Angiotensin II impairs endothelial function via tyrosine phosphorylation of the endothelial nitric oxide synthase

Proline-rich tyrosine kinase 2 (PYK2) can be activated by angiotensin II (Ang II) and reactive oxygen species. We report that in endothelial cells, Ang II enhances the tyrosine phosphorylation of endothelial NO synthase (eNOS) in an AT1-, H2O2-, and PYK2-dependent manner. Low concentrations (1–100 µmol/liter) of H2O2 stimulated the phosphorylation of eNOS Tyr657 without affecting that of Ser1177, and attenuated basal and agonist-induced NO production. In isolated mouse aortae, 30 µmol/liter H2O2 induced phosphorylation of eNOS on Tyr657 and impaired acetylcholine-induced relaxation. Endothelial overexpression of a dominant-negative PYK2 mutant protected against H2O2-induced endothelial dysfunction. Correspondingly, carotid arteries from eNOS−/− mice overexpressing the nonphosphorylatable eNOS Y657F mutant were also protected against H2O2. In vivo, 3 wk of treatment with Ang II considerably increased levels of Tyr657-phosphorylated eNOS in the aortae of wild-type but not Nox2y/− mice, and this was again associated with a clear impairment in endothelium-dependent vasodilatation in the wild-type but not in the Nox2y/− mice. Collectively, endothelial PYK2 activation by Ang II and H2O2 causes the phosphorylation of eNOS on Tyr657, attenuating NO production and endothelium-dependent vasodilatation. This mechanism may contribute to the endothelial dysfunction observed in cardiovascular diseases associated with increased activity of the renin–angiotensin system and elevated redox stress

AMP-Activated Protein Kinase alpha 2 in Neutrophils Regulates Vascular Repair via Hypoxia-Inducible Factor-1 alpha and a Network of Proteins Affecting Metabolism and Apoptosis

Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPK alpha 1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPK alpha 2 subunit in vascular repair. Objective: To determine the role of the AMPK alpha 2 subunit in vascular repair. Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPK alpha 2(-/-) versus wild-type mice, a phenotype reproduced in mice lacking AMPK alpha 2 in myeloid cells (AMPK alpha 2(Delta MC)). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPK alpha 2(Delta MC) mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPK alpha 2(Delta MC) mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPK alpha 2(Delta MC) hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1 alpha induction was attenuated in AMPK alpha 2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPK alpha 2(Delta MC) mice. Mechanistically, isocitrate dehydrogenase expression and the production of alpha-ketoglutarate, which negatively regulate hypoxia-inducible factor-1 alpha stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPK alpha 2(Delta MC) mice. Conclusions: AMPK alpha 2 regulates alpha-ketoglutarate generation, hypoxia-inducible factor-1 alpha stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPK alpha 2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia

Nitric Oxide-Induced Activation of the AMP-Activated Protein Kinase α2 Subunit Attenuates IκB Kinase Activity and Inflammatory Responses in Endothelial Cells

BACKGROUND: In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFκB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO). METHODOLOGY/PRINCIPAL FINDINGS: Overexpression of a dominant negative AMPKα2 mutant in tumor necrosis factor-α-stimulated human endothelial cells resulted in increased NFκB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKα2(-/-) mice the interleukin (IL)-1β induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFκB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKα2(-/-) mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IκB and p65, indicating a link between AMPK and the IκB kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPKα2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKα2(+/+) versus AMPKα2(-/-) mice. CONCLUSIONS: These results demonstrate that the IKK is a direct substrate of AMPKα2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFκB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK

Identification of kinetic triplets by results of derivatographic analysis

The method for identification of the triplet of kinetic parameters of a heterogeneous reaction using the data of the derivatographic analysis is proposed. This method is characterized by high accuracy and relative simplicity and it can be effectively realized using MS Excel software

Anaphylactic shock depends on endothelial Gq/G11

Anaphylactic shock is a severe allergic reaction involving multiple organs including the bronchial and cardiovascular system. Most anaphylactic mediators, like platelet-activating factor (PAF), histamine, and others, act through G protein–coupled receptors, which are linked to the heterotrimeric G proteins Gq/G11, G12/G13, and Gi. The role of downstream signaling pathways activated by anaphylactic mediators in defined organs during anaphylactic reactions is largely unknown. Using genetic mouse models that allow for the conditional abrogation of Gq/G11- and G12/G13-mediated signaling pathways by inducible Cre/loxP-mediated mutagenesis in endothelial cells (ECs), we show that Gq/G11-mediated signaling in ECs is required for the opening of the endothelial barrier and the stimulation of nitric oxide formation by various inflammatory mediators as well as by local anaphylaxis. The systemic effects of anaphylactic mediators like histamine and PAF, but not of bacterial lipopolysaccharide (LPS), are blunted in mice with endothelial Gαq/Gα11 deficiency. Mice with endothelium-specific Gαq/Gα11 deficiency, but not with Gα12/Gα13 deficiency, are protected against the fatal consequences of passive and active systemic anaphylaxis. This identifies endothelial Gq/G11-mediated signaling as a critical mediator of fatal systemic anaphylaxis and, hence, as a potential new target to prevent or treat anaphylactic reactions