Background-The renin-angiotensin system (RAS) is a key player in the progression of heart failure. is thought to modulate the activity of the RAS. Furthermore, this peptide may play a part in the beneficial effects of angiotensin-converting enzyme inhibitors in cardiovascular disease. We assessed the effects of angiotensin-(1-7) on the progression of heart failure. Methods and Results-Male Sprague-Dawley rats underwent either coronary ligation or sham surgery. Two weeks after induction of myocardial infarction, intravenous infusion of angiotensin-(1-7) (24 g/kg per hour) or saline was started by minipump. After 8 weeks of treatment, hemodynamic parameters were measured, endothelial function was assessed in isolated aortic rings, and plasma angiotensin-(1-7) levels were determined. Myocardial infarction resulted in a significant deterioration of left ventricular systolic and diastolic pressure, dP/dt, and coronary flow. Raising plasma levels 40-fold, angiotensin-(1-7) infusion attenuated this impairment to a nonsignificant level, markedly illustrated by a 40% reduction in left ventricular end-diastolic pressure. Furthermore, angiotensin-(1-7) completely preserved aortic endothelial function, whereas endothelium-dependent relaxation in aortas of saline-treated infarcted rats was significantly decreased. Conclusions-Angiotensin-(1-7) preserved cardiac function, coronary perfusion, and aortic endothelial function in a rat model for heart failure.

Background Vascular dysfunction in atherosclerosis and diabetes, as observed in the aging population of developed societies, is associated with vascular DNA damage and cell senescence. We hypothesized that cumulative DNA damage during aging contributes to vascular dysfunction. Methods and Results In mice with genomic instability due to the defective nucleotide excision repair genes ERCC1 and XPD (Ercc1d/− and XpdTTD mice), we explored age-dependent vascular function as compared to wild-type mice. Ercc1d/− mice showed increased vascular cell senescence, accelerated development of vasodilator dysfunction, increased vascular stiffness and elevated blood pressure at very young age. The vasodilator dysfunction was due to decreased endothelial eNOS levels as well as impaired smooth muscle cell function, which involved phosphodiesterase (PDE) activity. Similar to Ercc1d/− mice, age-related endothelium-dependent vasodilator dysfunction in XpdTTD animals was increased. To investigate the implications for human vascular disease, we explored associations between single nucleotide polymorphisms (SNPs) of selected nucleotide excision repair genes and arterial stiffness within the AortaGen Consortium, and found a significant association of a SNP (rs2029298) in the putative promoter region of DDB2 gene with carotid-femoral pulse wave velocity. Conclusions Mice with genomic instability recapitulate age-dependent vascular dysfunction as observed in animal models and in humans, but with an accelerated progression, as compared to wild type mice. In addition, we found associations between variations in human DNA repair genes and markers for vascular stiffness which is associated with aging. Our study supports the concept that genomic instability contributes importantly to the development of cardiovascular disease.

We conclude that the TRPV4 channel is involved in flow-induced, endothelium-dependent vasodilatation of murine carotid arteries. Moreover, the activation of the TRPV4 channel by flow requires an active CYP epoxygenase and the translocation of the channel to the cell membrane.

Abstract-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 (Ϸ50%), 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 Ϫ/Ϫ 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. (Circ Res. 2008;102:1520-1528.) Key Words: blood flow Ⅲ insulin Ⅲ mechanotransduction Ⅲ nitric oxide Ⅲ nitric oxide synthases Ⅲ phosphorylation O ver the last 10 years, it has become clear that the endothelial nitric oxide (NO) synthase (eNOS) is regulated both by changes in the intracellular concentration of free Ca 2ϩ , as well as by the phosphorylation of the enzyme. 1 Most is known about the role played by Ser1177 (human sequence), which is situated in the reductase domain of the enzyme and Thr495, located in the calmodulin (CaM)-binding domain, in the regulation of NO production, the phosphorylation of which appear to play a reciprocal role in the regulation of eNOS activity. 2,3 There are several potentially phosphorylatable tyrosine residues in eNOS, and there have been numerous reports showing that tyrosine kinase inhibitors attenuate endothelial NO production and flow-induced vasodilatation. 4 -6 It is clear that the enzyme can be tyrosine-phosphorylated in endothelial cells treated with tyrosine phosphatase inhibitors, 6,7 H 2 O 2 , 7,8 or exposed to fluid shear stress, 9 as well as in cells overexpressing v-Src. 8,10 Indeed, Src was reported to phosphorylate a tyrosine residue (Tyr83, bovine sequence; Tyr81, human sequence) in the oxygenase domain of eNOS in bovine aortic endotheli...

Abstract-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. (Circ Res. 2005;97:1236-1244.)Key Words: Akt Ⅲ blood flow Ⅲ endothelial nitric oxide synthase Ⅲ mechanotransduction Ⅲ protein kinase A A lthough the endothelial NO synthase (eNOS) was initially described as a Ca 2ϩ -dependent enzyme, it is now clear that the changes in the phosphorylation of several serine and threonine (and possibly also tyrosine) residues regulate NO production in response to Ca 2ϩ -elevating agonists as well as to hemodynamic stimuli, such as cyclic stretch and fluid shear stress (for reviews, see Fleming and Busse 1 and Boo and Jo 2 ). Most is known about the role played by Ser1177, which is situated in the reductase domain of the enzyme, and Thr495, which is situated in the calmodulin-binding domain, in the regulation of NO production. The phosphorylation of these sites appears to play a reciprocal role in the regulation of eNOS activity as Ser1177 becomes phosphorylated in response to endothelial cell activation, whereas Thr495 is constitutively phosphorylated but dephosphorylated on stimulation as a consequence of the activation of phosphatases. [3][4][5] The dephosphorylation of Thr495 facilitates the Ca 2ϩ -dependent association of calmodulin with eNOS, 5 whereas the phosphorylation of eNOS on Ser1177 increases NO output in an apparently Ca 2ϩ -independent manner. 6,7 As the maintained production of endothelium-derived NO in response...

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.

Abstract-Monoamine oxidases (MAOs) generate H 2 O 2 as a by-product of their catalytic cycle. Whether MAOs are mediators of endothelial dysfunction is unknown and was determined here in the angiotensin II and lipopolysaccharidemodels of vascular dysfunction in mice. Quantitative real-time polymerase chain reaction revealed that mouse aortas contain enzymes involved in catecholamine generation and MAO-A and MAO-B mRNA. MAO-A and -B proteins could be detected by Western blot not only in mouse aortas but also in human umbilical vein endothelial cells. Ex vivo incubation of mouse aorta with recombinant MAO-A increased H 2 O 2 formation and induced endothelial dysfunction that was attenuated by polyethylene glycol-catalase and MAO inhibitors. In vivo lipopolysaccharide (8 mg/kg IP overnight) or angiotensin II (1 mg/kg per day, 2 weeks, minipump) treatment induced vascular MAO-A and -B expressions and resulted in attenuated endothelium-dependent relaxation of the aorta in response to acetylcholine. MAO inhibitors reduced the lipopolysaccharide-and angiotensin II-induced aortic reactive oxygen species formation by 50% (ferrous oxidation xylenol orange assay) and partially normalized endothelium-dependent relaxation. MAO-A and MAO-B inhibitors had an additive effect; combined application completely restored endothelium-dependent relaxation. To determine how MAO-dependent H 2 O 2 formation induces endothelial dysfunction, cyclic GMP was measured. Histamine stimulation of human umbilical vein endothelial cells to activate endothelial NO synthase resulted in an increase in cyclic GMP, which was almost abrogated by MAO-A exposure. MAO inhibition prevented this effect, suggesting that MAO-induced H 2 O 2 formation is sufficient to attenuate endothelial NO release. Sturza et al MAOs in Endothelial Dysfunction 141MAO-A-mediated H 2 O 2 production has been shown to be relevant for ischemia-reperfusion injury of the kidney 11 and the heart. Moreover, MAO-A is thought to be involved in myocyte hypertrophy ex vivo 12,13 and also in maladaptive myocardial hypertrophy and transition to heart failure in vivo.14 The unfavorable effects of MAO activation are antagonized by a couple of relatively selective MAO inhibitors, which are either irreversible, such as clorgyline for MAO-A and selegiline for MAO-B, or reversible, such as moclobemide for MAO-A and lazabemide for MAO-B, respectively.8 Systemic MAO inhibition leads to the accumulation of catecholamines with subsequent increase in sympathetic activity and hypertension. 15 This aspect limits the use of MAO inhibitors in a vascular scenario, and therefore, MAO inhibitors have not been considered an approach to improve vascular function.Another prototypic hypertensive agent is angiotensin II (Ang II). Interestingly, potential interactions of Ang II and MAOs have been reported in the central nervous system. In coculture systems of hypothalamic and brain stem neurons, Ang II stimulated MAO activity, but the underlying mechanism was not studied. 16 More recently, it was reported that in...

Statins increase expression of mdr2 and mdr1b in rats, revealing a novel effect of these commonly used drugs.