formation alone is not sufficient and PKC is required for RhoA-induced ROCK activation leading to eNOS gene suppression.     

Elastokine-mediated up-regulation of MT1-MMP is triggered by nitric oxide in endothelial cells

Membrane-type I matrix metalloproteinase (MT1-MMP) has been previously reported to be up-regulated in human microvascular endothelial cell-1 line (HMEC) by elastin-derived peptides (elastokines). The aim of the present study was to identify the signaling pathways responsible for this effect. We showed that elastokines such as (VGVAPG)₃ peptide and kappa elastin induced nitric oxide (NO) production in a time-, concentration- and receptor-dependent manner as it could be abolished by lactose and a receptor-derived competitive peptide. As evidenced by the use of NO synthase inhibitors, elastokine-mediated up-regulation of MT1-MMP and pseudotube formation on Matrigel required NO production through activation of the PI₃-kinase/Akt/NO synthase and NO/cGMP/Erk1/2 pathways. Elastokines induced both PI₃-kinase p110γ sub-unit, Akt and Erk1/2 activation, as shown by a transient increase in phospho-Akt and phospho-Erk1/2, reaching a maximum after 5 and 15 min incubation, respectively. Inhibitors of PI₃-kinase and MEK1/2 suppressed elastokine-mediated MT1-MMP expression at both the mRNA and protein levels, and decreased the ability of elastokines to accelerate pseudotube formation. Besides, elastokines mediated a time- and concentration-dependent increase of cGMP, suggesting a link between NO and MT1-MMP expression. This was validated by the use of a guanylyl cyclase inhibitor, a NO donor and a cGMP analog. The guanylyl cyclase inhibitor abolished the stimulatory effect of elastokines on MT1-MMP expression. Inversely, the cGMP analog, mimicked the effect of both elastokines and NO donor in a concentration- and time-dependent manner. Overall, our results demonstrated that such elastokine properties through NO and MT1-MMP may be of importance in the context of tumour progression.     

Hyperketonemia increases monocyte adhesion to endothelial cells and is mediated by LFA-1 expression in monocytes and ICAM-1 expression in endothelial cells

Frequent episodes of hyperketonemia are associated with a higher incidence of vascular disease. The objective of this study was to examine the hypothesis that hyperketonemia increases monocyte-endothelial cell (EC) adhesion and the development of vascular disease in diabetes. Human U937 and THP-1 monocyte cell lines and human umbilical vein endothelial cells (HUVECs) were cultured with acetoacetate (AA) (0-10 mM) or β-hydroxybutyrate (BHB) (0-10 mM) for 24 h prior to evaluating adhesion and adhesion molecule expression. The results demonstrate a significant (P < 0.01) increase in both U937 and THP-1 adhesion to HUVEC monolayers treated with 4 mM AA compared with control. Equal concentrations of BHB resulted in similar increases in monocyte-EC adhesion. Similarly, treatments of AA or BHB to isolated monocytes from human blood also show increases in adhesion to endothelial cells. intercellular adhesion molecule-1 (ICAM-1) was significantly increased on the surface of HUVECs and an increase in total protein expression with AA treatment compared with control. The expression level of lymphocyte function-associated antigen-1 (LFA-1) was increased in monocytes treated with AA, and LFA-1 affinity was altered from low to high affinity following treatment with both AA and BHB. Monocyte adhesion could be blocked when cells were preincubated with an antibody to ICAM-1 or LFA-1. Results also show a significant increase in IL-8 and MCP-1 secretion in monocytes and HUVECs treated with 0-10 mM AA. These results suggest that hyperketonemia can induce monocyte adhesion to endothelial cells and that it is mediated via increased ICAM-1 expression in endothelial cells and increased expression and affinity of LFA-1 in monocytes.     

eNOS activation mediated by AMPK after stimulation of endothelial cells with histamine or thrombin is dependent on LKB1

Reports on the role of AMP-activated protein kinase (AMPK) in thrombin-mediated activation of endothelial nitric-oxide synthase (eNOS) in endothelial cells have been conflicting. Previously, we have shown that under culture conditions that allow reduction of ATP-levels after stimulation, activation of AMPK contributes to eNOS phosphorylation and activation in endothelial cells after treatment with thrombin. In this paper we examined the signaling pathways mediating phosphorylation and activation of eNOS after stimulation of cultured human umbilical vein endothelial cells (HUVEC) with histamine and the role of LKB1-AMPK in the signaling. In Morgan's medium 199 intracellular ATP was lowered by treatment with histamine or the ionophore A23187 while in medium RMPI 1640 ATP was unchanged after identical treatment. In medium 199 inhibition of Ca^+ 2/CaM kinase kinase (CaMKK) by STO-609 only partially inhibited AMPK phosphorylation but after gene silencing of LKB1 with siRNA there was a total inhibition of AMPK phosphorylation by STO-609 after treatment with either histamine or thrombin, demonstrating phosphorylation of AMPK by both upstream kinases, LKB1 and CaMKK. Downregulation of AMPK with siRNA partially inhibited eNOS phosphorylation caused by histamine in cells maintained in medium 199. Downregulation of LKB1 by siRNA inhibited both phosphorylation and activity of eNOS and addition of the AMPK inhibitor Compound C had no further effect on eNOS phosphorylation. When experiments were carried out in medium 1640, STO-609 totally prevented the phosphorylation of AMPK without affecting eNOS phosphorylation. AMPKα2 downregulation resulted in a loss of the integrity of the endothelial monolayer and increased expression of GRP78, indicative of endoplasmic reticular (ER) stress. Downregulation of AMPKα1 had no such effect. The results show that culture conditions affect endothelial signal transduction pathways after histamine stimulation. Under conditions where intracellular ATP is lowered by histamine, AMPK is activated by both LKB1 and CaMKK and, in turn, mediates eNOS phosphorylation in an LKB1 dependent manner. Both AMPKα1 and − α2 are involved in the signaling. Under conditions where intracellular ATP is unchanged after histamine treatment, CaMKK alone activates AMPK and eNOS is phosphorylated and activated independent of AMPK.     

Mitochondrial fragmentation in excitotoxicity requires ROCK activation

Mitochondria morphology constantly changes through fission and fusion processes that regulate mitochondrial function, and it therefore plays a prominent role in cellular homeostasis. Cell death progression is associated with mitochondrial fission. Fission is mediated by the mainly cytoplasmic Drp1, which is activated by different post-translational modifications and recruited to mitochondria to perform its function. Our research and other studies have shown that in the early moments of excitotoxic insult Drp1 must be nitrosylated to mediate mitochondrial fragmentation in neurons. Nonetheless, mitochondrial fission is a multistep process in which filamentous actin assembly/disassembly and myosin-mediated mitochondrial constriction play prominent roles. Here we establish that in addition to nitric oxide production, excitotoxicity-induced mitochondrial fragmentation also requires activation of the actomyosin regulator ROCK. Although ROCK1 has been shown to phosphorylate and activate Drp1, experiments using phosphor-mutant forms of Drp1 in primary cortical neurons indicate that in excitotoxic conditions, ROCK does not act directly on Drp1 to mediate fission, but may act on the actomyosin complex. Thus, these data indicate that a wider range of signaling pathways than those that target Drp1 are amenable to be inhibited to prevent mitochondrial fragmentation as therapeutic option.     

Sirt6 deficiency contributes to mitochondrial fission and oxidative damage in podocytes via ROCK1‐Drp1 signalling pathway

ObjectivesIncreasing evidence suggests that mitochondrial dysfunction is the key driver of angiotensin II (Ang II)‐induced kidney injury. This study was designed to investigate whether Sirtuin 6 (Sirt6) could affect Ang II‐induced mitochondrial damage and the potential mechanisms.Materials and MethodsPodocyte‐specific Sirt6 knockout mice were infused with Ang II and cultured podocytes were stimulated with Ang II to evaluate the effects of Sirt6 on mitochondrial structure and function in podocytes. Immunofluorescence staining was used to detect protein expression and mitochondrial morphology in vitro. Electron microscopy was used to assess mitochondrial morphology in mice. Western blotting was used to quantify protein expression.ResultsMitochondrial fission and decreased Sirt6 expression were observed in podocytes from Ang II‐infused mice. In Sirt6‐deficient mice, Ang II infusion induced increased apoptosis and mitochondrial fragmentation in podocytes than that in Ang II‐infused wild‐type mice. In cultured human podocytes, Sirt6 knockdown exacerbated Ang II‐induced mitochondrial fission, whereas Sirt6 overexpression ameliorated the Ang II‐induced changes in the balance between mitochondrial fusion and fission. Functional studies revealed that Sirt6 deficiency exacerbated mitochondrial fission by promoting dynamin‐related protein 1 (Drp1) phosphorylation. Furthermore, Sirt6 mediated Drp1 phosphorylation by promoting Rho‐associated coiled coil‐containing protein kinase 1 (ROCK1) expression.ConclusionOur study has identified Sirt6 as a vital factor that protects against Ang II‐induced mitochondrial fission and apoptosis in podocytes via the ROCK1‐Drp1 signalling pathway.

Schematic of the molecular action proposed in this study. Schematic depicting that Ang II‐induced sirt6 expression decline promotes mitochondrial fission and podocyte injury through ROCK1‐Drp1 signalling. SIRT6 reduction leads to increased levels of ROCK1, thereby enhancing Drp1 phosphorylation at the Ser637 site. The Drp1 phosphorylation finally results in podocyte injury by inducing mitochondrial fission and apoptosis.     

High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases

High density lipoprotein (HDL) activates endothelial nitric-oxide synthase (eNOS), leading to increased production of the antiatherogenic molecule NO. A variety of stimuli regulate eNOS activity through signaling pathways involving Akt kinase and/or mitogen-activated protein (MAP) kinase. In the present study, we investigated the role of kinase cascades in HDL-induced eNOS stimulation in cultured endothelial cells and COS M6 cells transfected with eNOS and the HDL receptor, scavenger receptor B-I. HDL (10-50 microg/ml, 20 min) caused eNOS phosphorylation at Ser-1179, and dominant negative Akt inhibited both HDL-mediated phosphorylation and activation of the enzyme. Phosphoinositide 3-kinase (PI3 kinase) inhibition or dominant negative PI3 kinase also blocked the phosphorylation and activation of eNOS by HDL. Studies with genistein and PP2 showed that the nonreceptor tyrosine kinase, Src, is an upstream stimulator of the PI3 kinase-Akt pathway in this paradigm. In addition, HDL activated MAP kinase through PI3 kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition fully attenuated eNOS stimulation by HDL without affecting Akt or eNOS Ser-1179 phosphorylation. Conversely, dominant negative Akt did not alter HDL-induced MAP kinase activation. These results indicate that HDL stimulates eNOS through common upstream, Src-mediated signaling, which leads to parallel activation of Akt and MAP kinases and their resultant independent modulation of the enzyme.     

Early response of endothelial cells to flow is mediated by VE-cadherin

Endothelial cells are known to respond to flow onset by increasing actin turnover rate. Current models assume that an increase in the actin turnover rate should result in a rise in cell crawling speed. Here we report that confluent endothelial monolayer shows an unexpected behavior: cell crawling speed decreases by approximately 40% within the first 30 min of flow onset. A drop in crawling speed has not been observed in either subconfluent endothelial cells or in VE-cadherin-deficient cells. We found that flow onset caused an increase in the number of VE-cadherin-GFP molecules in the junctions and elicited changes in the cytoskeleton-associated fractions of alpha, beta -catenins and VE-cadherin. Flow application also increased the strength of interactions of endothelial cells with surfaces coated with recombinant VE-cadherin. These observations suggest that endothelial cell junctional proteins respond to flow transiently by increasing the strength of intercellular attachments early after flow onset and support the view on the active role of intercellular adhesions in mechanotransduction.     

Synthesis of an endogeneous lectin, galectin-1, by human endothelial cells is up-regulated by endothelial cell activation

The pattern of expression of an endogenous lectin, galectin-1, was examined in human lymphoid tissue. Galectin-1 was detected in the endothelial cells lining specialized vessels, termed high endothelial venules, in activated lymphoid tissue, but not in a resting lymph node. Cultured endothelial cells (human aortic and umbilical vein endothelial cells (HAECs and HUVECs)) expressed galectin-1. Activation of the cultured endothelial cells increased the level of galectin-1 expression, as determined by ELISA, Northern blot analysis and high throughput cDNA sequencing. These results suggest that galectin-1 expressed by endothelial cells may bind to and affect the trafficking of cells emigrating from blood into tissues.     

Mitochondrial Dok-4 recruits Src kinase and regulates NF-kappaB activation in endothelial cells

The downstream of kinase (Dok) family of adapter proteins consists of at least five members structurally characterized by an NH2-terminal tandem of conserved pleckstrin homology and phosphotyrosine binding domains linked to a unique COOH-terminal region. To determine the role of the novel adapter protein Dok-4 in endothelial cells, we first investigated the cell localization of Dok-4. Most surprisingly, immunofluorescence microscopy, cell fractionation studies, and studies with enhanced green fluorescent protein chimeras showed that wild type Dok-4 (Dok-4-WT) specifically localized in mitochondria. An NH2-terminal deletion mutant of Dok-4 (Dok-4-(deltaN11-29)), which lacks the mitochondrial targeting sequence, could not accumulate in mitochondria. Co-immunoprecipitation revealed an interaction of c-Src with Dok-4-WT in endothelial cells. Most interestingly, overexpression of Dok-4-WT, but not Dok-4-(deltaN1-99), increased mitochondrial c-Src expression, whereas knock-down of endogenous Dok-4 with a small interfering RNA vector greatly inhibited mitochondrial localization of c-Src, suggesting a unique function for Dok-4 as an anchoring protein for c-Src in mitochondria. Dok-4-WT significantly decreased 39-kDa subunit complex I expression. PP2, a specific Src kinase inhibitor, prevented the Dok-4-mediated complex I decrease, suggesting the involvement of Src kinase in regulation of complex I expression. Dok-4-WT enhanced tumor necrosis factor-alpha (TNF-alpha)-mediated reactive oxygen species (ROS) production, supporting the functional relevance of a Dok-4-Src-complex I/ROS signaling pathway in mitochondria. Finally, Dok-4 enhanced TNF-alpha-mediated NF-kappaB activation, whereas this was inhibited by transfection with Dok-4 small interfering RNA. In addition, Dok-4-induced NF-kappaB activation was also inhibited by transfection of a dominant negative form of c-Src. These data suggest a role for mitochondrial Dok-4 as an anchoring molecule for the tyrosine kinase c-Src, and in turn as a regulator of TNF-alpha-mediated ROS production and NF-kappaB activation.     

Downregulation of VEGF-D expression by interleukin-1beta in cardiac microvascular endothelial cells is mediated by MAPKs and PKCalpha/beta1

Interleukin-1beta (IL-1beta) is a proinflammatory cytokine increased in the heart following myocardial infarction. Vascular endothelial growth factors (VEGFs) are implicated in angiogenesis due to their involvement in the recruitment and proliferation of endothelial cells. Here we studied expression of VEGFs in response to IL-1beta in rat cardiac microvascular endothelial cells (CMECs) and investigated the signaling pathways involved in the regulation of VEGF-D. cDNA array analysis indicated that IL-1beta modulates the expression of numerous angiogenesis-related genes, notably decreasing the expression of VEGF-D. RT-PCR and Western blot analyses confirmed decreased expression of VEGF-D in response to IL-1beta. IL-1beta decreased the expression of VEGF-C to a lesser extent with no effects on VEGF-A or -B. Inhibition of ERK1/2, JNKs, or PKCalpha/beta1 alone partially inhibited IL-1beta-induced VEGF-D downregulation. Concurrent inhibition of ERK1/2 or JNKs and PKCalpha/beta1 resulted in a synergistic inhibition of IL-1beta-induced decreases in VEGF-D. Inhibition of ERK1/2 partially inhibited IL-1beta-stimulated inactivation of GSK-3beta with no effect on beta-catenin levels. Inhibition of GSK-3beta using SB216763 inhibited basal VEGF-D expression. We conclude that IL-1beta downregulates VEGF-D expression in CMECs via the involvement of ERK1/2, JNKs, and PKCalpha/beta(1). This is the first report to indicate inhibition of VEGF-D gene expression in response to IL-1beta in cardiac microvascular endothelial cells, a cell type of central interest in angiogenesis.     

The mammalian formin FHOD1 is activated through phosphorylation by ROCK and mediates thrombin-induced stress fibre formation in endothelial cells

Formin-family proteins, in the active state, form actin-based structures such as stress fibres. Their activation mechanisms, however, are largely unknown except that mDia and its closely related formins can be activated by direct binding of the small GTPase Rho or Cdc42. Here we show that the Rho-dependent protein kinase ROCK phosphorylates the C-terminal residues Ser1131, Ser1137, and Thr1141 of formin homology domain protein 1 (FHOD1), a major endothelial formin that is normally autoinhibited by intramolecular interaction between the N- and C-terminal regions. Phosphorylation of FHOD1 at the three residues fully disrupts the autoinhibitory interaction, which culminates in formation of stress fibres. We also demonstrate that, in vascular endothelial cells, thrombin, a vasoactive substance leading to Rho activation, elicits both FHOD1 phosphorylation and stress fibre formation in a ROCK-dependent manner, and that FHOD1 depletion by RNA interference impairs thrombin-induced stress fibre formation. Based on these findings we propose a novel mechanism for activation of formin-family proteins: ROCK, activated by G protein-coupled receptor ligands such as thrombin, directly phosphorylates FHOD1 at the C-terminal region, which renders this formin in the active form, leading to stress fibre formation.     

Angiotensin II-induced MMP-2 release from endothelial cells is mediated by TNF-alpha

Angiotensin II (ANG II) has been etiologically linked to vascular disease; however, its role in the alterations of endothelial function that occur in vascular disorders is not completely understood. Matrix metalloproteinases (MMPs) and proinflammatory cytokines are involved in the pathological remodeling of blood vessels that occurs in vascular disease. In this study we evaluated the effects of ANG II on tumor necrosis factor (TNF)- and MMP-2 production in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with ANG II (0.1–10 µM) for 24 h, in the presence or absence of antagonists of ANG II type 1 (AT₁R) and type 2 (AT₂R) receptors, and the production and release of TNF- and MMP-2 were assessed. ANG II increased TNF- mRNA and protein expression and the release of bioactive TNF-. Moreover, ANG II induced MMP-2 release and reduced the secretion of tissue inhibitor of MMP (TIMP)-2 from endothelial cells. To elucidate whether endogenous TNF- could mediate the effects of ANG II on MMP-2 release, cells were pretreated with anti-TNF- neutralizing antibodies or pentoxifylline (an inhibitor of TNF- synthesis). TNF- inhibition prevented the secretion of MMP-2 induced by ANG II. Furthermore, AT₁R antagonism with candesartan prevented the formation of MMP-2 and TNF- and the reduction of TIMP-2 induced by ANG II. These results indicate that ANG II, via AT₁R, modulates the secretion of TNF- and MMP-2 from endothelial cells and that TNF- mediates the effects of ANG II on MMP-2 release. remodeling; vasoactive mediators; inflammation     

Mitochondrial fusion in human cells is efficient,requires the inner membrane potential,and is mediated by mitofusins

Mitochondrial fusion remains a largely unknown process despite its observation by live microscopy and the identification of few implicated proteins. Using green and red fluorescent proteins targeted to the mitochondrial matrix, we show that mitochondrial fusion in human cells is efficient and achieves complete mixing of matrix contents within 12 h. This process is maintained in the absence of a functional respiratory chain, despite disruption of microtubules or after significant reduction of cellular ATP levels. In contrast, mitochondrial fusion is completely inhibited by protonophores that dissipate the inner membrane potential. This inhibition, which results in rapid fragmentation of mitochondrial filaments, is reversible: small and punctate mitochondria fuse to reform elongated and interconnected ones upon withdrawal of protonophores. Expression of wild-type or dominant-negative dynamin-related protein 1 showed that fragmentation is due to dynamin-related protein 1-mediated mitochondrial division. On the other hand, expression of mitofusin 1 (Mfn1), one of the human Fzo homologues, increased mitochondrial length and interconnectivity. This process, but not Mfn1 targeting, was dependent on the inner membrane potential, indicating that overexpressed Mfn1 stimulates fusion. These results show that human mitochondria represent a single cellular compartment whose exchanges and interconnectivity are dynamically regulated by the balance between continuous fusion and fission reactions.     

血管内皮细胞介导的纤溶作用

血管内皮细胞可合成和释放tPA、uPA和PAI-1,也提供纤溶成份之间相互作用的活性表面,在纤溶的启动和调节中起重要作用。内皮细胞介导的纤溶作用受到神经激素,生长因子、血浆成份、血细胞、血管壁基质和许多外源性物质的调控。     

VEGF-induced Rac1 activation in endothelial cells is regulated by the guanine nucleotide exchange factor Vav2

Vascular endothelial growth factor (VEGF) signaling is critical for both normal and disease-associated vascular development. Dysregulated VEGF signaling has been implicated in ischemic stroke, tumor angiogenesis, and many other vascular diseases. VEGF signals through several effectors, including the Rho family of small GTPases. As a member of this family, Rac1 promotes VEGF-induced endothelial cell migration by stimulating the formation of lamellipodia and membrane ruffles. To form these membrane protrusions, Rac1 is activated by guanine nucleotide exchange factors (GEFs) that catalyze the exchange of GDP for GTP. The goal of this study was to identify the GEF responsible for activating Rac1 in response to VEGF stimulation. We have found that VEGF stimulates biphasic activation of Rac1 and for these studies we focused on the peak of activation that occurs at 30 min. Inhibition of VEGFR-2 signaling blocks VEGF-induced Rac1 activation. Using a Rac1 nucleotide-free mutant (G15ARac1), which has a high affinity for binding activated GEFs, we show that the Rac GEF Vav2 associates with G15ARac1 after VEGF stimulation. Additionally, we show that depleting endothelial cells of endogenous Vav2 with siRNA prevents VEGF-induced Rac1 activation. Moreover, Vav2 is tyrosine phosphorylated upon VEGF treatment, which temporally correlates with Rac1 activation and requires VEGFR-2 signaling and Src kinase activity. Finally, we show that depressing Vav2 expression by siRNA impairs VEGF-induced endothelial cell migration. Taken together, our results provide evidence that Vav2 acts downstream of VEGF to activate Rac1.