Insulin induces the release of vasodilator compounds from platelets by a nitric oxide-G kinase-VAMP-3-dependent pathway.

Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process. Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS-/- mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of alphaIIbbeta3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO-G kinase-dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.

Inhibition of endothelial nitric oxide synthase activity by proline-rich tyrosine kinase 2 in response to fluid shear stress and insulin.

In native and primary cultures of endothelial cells, fluid shear stress elicits the tyrosine phosphorylation of the endothelial NO synthase (eNOS), however, the consequences of this modification on enzyme activity are unclear. We found that fluid shear stress induces the association of eNOS with the proline-rich tyrosine kinase 2 (PYK2) in endothelial cells and that the eNOS immunoprecipitated from eNOS- and PYK2-overexpressing HEK293 cells was tyrosine-phosphorylated on Tyr657. In mouse carotid arteries, the overexpression of wild-type PYK2, but not a dominant-negative PYK2, decreased eNOS activity (approximately 50%), whereas in murine lung endothelial cells, the downregulation of PYK2 (small interfering RNA) increased ionomycin-induced NO production. Mutation of Tyr657 to the phosphomimetic residues aspartate (D) or glutamate (E) abolished enzyme activity, whereas a nonphosphorylatable mutant (phenylalanine [F]) showed activity comparable to the wild-type enzyme. Moreover, normal flow-induced vasodilatation was apparent in carotid arteries from eNOS(-/-) mice overexpressing either the wild-type eNOS or the Y657F mutant, whereas no flow-induced vasodilatation was apparent in arteries expressing the Y657E eNOS mutant. Insulin also activated PYK2 and stimulated eNOS in endothelial cells expressing the Y657F mutant but not wild-type eNOS. These data indicate that PYK2 mediates the tyrosine phosphorylation of eNOS on Tyr657 in response to fluid shear stress and insulin stimulation and that this modification attenuates the activity of the enzyme. The PYK2-dependent inhibition of NO production may serve to keep eNOS activity low and limit the detrimental consequences of maintained high NO output, ie, the generation of peroxynitrite.

Platelet sarcoplasmic endoplasmic reticulum Ca2+-ATPase and mu-calpain activity are altered in type 2 diabetes mellitus and restored by rosiglitazone.

BACKGROUND: Platelets from patients with type 2 diabetes mellitus display hyperaggregability and increased thrombogenic potential. METHODS AND RESULTS: In platelets from patients with type 2 diabetes mellitus, we found enhanced tyrosine nitration and inactivation of the sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA-2), elevated platelet [Ca2+]i, and activation of mu-calpain. The tyrosine nitration of SERCA-2 and the activation of mu-calpain in vitro in platelets from healthy volunteers could be evoked in vitro by peroxynitrite. Platelet endothelial cell adhesion molecule-1 was identified as a mu-calpain substrate; its in vitro degradation was stimulated by peroxynitrite and prevented by calpain inhibitors. Calpain activation also was linked to hyperresponsiveness to thrombin and the loss of platelet sensitivity to nitric oxide synthase inhibitors. Platelets from patients with type 2 diabetes mellitus (hemoglobin A1c >6.6%) contained little or no intact platelet endothelial cell adhesion molecule-1, whereas degradation products were detectable. The peroxisome proliferator-activated receptor-gamma agonist rosiglitazone increased SERCA-2 expression in megakaryocytes, and treating patients with type 2 diabetes mellitus with rosiglitazone for 12 weeks increased platelet SERCA-2 expression and Ca2+-ATPase activity, decreased SERCA-2 tyrosine nitration, and normalized platelet [Ca2+]i. Rosiglitazone also reduced mu-calpain activity, normalized platelet endothelial cell adhesion molecule-1 levels, and partially restored platelet sensitivity to nitric oxide synthase inhibition. CONCLUSIONS: These data identify megakaryocytes/platelets as additional cellular targets for peroxisome proliferator-activated receptor-gamma agonists and highlight potential benefits of rosiglitazone therapy in cardiovascular diseases.

Epoxyeicosatrienoic acids and the soluble epoxide hydrolase are determinants of pulmonary artery pressure and the acute hypoxic pulmonary vasoconstrictor response.

Recent findings have indicated a role for cytochrome P-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in acute hypoxic pulmonary vasoconstriction (HPV). Given that the intracellular concentration of EETs is determined by the soluble epoxide hydrolase (sEH), we assessed the influence of the sEH and 11,12-EET on pulmonary artery pressure and HPV in the isolated mouse lung. In lungs from wild-type mice, HPV was significantly increased by sEH inhibition, an effect abolished by pretreatment with CYP epoxygenase inhibitors and the EET antagonist 14,15-EEZE. HPV and EET production were greater in lungs from sEH(-/-) mice than from wild-type mice and sEH inhibition had no further effect on HPV, while MSPPOH and 14,15-EEZE decreased the response. 11,12-EET increased pulmonary artery pressure in a concentration-dependent manner and enhanced HPV via a Rho-dependent mechanism. Both 11,12-EET and hypoxia elicited the membrane translocation of a transient receptor potential (TRP) C6-V5 fusion protein, the latter effect was sensitive to 14,15-EEZE. Moreover, while acute hypoxia and 11,12-EET increased pulmonary pressure in lungs from TRPC6(+/-) mice, lungs from TRPC6(-/-) mice did not respond to either stimuli. These data demonstrate that CYP-derived EETs are involved in HPV and that EET-induced pulmonary contraction under normoxic and hypoxic conditions involves a TRPC6-dependent pathway.

Fluid shear stress and NO decrease the activity of the hydroxy-methylglutaryl coenzyme A reductase in endothelial cells via the AMP-activated protein kinase and FoxO1.

The rate-limiting enzyme for cholesterol synthesis, the hydroxy-methylglutaryl coenzyme A reductase (HCR), is phosphorylated by the AMP-activated protein kinase (AMPK). As shear stress activates the AMPK in endothelial cells, we determined whether it affects HCR activity and subsequent HCR-dependent signaling. Shear stress (12 dynes cm(-2)) rapidly increased the phosphorylation and activity (6.5- and 4-fold, respectively) of the AMPK in cultured endothelial cells and the activated AMPK phosphorylated the HCR in vitro. Moreover, shear stress and the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) attenuated endothelial HCR activity by 37% and 33%, respectively. Inhibition of NO production attenuated the acute shear stress-induced phosphorylation of the AMPK and the decrease in HCR activity. Prolonged shear stress (18 hours) led to a significant (50%) decrease in HCR mRNA expression that was dependent on NO, AMPK, and the subsequent phosphorylation and degradation of FoxO1a. Correspondingly, the downregulation of FoxO (small interfering RNA) decreased HCR expression. Prolonged shear stress also attenuated the bradykinin-induced activation of Ras and extracellular signal-regulated kinase 1/2, a phenomenon that was comparable to the effects of cerivastatin and that was reversed by mevalonate and thus attributed to HCR inhibition. A decrease (35%) in HCR expression was also detected in femoral arteries from mice following voluntary exercise, and the bradykinin-induced vasodilatation of the mouse hindlimb was attenuated by both exercise and the HCR inhibitor cerivastatin. These data indicate that fluid shear stress regulates the activity and expression of the HCR in endothelial cells and determines responsiveness to stimuli, such as bradykinin via a mechanism involving NO, AMPK, FoxO1a, and p21Ras.

Cytochrome P450 2C9-induced angiogenesis is dependent on EphB4.

OBJECTIVE: Cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) are known to stimulate angiogenesis, but the mechanisms involved are incompletely understood. Because EphB4 is involved in vascular development, the aim of this study was to investigate whether, and to what extent, EphB4 is part of the signaling cascade that results in CYP2C9-mediated angiogenesis. METHODS AND RESULTS: CYP2C9 overexpression as well as stimulation with 11,12-EET (up to 48 hours) time-dependently increased EphB4 expression in endothelial cells. This effect and the activation of the EphB4 promoter were mediated by the phosphatidylinositol-3-kinase (P13-K)/Akt pathway and sensitive to the P13-K inhibitor LY 294002 as well as to simultaneous transfection with dominant-negative Akt. 11,12-EET treatment also increased EphB4 expression in isolated mouse mesenteric arteries as well as in the vessels that developed in 11,12-EET-impregnated Matrigel plugs. Moreover, the CYP2C9-stimulated formation of capillary-like structures in a modified spheroid assay was markedly attenuated by EphB4 downregulation (antisense oligonucleotides). Using a parallel approach in vivo, the inclusion of siRNA directed against EphB4 in EET-impregnated Matrigel plugs prevented endothelial cell invasion and vascularization. CONCLUSIONS: Our data indicate that EphB4 is a critical component of the CYP2C9- and 11,12-EET-activated signaling cascade that promotes angiogenesis in vitro as well as in vivo.

Shear stress-induced activation of the AMP-activated protein kinase regulates FoxO1a and angiopoietin-2 in endothelial cells.

AIMS: Phosphorylation of forkhead box O (FoxO) transcription factors induces their nuclear exclusion and proteosomal degradation. Here, we investigated the effect of fluid shear stress on FoxO1a in primary cultures of human endothelial cells and the kinases that regulate its phosphorylation. METHODS AND RESULTS: Shear stress (12 dynes/cm2) elicited the phosphorylation, nuclear exclusion, and degradation of FoxO1a. Inhibition of Akt signalling using either a dominant negative (DN) mutant of Akt or downregulation of Gab1 largely failed to affect the shear stress-induced changes in FoxO1a, while a DN-AMP-activated protein kinase (AMPK) abrogated its shear stress-induced phosphorylation and degradation. Similar effects were observed using the AMPK inhibitor compound C. Moreover, in an in vitro assay, the AMPK directly phosphorylated FoxO1a. As FoxO1a regulates the expression of angiopoietin-2 (Ang-2), we determined the role of shear stress and the AMPK in this phenomenon. Not only did the DN-AMPK increase the expression of Ang-2 in cells maintained under static conditions, it also abrogated the shear stress-induced decrease in FoxO1a and Ang-2 protein levels. Functionally, Ang-2 sensitizes endothelial cells to the effects of tumour necrosis factor (TNF)-alpha, and DN-AMPK increased basal endothelial cell E-selectin expression and permeability as well as the increase induced by TNF-alpha. CONCLUSION: These data indicate that the AMPK activated by fluid shear stress is a novel regulator of FoxO1a phosphorylation and protein levels. Moreover, as the AMPK-dependent phosphorylation and degradation of FoxO1a attenuates Ang-2 expression and protects against the pro-inflammatory actions of TNF-alpha, this kinase may be a useful target to prevent the progression of vascular diseases.

Chemotaxis and differentiation of human adipose tissue CD34+/CD31- progenitor cells: role of stromal derived factor-1 released by adipose tissue capillary endothelial cells.

The native CD34+/CD31- cell population present in the stroma-vascular fraction of human adipose tissue (hAT) displays progenitor cell properties since they exhibit adipocyte- and endothelial cell-like phenotypes under appropriate stimuli. To analyze the signals within hAT regulating their phenotypes, the influence of hAT-derived capillary endothelial cells (CECs) was studied on the chemotaxis and differentiation of the hAT-CD34+/CD31- cells. Conditioned medium from hAT-CECs led to a strong chemotaxis of the hAT-CD34+/CD31- cells that was inhibited with pretreatments with pertussis toxin, CXCR-4 antagonist, or neutralizing antibodies. Furthermore, hAT-CECs produced and secreted the CXCR-4 ligand, that is, the stromal derived factor-1 (SDF-1). Finally, hAT-CECs induced the differentiation of hAT-CD34+/CD31- cells toward an endothelial cell (EC) phenotype. Indeed, hAT-CECs and -CD34+/CD31- cell coculture stimulated in a two-dimensional system the expression of the EC CD31 marker by the hAT-progenitor cells and, in a three-dimensional approach, the formation of capillary-like structures via a SDF-1/CXCR-4 dependent pathway. Thus, the migration and differentiation of hAT progenitor cells are modulated by hAT-CEC-derived factors. SDF-1, which is secreted by hAT-derived CECs, and its receptor CXCR-4, expressed by hAT-derived progenitor cells, may promote chemotaxis and differentiation of hAT-derived progenitor cells and thus contribute to the formation of the vascular network during the development of hAT.

Inactivation of extracellular superoxide dismutase contributes to the development of high-volume hypertension.

OBJECTIVES: Extracellular superoxide dismutase (ecSOD) lowers superoxide anions and maintains vascular nitric oxide level. We studied the function of ecSOD in high-volume hypertension induced by the 1-kidney-1-clip model in wild-type, ecSOD-/- mice, and endothelial nitric oxide synthase (eNOS)-/- mice. METHODS AND RESULTS: The 1-kidney-1-clip model resulted in impaired endothelium-dependent relaxation and hypertension and vascular oxidative stress in wild-type and ecSOD-/- mice. Recombinant ecSOD lowered the blood pressure and improved aortic nitric oxide bioavailability in wild-type and ecSOD-/- but not eNOS-/- mice. ecSOD had no effect on blood pressure in eNOS-/- or wild-type mice treated with a nitric oxide synthase inhibitor. The 1-kidney-1-clip model markedly induced ecSOD protein expression, whereas activity was increased by only 25%, suggesting a partial inactivation of ecSOD in high-volume hypertension. Incubation of aortic segments with peroxynitrite or hydrogen peroxide attenuated ecSOD activity, but peroxynitrite did not induce tyrosine nitration of ecSOD, suggesting oxidative inactivation of the enzyme. Administration of polyethyleneglycol-catalase for 3 days selectively lowered the blood pressure in ecSOD+/+ but not ecSOD-/- mice and improved nitric oxide bioavailability. In contrast, acute application of catalase had no effect. CONCLUSIONS: Nitric oxide mediates the vascular effects of ecSOD. Vascular dysfunction in 1-kidney-1-clip model hypertension is partially a consequence of inactivation of ecSOD by reactive oxygen species.

Nox1 mediates basic fibroblast growth factor-induced migration of vascular smooth muscle cells.

OBJECTIVE: Basic fibroblast growth factor (bFGF) stimulates vascular smooth muscle cell (SMC) migration. We determined whether bFGF increases SMC reactive oxygen-species (ROS) and studied the role of ROS for SMC migration. METHODS AND RESULTS: bFGF rapidly increased rat SMC ROS formation and migration through pathways sensitive to inhibition of NADPH oxidases, PI3-kinase, protein kinase C, and Rac-1. SiRNA directed against the NADPH oxidase Nox4 impaired basal but not bFGF-induced ROS formation and did not affect migration. In contrast, siRNA against Nox1 blocked the agonist-induced ROS generation as well as the bFGF-induced migration. Agonist-induced migration was also attenuated in SMC derived from Nox1 y/- mice and transduction of Nox1 restored normal migration. Likewise, SMC outgrowth in response to bFGF was attenuated in aortic segments from Nox1 y/- mice as compared with Nox1 y/+ mice. bFGF activated JNK but not Src in a Nox1-dependent manner. Consequently, phosphorylation of the adaptor protein paxillin, which is central for migration and secretion of matrix-metalloproteinases, were dependent on Nox1 as well as JNK but not Src. CONCLUSIONS: These data demonstrate that bFGF activates the Nox1-containing NADPH oxidase and that bFGF through a pathway involving ROS and JNK stimulates SMC migration.

Epoxyeicosatrienoic acids regulate Trp channel dependent Ca2+ signaling and hyperpolarization in endothelial cells.

OBJECTIVE: An initial step in endothelium-derived hyperpolarizing factor-mediated responses is endothelial cell hyperpolarization. Here we address the mechanisms by which cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) contribute to this effect in native and cultured endothelial cells. METHODS AND RESULTS: In native CYP2C-expressing endothelial cells, bradykinin elicited a Ca(2+) influx that was potentiated by the soluble epoxide hydrolase inhibitor, 1-adamantyl-3-cyclohexylurea (ACU), and attenuated by CYP inhibition. Similar effects were observed in cultured endothelial cells overexpressing CYP2C9, but not in CYP2C9-deficient cells, and were prevented by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid as well as by the cAMP antagonist, Rp-cAMPS. The effects on Ca(2+) were mirrored by prolongation of the bradykinin-induced hyperpolarization. Ruthenium red and the combination of charybdotoxin and apamin prevented the latter effect, suggesting that Trp channel activation increases Ca(2+) influx and prolongs the activation of Ca(2+)-dependent K(+) (K(Ca)) channels. Indeed, overexpression of CYP2C9 enhanced the agonist-induced translocation of a TrpC6-V5 fusion protein to caveolin-1-rich areas of the endothelial cell membrane, which was prevented by Rp-cAMPS and mimicked by 11,12-EET. CONCLUSIONS: Elevated EET levels regulate Ca(2+) influx into endothelial cells and the subsequent activation of K(Ca) channels, via a cAMP/PKA-dependent mechanism that involves the intracellular translocation of Trp channels.

The effects of COX-2 selective and non-selective NSAIDs on the initiation and progression of atherosclerosis in ApoE-/- mice.

In this study, we investigated the effects of prolonged administration of the selective COX-2 inhibitors celecoxib and rofecoxib and the non-selective NSAID naproxen on the initiation and progression of atherosclerosis. ApoE(-/-) mice, as well as corresponding wild-type mice, were fed either a normal chow or a high fat Western diet with or without addition of the respective drugs over a period of 16 weeks. Thereafter, aortic lesion size, plasma lipid levels, and COX-2 expression in the plaques were determined. The results showed that neither the COX-2 selective inhibitors nor naproxen had a significant impact on the initiation and progression of atherosclerosis in diet-fed ApoE(-/-) mice, although both celecoxib and rofecoxib showed a tendency to reduce plaque size. This slight effect may be due to selective inhibition of COX-2 activity because the COX-2 expression was not altered in the plaque. Plasma lipid levels were also not significantly influenced by these drugs. Interestingly, in ApoE(-/-) mice that have been fed with normal chow, we found an increased incidence of plaque formation after treatment with celecoxib and rofecoxib, indicating that coxibs may promote the initiation of atherosclerosis. This effect was probably masked in diet-fed mice by the more pronounced effects of the high cholesterol diet. In conclusion, the reduction in diet-induced plaque size in animals fed a high fat diet and the promotion of atherosclerosis in mice on a normal diet indicate a dual role of the coxibs. In advanced stages of atherosclerosis, they may exert antithrombotic properties due to their COX-2 inhibiting activity, whereas in very early stages they may favor the initiation of atherogenesis. However, because these results were only observed in ApoE(-/-) and not in wild-type animals, coxibs may increase the risk of thrombosis in patients with a predisposition for thrombotic complications.

NADPH oxidase plays a central role in blood-brain barrier damage in experimental stroke.

BACKGROUND AND PURPOSE: Cerebral ischemia/reperfusion is associated with reactive oxygen species (ROS) generation, and NADPH oxidases are important sources of ROS. We hypothesized that NADPH oxidases mediate blood-brain barrier (BBB) disruption and contribute to tissue damage in ischemia/reperfusion. METHODS: Ischemia was induced by filament occlusion of the middle cerebral artery in mice for 2 hours followed by reperfusion. BBB permeability was measured by Evans blue extravasation. Monolayer permeability was determined from transendothelial electrical resistance of cultured porcine brain capillary endothelial cells. RESULTS: BBB permeability was increased in the ischemic hemisphere 1 hour after reperfusion. In NADPH oxidase-knockout (gp91phox(-/-)) mice, middle cerebral artery occlusion-induced BBB disruption and lesion volume were largely attenuated compared with those in wild-type mice. Inhibition of NADPH oxidase by apocynin prevented BBB damage. In porcine brain capillary endothelial cells, hypoxia/reoxygenation induced translocation of the NADPH oxidase activator Rac-1 to the membrane. In vivo inhibition of Rac-1 by the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin or Clostridium difficile lethal toxin B also prevented the ischemia/reperfusion-induced BBB disruption. Stimulation of porcine brain capillary endothelial cells with H(2)O(2) increased permeability, an effect attenuated by inhibition of phosphatidyl inositol 3-kinase or c-Jun N-terminal kinase but not blockade of extracellular signal-regulated kinase-1/2 or p38 mitogen-activated protein kinase. Inhibition of Rho kinase completely prevented the ROS-induced increase in permeability and the ROS-induced polymerization of the actin cytoskeleton. CONCLUSIONS: Activation of Rac and subsequently of the gp91phox containing NADPH oxidase promotes cerebral ROS formation, which then leads to Rho kinase-mediated endothelial cell contraction and disruption of the BBB. Inhibition of NADPH oxidase is a promising approach to reduce brain injury after stroke.

Cytochrome P450 2C9 is involved in flow-dependent vasodilation of peripheral conduit arteries in healthy subjects and in patients with chronic heart failure.

BACKGROUND: Flow-mediated dilation (FMD) of human conduit arteries is, in part, related to shear stress-induced release of endothelium-derived nitric oxide (NO). However, NO synthase inhibitors do not completely abolish this FMD-response. Recently, a cytochrome P450 (CYP) epoxygenase of the 2C family was linked to NO- and prostacyclin-independent relaxation of conduit arteries. We therefore evaluated the contribution of CYP 2C9 to FMD in humans. METHODS AND RESULTS: FMD of the radial artery was determined in 12 healthy volunteers by high-resolution ultrasound and analyzed before and after intra-arterial infusion of sulfaphenazole, a specific CYP 2C9 inhibitor, L-NMMA (NO synthase inhibitor) and co-infusion of both. Endothelium-independent vasodilation was characterized after intra-arterial infusion of SNP. FMD was reduced after sulfaphenazole (11.5+/-0.87% vs. 7.4+/-0.95%, p<0.01), after L-NMMA (6.0+/-0.71%; p<0.01), and after co-infusion 3.9+/-0.73% (p<0.05 vs. L-NMMA; p<0.01 vs. sulfaphenazole). Sulfaphenazole had no effect on endothelium-independent vasodilation. In patients with chronic heart failure, the portion of FMD blocked by sulfaphenazole was not affected. CYP 2C was detected by immunohistochemistry in radial artery samples obtained from patients undergoing coronary bypass surgery. CONCLUSIONS: FMD in human conductance arteries is reduced after inhibition of CYP 2C9, supporting the concept that CYP 2C metabolites contribute to endothelium-mediated vasodilation of peripheral conduit arteries in vivo. In patients with heart failure, the CYP-dependent FMD appears to be preserved.

Gab1, SHP2, and protein kinase A are crucial for the activation of the endothelial NO synthase by fluid shear stress.

Fluid shear stress enhances NO production in endothelial cells by a mechanism involving the activation of the phosphatidylinositol 3-kinase and the phosphorylation of the endothelial NO synthase (eNOS). We investigated the role of the scaffolding protein Gab1 and the tyrosine phosphatase SHP2 in this signal transduction cascade in cultured and native endothelial cells. Fluid shear stress elicited the phosphorylation and activation of Akt and eNOS as well as the tyrosine phosphorylation of Gab1 and its association with the p85 subunit of phosphatidylinositol 3-kinase and SHP2. Overexpression of a Gab1 mutant lacking the pleckstrin homology domain abrogated the shear stress-induced phosphorylation of Akt but failed to affect the phosphorylation or activity of eNOS. The latter response, however, was sensitive to a protein kinase A (PKA) inhibitor. Mutation of Gab1 Tyr627 to phenylalanine (YF-Gab1) to prevent the binding of SHP2 completely prevented the shear stress-induced phosphorylation of eNOS, leaving the Akt response intact. A dominant-negative SHP2 mutant prevented the activation of PKA and phosphorylation of eNOS without affecting that of Akt. Moreover, shear stress elicited the formation of a signalosome complex including eNOS, Gab1, SHP2 and the catalytic subunit of PKA. In isolated murine carotid arteries, flow-induced vasodilatation was prevented by a PKA inhibitor as well as by overexpression of either the YF-Gab1 or the dominant-negative SHP2 mutant. Thus, the shear stress-induced activation of eNOS depends on Gab1 and SHP2, which, in turn, regulate the phosphorylation and activity of eNOS by a PKA-dependent but Akt-independent mechanism.

Noxa1 is a central component of the smooth muscle NADPH oxidase in mice.

NADPH oxidase is the most important source of oxygen-derived radicals (ROS) in the vascular wall. In vascular smooth muscle cells (VSMC), NADPH oxidase is characterized by the expression of the membrane subunit Nox1, which is activated by cytoplasmic proteins binding to its activation domain. We set out to identify the cytoplasmic protein involved in NADPH oxidase activation in mouse VSMC. Western blot analysis revealed that human endothelial cells and leukocytes but not VSMC from the aorta of the rat and the mouse express the classic NADPH oxidase activator p67phox. In mouse VSMC, however, the p67phox homologue Noxa1 was detected. Using antibodies generated against mouse Noxa1, the protein was observed in the cytosolic fraction of mouse VSMC with a molecular weight of about 51 kDa. Immunohistochemistry revealed that Noxa1 is expressed in the smooth muscle layer but not in endothelium or the adventitia of the mouse carotid artery. Fluorescent fusion proteins of Noxa1 were observed to be expressed in the cytoplasm of VSMC and coexpression of the NADPH oxidase organizer Noxo1 targeted the complex to membrane. An antisense plasmid of Noxa1 attenuated the endogenous Noxa1 protein expression in VSMC. This plasmid attenuated the ROS formation in mouse VSMC as detected using L012 chemiluminescence and prevented the agonist-induced ROS production in response to basic fibroblast growth factor and epidermal growth factor. In conclusion, these data indicate that Noxa1 replaces p67phox in VSMC and plays a central role in the activation of the NADPH oxidase in the vascular wall.

Xanthine oxidase inhibitor tungsten prevents the development of atherosclerosis in ApoE knockout mice fed a Western-type diet.

Hyperlipidemia enhances xanthine oxidase (XO) activity. XO is an important source of reactive oxygen species (ROS). Since ROS are thought to promote atherosclerosis, we hypothesized that XO is involved in the development of atherosclerosis. ApoE(-/-) mice were fed a Western-type (WD) or control diet. In subgroups, tungsten (700 mg/L) was administered to inhibit XO. XO is a secreted enzyme which is formed in the liver as xanthine dehydrogenase (XDH) and binds to the vascular endothelium. High expression of XDH was found in the liver and WD increased liver XDH mRNA and XDH protein expression. WD induced the conversion of XDH to the radical-forming XO. Moreover, WD increased the hepatic expression of CD40, demonstrating activation of hepatic cells. Aortic tissue of ApoE(-/-) mice fed a WD for 6 months exhibited marked atherosclerosis, attenuated endothelium-dependent relaxation to acetylcholine, increased vascular oxidative stress, and mRNA expression of the chemokine KC. Tungsten treatment had no effect on plasma lipids but lowered the plasma XO activity. In animals fed a control diet, tungsten had no effect on radical formation, endothelial function, or atherosclerosis development. In mice fed a WD, however tungsten attenuated the vascular superoxide anion formation, prevented endothelial dysfunction, and attenuated KC mRNA expression. Most importantly, tungsten treatment largely prevented the development of atherosclerosis in the aorta of ApoE(-/-) mice on WD. Therefore, tungsten, potentially via the inhibition of XO, prevents the development of endothelial dysfunction and atherosclerosis in ApoE(-/-) mice on WD.

Dynamic modulation of interendothelial gap junctional communication by 11,12-epoxyeicosatrienoic acid.

Functional gap junctional communication between vascular cells has been implicated in ascending dilatation and the cytochrome P-450 (CYP) inhibitor-sensitive and NO- and prostacyclin-independent dilatation of many vascular beds. Here, we assessed the mechanisms by which the epoxyeicosatrienoic acids (EETs) generated by a CYP 2C enzyme control interendothelial gap junctional communication. In CYP 2C-expressing porcine coronary endothelial cells, bradykinin, which enhances EET formation, elicited a biphasic effect on the electrical coupling and transfer of Lucifer yellow between endothelial cells, consisting of a transient increase in coupling followed by a sustained uncoupling. The initial phase was sensitive to the CYP 2C9 inhibitor sulfaphenazole and the protein kinase A (PKA) inhibitors Rp-cAMPS and KT5720 and could be mimicked by forskolin and caged cAMP as well as by the PKA activators 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphorothioate sodium salt and Sp-cAMPS. Gap junction uncoupling in bradykinin-stimulated porcine coronary endothelial cells was prevented by inhibiting the activation of extracellular signal-regulated kinase (ERK)1/2. In human endothelial cells, which express little CYP 2C, bradykinin elicited only an ERK1/2-mediated inhibition of intercellular communication. The CYP 2C9 product, 11,12-EET, also exerted a dual effect on the electrical and dye coupling of human endothelial cells, which was sensitive to PKA inhibition. These results demonstrate that an agonist-activated CYP-dependent pathway as well as 11,12-EET can positively regulate interendothelial gap junctional communication, most probably via the activation of PKA, an effect that is curtailed by the subsequent activation of ERK1/2.

CK2 phosphorylates the angiotensin-converting enzyme and regulates its retention in the endothelial cell plasma membrane.

Soluble angiotensin-converting enzyme (ACE) is derived from the membrane-bound form by proteolytic cleavage of its C-terminal domain. Because intracellular events might be involved in the regulation of the cleavage process, we determined whether the cytoplasmic tail of ACE is phosphorylated and whether this process regulates secretion. Immunoprecipitation of ACE (180 kDa) from (32)P-labeled endothelial cells revealed that ACE is phosphorylated. Phosphorylation was not observed in endothelial cells overexpressing a mutant form of ACE (ACEDeltaS, all five cytoplasmic serine residues replaced by alanine). CK2 coprecipitated with ACE from endothelial cells, and CK2 phosphorylated both ACE and a peptide corresponding to the cytoplasmic tail. Mutation of serine(1270) within the CK2 consensus sequence almost abolished ACE phosphorylation. In ACE-overexpressing endothelial cells, ACE was mostly localized to the plasma membrane. However, no ACE was detected in the plasma membrane of ACEDeltaS-overexpressing cells, although a precursor ACE (170 kDa) was prominent in the endoplasmic reticulum and the cell supernatant contained substantial amounts of the soluble protein (175 kDa). A correlation between ACE-phosphorylation and secretion was confirmed in endothelial cells treated with the CK2-inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, which time-dependently decreased the phosphorylation of ACE and increased its shedding. These results indicate that the CK2-mediated phosphorylation of ACE regulates its retention in the plasma membrane and may determine plasma ACE levels.

Angiotensin-converting enzyme is involved in outside-in signaling in endothelial cells.

Not all of the cardiovascular effects of angiotensin-converting enzyme (ACE) inhibitors can be attributed to changes in angiotensin II and bradykinin levels. Because the cytoplasmic tail of ACE is phosphorylated, we determined whether ACE inhibitors affect the phosphorylation of ACE and whether ACE possesses the characteristics of a signal transduction molecule. The ACE inhibitors ramiprilat and perindoprilat, and the substrate bradykinin (but not angiotensin I), enhanced the activity of ACE-associated CK2 and the phosphorylation of ACE Ser1270 in cultured endothelial cells. Mitogen-activated protein kinase kinase 7 and c-Jun N-terminal kinase (JNK) coprecipitated with ACE, and stimulation of endothelial cells with ACE inhibitors increased the activity of ACE-associated JNK and elicited the accumulation of phosphorylated c-Jun in the nucleus. Ramiprilat was however unable to activate JNK or to stimulate the nuclear accumulation of c-Jun in endothelial cells expressing a S1270A ACE mutant or in ACE-deficient cells. Because the ACE inhibitor-induced increase in ACE expression has been linked to the formation of c-Jun homodimers, we investigated whether ACE signaling via JNK contributes to this response in vitro and in vivo. Prolonged ramiprilat treatment increased ACE expression in primary cultures of human endothelial cells and in vivo (mouse lung), a response that was prevented by pretreatment with the JNK inhibitor SP600125. Thus, ACE is involved in outside-in signaling in endothelial cells and "ACE signaling" may be an important cellular mechanism contributing to the beneficial effects of ACE inhibitors.