Contribution of arginase activation to vascular dysfunction in cigarette smoking

Background

Cigarette smoke increases the risk of several cardiovascular diseases and has synergistic detrimental effects when present with other risks that contribute to its pathogenesis. Oxidative injury to the endothelium via reactive oxygen species (ROS) and nitric oxide (NO) dysregulation is a common denominator of smoking-induced alterations in vascular function. However, the mechanisms underlying ROS and NO dysregulation due to smoking remain unclear. We tested if arginase (Arg) activation/upregulation contributes to this phenomenon by constraining nitric oxide synthase (NOS) activity.

Methods

Arg2 knockout (Arg2^−/−) and control C57BL/6J mice were either exposed to cigarette smoke, 6 h/day/2 weeks (Second Hand Smoking; SHS) or housed in normal environment (Non Smoking; NS). Arg activity, NO and ROS levels were determined by measuring urea production, fluorescent dye (DAF), and dihydroethedium (DHE) respectively in isolated mouse aorta.

Results

Arg activity and ROS levels were higher NO lower in SHS compared to NS mice. SHS failed to lower NO levels in Arg2^−/− mice. Endothelial dependent vasodilation (EDV) was attenuated in SHS mice as compared to controls (78.80% ± 8 vs 46.58% ± 5). This impaired EDV was abolished in Arg2^−/− mice (67.48% ± 7 in SHS vs. 78.80% ± 8 in NS). Vascular stiffness was increased in SHS mice as compared to NS controls but remained unchanged in Arg2^−/− mice.

Conclusion

Endothelial NOS is uncoupled by smoking exposure, leading to endothelial dysfunction and vascular stiffness, a process that is prevented by Arg2 deletion. Hence, we identify Arg2 upregulation as a critical pathogenic factor and target for therapy in oxidative injury following smoking exposure through reciprocal regulation of endothelial NOS.     

Inducible nitric oxide synthase is involved in endothelial dysfunction of mesenteric small arteries from hypothyroid rats

The time-dependent effects of mild hypothyroidism on endothelial function were assessed in rat mesenteric arteries. Male Wistar rats were treated with methimazole (MMI; 0.003%) or placebo up to 16 wk. Endothelial function of mesenteric small arteries was assessed by pressurized myograph. MMI-treated animals displayed a decrease in serum thyroid hormones, an increment of plasma TSH and inflammatory cytokines, and a blunted vascular relaxation to acetylcholine, as compared with controls. Endothelial dysfunction resulted from a reduced nitric oxide (NO) availability caused by oxidative excess. Vascular-inducible NO synthase (iNOS) expression was up-regulated. S-methylisothiourea (an iNOS inhibitor) normalized endothelium-dependent relaxations and restored NO availability in arteries from 8-wk MMI-animals and partly ameliorated these alterations in 16-wk MMI rats. Similar results were obtained when MMI-induced hypothyroidism was prevented by T(4) replacement. Among controls, an impaired NO availability, secondary to oxidative excess, occurred at 16 wk, and it was less pronounced than in age-matched MMI animals. Both endothelial dysfunction and oxidant excess secondary to aging were prevented by apocynin (nicotinamide adenine dinucleotide phosphate oxidase inhibitor). Mesenteric superoxide production was reduced by S-methylisothiourea and T(4) replacement in MMI animals and abolished by apocynin in controls (dihydroethidium staining). MMI-induced mild hypothyroidism is associated with endothelial dysfunction caused by a reduced NO availability, secondary to oxidative excess. It is suggested that in this animal model, characterized by TSH elevation and low-grade inflammation, an increased expression and function of iNOS, resulting in superoxide generation, accounts for an impaired NO availability.     

Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells

Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression.     

Variant estrogen receptor-c-Src molecular interdependence and c-Src structural requirements for endothelial NO synthase activation

Little is known about the tyrosine kinase c-Src's function in the systemic circulation, in particular its role in arterial responses to hormonal stimuli. In human aortic and venous endothelial cells, c-Src is indispensable for 17beta-estradiol (E2)-stimulated phosphatidylinositol 3-kinase/Akt/endothelial NO synthase (eNOS) pathway activation, a possible mechanism in E2-mediated vascular protection. Here we show that c-Src supports basal and E2-stimulated NO production and is required for E2-induced vasorelaxation in murine aortas. Only membrane c-Src is structurally and functionally involved in E2-induced eNOS activation. Independent of c-Src kinase activity, c-Src is associated with an N-terminally truncated estrogen receptor alpha variant (ER46) and eNOS in the plasma membrane through its "open" (substrate-accessible) conformation. In the presence of E2, c-Src kinase is activated by membrane ER46 and in turn phosphorylates ER46 for subsequent ER46 and c-Src membrane recruitment, the assembly of an eNOS-centered membrane macrocomplex, and membrane-initiated eNOS activation. Overall, these results provide insights into a critical role for the tyrosine kinase c-Src in estrogen-stimulated arterial responses, and in membrane-initiated rapid signal transduction, for which obligate complex assembly and localization require the c-Src substrate-accessible structure.     

Endothelial nitric oxide synthase and endothelial dysfunction

Nitric oxide (NO) regulates vascular tone and local blood flow, platelet aggregation and adhesion, and leukocyte-endothelial cell interactions. Abnormalities in NO production by the vascular endothelium result in endothelial dysfunction, which occurs in hypertension, diabetes, aging, and as a prelude to atherosclerosis. The common feature of endothelial dysfunction is a decrease in the amount of bioavailable NO. In this article, the physiologic roles of NO and the mechanisms of endothelial dysfunction are reviewed. Regulation of endothelial NO synthase (eNOS) activity by fatty acid modifications, intracellular localization, interactions with heat shock protein 90 (hsp90) and caveolin, substrate and cofactor dependence, and phosphorylation might all affect the level of bioavailable NO. A hypothesis is proposed that the final common pathway of diverse causes of endothelial dysfunction involves abnormalities in eNOS phosphorylation at Ser 1179 and other key phosphorylation sites     

OxLDL-dependent activation of arginase II is dependent on the LOX-1 receptor and downstream RhoA signaling

AimsArginase II regulates NOS activity by competing for the substrate l-arginine. Oxidized LDL (OxLDL) is a proatherogenic molecule that activates arginase II. We tested the hypotheses that OxLDL-dependent arginase II activation occurs through a specific receptor, and via a Rho GTPase effector mechanism that is inhibited by statins.Methods and resultsArginase II activation by OxLDL was attenuated following preincubation with the LOX-1 receptor-blocking antibody JTX92. This also prevented the dissociation of arginase II from microtubules. LOX-1^?/? mice failed to exhibit the increased arginase II activity seen in WT mice fed a high cholesterol diet. Furthermore, endothelium from LOX-1^?/? mice failed to demonstrate the diet-dependent reduction in NO and increase in ROS that were observed in WT mice. OxLDL induced Rho translocation to the membrane and Rho activation, and these effects were inhibited by pretreatment with JTX92 or statins. Transfection with siRNA for RhoA, or inhibition of ROCK both decreased OxLDL-stimulated arginase II activation. Preincubation with simvastatin or lovastatin blocked OxLDL-induced dissociation of arginase II from microtubules and prevented microtubule depolymerization.ConclusionsThis study provides a new focus for preventive therapy for atherosclerotic disease by delineating a clearer path from OxLDL through the endothelial cell LOX-1 receptor, RhoA, and ROCK, to the activation of arginase II, downregulation of NO, and vascular dysfunction.     

S-nitrosylation of endothelial nitric oxide synthase is associated with monomerization and decreased enzyme activity

Endothelial nitric oxide synthase (eNOS) is active only as a homodimer. Recent data has demonstrated that exogenous NO can act as an inhibitor of eNOS activity both in intact animals and vascular endothelial cells. However, the exact mechanism by which NO exerts its inhibitory action is unclear. Our initial experiments in bovine aortic endothelial cells indicated that exogenous NO decreased NOS activity with an associated decrease in eNOS dimer levels. We then undertook a series of studies to investigate the mechanism of dimer disruption. Exposure of purified human eNOS protein to NO donors or calcium-mediated activation of the enzyme resulted in a shift in eNOS from a predominantly dimeric to a predominantly monomeric enzyme. Further studies indicated that endogenous NOS activity or NO exposure caused S-nitrosylation of eNOS and that the presence of the thioredoxin and thioredoxin reductase system could significantly protect eNOS dimer levels and prevent the resultant monomerization and loss of activity. Further, exogenous NO treatment caused zinc tetrathiolate cluster destruction at the dimer interface. To further determine whether S-nitrosylation within this region could explain the effect of NO on eNOS, we purified a C99A eNOS mutant enzyme lacking the tetrathiolate cluster and analyzed its oligomeric state. This enzyme was predominantly monomeric, implicating a role for the tetrathiolate cluster in dimer maintenance and stability. Therefore, this study links the inhibitory action of NO with the destruction of zinc tetrathiolate cluster at the dimeric interface through S-nitrosylation of the cysteine residues.     

Endothelin 1 activation of endothelin A receptor/NADPH oxidase pathway and diminished antioxidants critically contribute to endothelial progenitor cell reduction and dysfunction in salt-sensitive hypertension

Circulating endothelial progenitor cells (EPCs) are reduced in hypertension, which inversely correlates with its mortality. Deoxycorticosterone acetate (DOCA)-salt hypertension features elevated endothelin (ET) 1 and oxidative stress. We tested the hypothesis that ET-1 induces EPC dysfunction by elevating oxidative stress through the ET(A)/NADPH oxidase pathway in salt-sensitive hypertension. Both ET(A) and ET(B) receptors were expressed in EPCs, but only ET(A) receptors were significantly increased in EPCs of DOCA-salt rats. EPC number and function were reduced in DOCA-salt rats compared with sham controls, and both were reversed by in vivo blockade of ET(A) receptors or NADPH oxidase. The enzymatic activities of NAPDH oxidase and its subunits gp91(phox), p22(phox), and Rac1 were augmented in EPCs of DOCA-salt rats, with concomitantly decreased antioxidant enzymes manganese superoxide dismutase, copper-zinc superoxide dismutase, and glutathione peroxidase 1. Reactive oxygen species level was elevated in EPCs from DOCA-salt rats, accompanied by increased EPC telomerase inactivation, senescence, and apoptosis, which were rescued by ET(A) or NADPH oxidase blockade. Cell therapy of normal or treated DOCA EPCs, but not untreated DOCA EPCs, significantly increased capillary density and blood perfusion in ischemic hindlimbs of DOCA-salt rats. p53 and Bax/Bcl-2 ratios were increased in EPCs of DOCA-salt rats, which were reversed by ET(A) antagonist, NADPH oxidase inhibitor, or polyethylene glycol-superoxide dismutase. Finally, in ET(B)-deficient rats, plasma ET-1 was elevated, and EPC number and telomerase activity were diminished. These results demonstrate, for the first time, that both ET-1 activation of ET(A)/NADPH oxidase pathway and diminished antioxidants critically contribute to EPC reduction and dysfunction via increased oxidative stress in salt-sensitive hypertension.     

Up-regulation of arginase II contributes to pulmonary vascular endothelial cell dysfunction during experimental pulmonary embolism

Pulmonary embolism (PE) causes pulmonary hypertension by mechanical obstruction and constriction of non-obstructed vasculature. We tested if experimental PE impairs pulmonary vascular endothelium-dependent dilation via activation of arginase II. Experimental PE was induced in male Sprague-Dawley rats by infusing 25 μm microspheres in the right jugular vein, producing moderate pulmonary hypertension. Shams received vehicle injection. Pulmonary arterial rings were isolated after 18 h and isometric tensions were determined. Dilations were induced with acetylcholine, calcium ionophore A23187 or nitroglycerin (NTG) in pre-contracted rings (phenylephrine). Protein expression was assessed by Western blot and immunohistochemistry. Arginase activity was inhibited by intravenous infusion of N(w)-hydroxy-nor-l-arginine (nor-NOHA). l-Arginine supplementation was also given. Endothelium-dependent dilation responses were significantly reduced in PE vs. vehicle-treated animals (ACh: 50 ± 9% vs. 93 ± 3%; A23187: 19 ± 7% vs. 85 ± 7%, p < 0.05), while endothelium-independent dilations (NTG) were unchanged. Endothelial nitric oxide synthase (eNOS) protein content was unchanged by PE. Expression of arginase II increased 4.5-fold and immunohistochemistry revealed increased arginase II staining. Nor-NOHA treatment and l-arginine supplementation significantly improved pulmonary artery ring endothelium-dependent dilation in PE (ACh: 58 ± 6% PE, 88 ± 6% PE + nor-NOHA, 84 ± 4% PE + l-arginine). Experimental PE impairs endothelium-dependent pulmonary artery dilation, while endothelium-independent dilation remains unchanged. The data support the conclusion that up-regulation of arginase II protein expression contributes to pulmonary artery endothelial dysfunction in this model of experimental PE.     

Nitroglycerin-induced S-nitrosylation and desensitization of soluble guanylyl cyclase contribute to nitrate tolerance

Nitrates such as nitroglycerin (GTN) and nitric oxide donors such as S-nitrosothiols are clinically vasoactive through stimulation of soluble guanylyl cyclase (sGC), which produces the second messenger cGMP. Development of nitrate tolerance, after exposure to GTN for several hours, is a major drawback to a widely used cardiovascular therapy. We recently showed that exposure to nitric oxide and to S-nitrosothiols causes S-nitrosylation of sGC, which directly desensitizes sGC to stimulation by nitric oxide. We tested the hypothesis that desensitization of sGC by S-nitrosylation is a mechanism of nitrate tolerance. Our results established that vascular tolerance to nitrates can be recapitulated in vivo by S-nitrosylation through exposure to cell membrane-permeable S-nitrosothiols and that sGC is S-nitrosylated and desensitized in the tolerant, treated tissues. We next determined that (1) GTN treatment of primary aortic smooth muscle cells induces S-nitrosylation of sGC and its desensitization as a function of GTN concentration; (2) S-nitrosylation and desensitization are prevented by treatment with N-acetyl-cysteine, a precursor of glutathione, used clinically to prevent development of nitrate tolerance; and (3) S-nitrosylation and desensitization are reversed by cessation of GTN treatment. Finally, we demonstrated that in vivo development of nitrate tolerance and crosstolerance by 3-day chronic GTN treatment correlates with S-nitrosylation and desensitization of sGC in tolerant tissues. These results suggest that in vivo nitrate tolerance is mediated, in part, by desensitization of sGC through GTN-dependent S-nitrosylation.     

FcgammaRIIB mediates C-reactive protein inhibition of endothelial NO synthase

C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk and endothelial dysfunction. Whether CRP has direct actions on endothelium and the mechanisms underlying such actions are unknown. Here we show in cultured endothelium that CRP prevents endothelial NO synthase (eNOS) activation by diverse agonists, resulting in the promotion of monocyte adhesion. CRP antagonism of eNOS occurs nongenomically and is attributable to blunted eNOS phosphorylation at Ser1179. Okadaic acid or knockdown of PP2A by short-interference RNA reverses CRP antagonism of eNOS, indicating a key role for the phosphatase. Aggregated IgG, the known ligand for Fcgamma receptors, causes parallel okadaic acid-sensitive loss of eNOS function, FcgammaRIIB expression is demonstrable in endothelium, and heterologous expression studies reveal that CRP antagonism of eNOS requires FcgammaRIIB. In FcgammaRIIB(+/+) mice, CRP blunts acetylcholine-induced increases in carotid artery vascular conductance; in contrast, CRP enhances acetylcholine responses in FcgammaRIIB(-/-) mice. Thus FcgammaRIIB mediates CRP inhibition of eNOS via PP2A, providing a mechanistic link between CRP and endothelial dysfunction.