Activated Platelets from Diabetic Rats Cause Endothelial Dysfunction by Decreasing Akt/Endothelial NO Synthase Signaling Pathway

Diabetes is associated with endothelial dysfunction and platelet activation, both of which may contribute to increased cardiovascular risk. The purpose of this study was to characterize circulating platelets in diabetes and clarify their effects on endothelial function. Male Wistar rats were injected with streptozotocin (STZ) to induce diabetes. Each experiment was performed by incubating carotid arterial rings with platelets (1.65×10⁷ cells/mL; 30 min) isolated from STZ or control rats. Thereafter, the vascular function was characterized in isolated carotid arterial rings in organ bath chambers, and each expression and activation of enzymes involved in nitric oxide and oxidative stress levels were analyzed. Endothelium-dependent relaxation induced by acetylcholine was significantly attenuated in carotid arteries treated with platelets isolated from STZ rats. Similarly, treatment with platelets isolated from STZ rats significantly reduced ACh-induced Akt/endothelial NO synthase signaling/NO production and enhanced TXB₂ (metabolite of TXA₂), while CD61 (platelet marker) and CD62P (activated platelet marker) were increased in carotid arteries treated with platelets isolated from STZ rats. Furthermore, the platelets isolated from STZ rats decreased total eNOS protein and eNOS dimerization, and increased oxidative stress. These data provide direct evidence that circulating platelets isolated from diabetic rats cause dysfunction of the endothelium by decreasing NO production (via Akt/endothelial NO synthase signaling pathway) and increasing TXA₂. Moreover, activated platelets disrupt the carotid artery by increasing oxidative stress.     

Tempol-nebivolol therapy potentiates hypotensive effect increasing NO bioavailability and signaling pathway

Abstract

Nebivolol is a third generation beta blocker with endothelial nitric oxide synthase (eNOS) agonist properties. Considering the role of reactive oxygen species (ROS) in the uncoupling of eNOS, we hypothesized that the preadministration of an antioxidant as tempol, could improve the hypotensive response of nebivolol in normotensive animals increasing the nitric oxide (NO) bioavailability by a reduction of superoxide (O₂^??) basal level production in the vascular tissue.

Male Sprague Dawley rats were given tap water to drink (control group) or tempol (an antioxidant scavenger of superoxide) for 1 week. After 1 week, Nebivolol, at a dose of 3 mg/kg, was injected intravenously to the control group or to the tempol-treated group. Mean arterial pressure, heart rate, and blood pressure variability were evaluated in the control, tempol, nebivolol, and tempol nebivolol groups, as well as, the effect of different inhibitor as Nβ-nitro-l-arginine methyl ester (L-NAME, a Nitric oxide synthase blocker) or glybenclamide, a K_ATP channel inhibitor. Also, the expression of α,β soluble guanylate cyclase (sGC), phospho-eNOS, and phospho-vasodilator-stimulated phosphoprotein (P-VASP) were evaluated by Western Blot and cyclic guanosine monophosphate (cGMP) levels by an enzyme-linked immunosorbent assay (ELISA) commercial kit assay.

We showed that pretreatment with tempol in normotensive rats produces a hypotensive response after nebivolol administration through an increase in the NO bioavailability and sGC, improving the NO/cGMP/protein kinase G (PKG) pathway compared to that of the nebivolol group.

We demonstrated that tempol preadministration beneficiates the response of a third-generation beta blocker with eNOS stimulation properties, decreasing the basal uncoupling of eNOS, and improving NO bioavailability. Our results clearly open a possible new strategy therapeutic for treating hypertension.     

Altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension

Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have developed a model (shunt) of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Our previous studies have identified a progressive development of endothelial dysfunction in shunt lambs that is dependent, at least in part, on decreased nitric oxide (NO) signaling. The purpose of this study was to evaluate the possible role of a disruption in carnitine metabolism in shunt lambs and to determine the effect on NO signaling. Our data indicate that at 2 wk of age, shunt lambs have significantly reduced expression (P < 0.05) of the key enzymes in carnitine metabolism: carnitine palmitoyltransferases 1 and 2 as well as carnitine acetyltransferase (CrAT). In addition, we found that CrAT activity was inhibited due to increased nitration. Furthermore, free carnitine levels were significantly decreased whereas acylcarnitine levels were significantly higher in shunt lambs (P < 0.05). We also found that alterations in carnitine metabolism resulted in mitochondrial dysfunction, since shunt lambs had significantly decreased pyruvate, increased lactate, and a reduced pyruvate/lactate ratio. In pulmonary arterial endothelial cells cultured from juvenile lambs, we found that mild uncoupling of the mitochondria led to a decrease in cellular ATP levels and a reduction in both endothelial NO synthase-heat shock protein 90 (eNOS-HSP90) interactions and NO signaling. Similarly, in shunt lambs we found a loss of eNOS-HSP90 interactions that correlated with a progressive decrease in NO signaling. Our data suggest that mitochondrial dysfunction may play a role in the development of endothelial dysfunction and pulmonary hypertension and increased pulmonary blood flow.     

Endothelial NO and O2− production rates differentially regulate oxidative,nitroxidative, and nitrosative stress in the microcirculation

Endothelial dysfunction causes an imbalance in endothelial NO and O₂⁻ production rates and increased peroxynitrite formation. Peroxynitrite and its decomposition products cause multiple deleterious effects including tyrosine nitration of proteins, superoxide dismutase (SOD) inactivation, and tissue damage. Studies have shown that peroxynitrite formation during endothelial dysfunction is strongly dependent on the NO and O₂⁻ production rates. Previous experimental and modeling studies examining the role of NO and O₂⁻ production imbalance on peroxynitrite formation showed different results in biological and synthetic systems. However, there is a lack of quantitative information about the formation and biological relevance of peroxynitrite under oxidative, nitroxidative, and nitrosative stress conditions in the microcirculation. We developed a computational biotransport model to examine the role of endothelial NO and O₂⁻ production on the complex biochemical NO and O₂⁻ interactions in the microcirculation. We also modeled the effect of variability in SOD expression and activity during oxidative stress. The results showed that peroxynitrite concentration increased with increase in either O₂⁻ to NO or NO to O₂⁻ production rate ratio (Q_O2⁻/Q_NO or Q_NO/Q_O2⁻, respectively). The peroxynitrite concentrations were similar for both production rate ratios, indicating that peroxynitrite-related nitroxidative and nitrosative stresses may be similar in endothelial dysfunction or inducible NO synthase (iNOS)-induced NO production. The endothelial peroxynitrite concentration increased with increase in both Q_O2⁻/Q_NO and Q_NO/Q_O2⁻ ratios at SOD concentrations of 0.1–100 μM. The absence of SOD may not mitigate the extent of peroxynitrite-mediated toxicity, as we predicted an insignificant increase in peroxynitrite levels beyond Q_O2⁻/Q_NO and Q_NO/Q_O2⁻ ratios of 1. The results support the experimental observations of biological systems and show that peroxynitrite formation increases with increase in either NO or O₂⁻ production, and excess NO production from iNOS or from NO donors during oxidative stress conditions does not reduce the extent of peroxynitrite mediated toxicity.     

Increased microparticle production and impaired microvascular endothelial function in aldosterone-salt-treated rats: protective effects of polyphenols

We aimed to characterize circulating microparticles in association with arterial stiffness, inflammation and endothelial dysfunction in aldosterone-salt-induced hypertension in rats and to investigate the preventive effects of red wine polyphenols. Uninephrectomized male Sprague-Dawley rats were treated with aldosterone-salt (1 µg.h⁻¹), with or without administration of either red wine polyphenols, Provinols™ (20 mg.kg⁻¹.day⁻¹), or spironolactone (30 mg.kg⁻¹.day⁻¹) for 4 weeks. Microparticles, arterial stiffness, nitric oxide (NO) spin trapping, and mesenteric arterial function were measured. Aldosterone-salt rats showed increased microparticle levels, including those originating from platelets, endothelium and erythrocytes. Hypertension resulted in enhanced aortic stiffness accompanied by increased circulating and aortic NO levels and an upregulation of aortic inducible NO-synthase, NFκB, superoxide anions and nitrotyrosine. Flow-induced dilatation was reduced in mesenteric arteries. These effects were prevented by spironolactone. Provinols™ did not reduce arterial stiffness or systolic hypertension but had effects similar to those of spironolactone on endothelial function assessed by flow-mediated vasodilatation, microparticle generation, aortic NO levels and oxidative stress and apoptosis in the vessel wall. Neither the contractile response nor endothelium-dependent relaxation in mesenteric arteries differed between groups. The in vivo effects of Provinols™ were not mediated by mineralocorticoid receptors or changes in shear stress. In conclusion, vascular remodelling and endothelial dysfunction in aldosterone-salt-mediated hypertension are associated with increased circulating microparticles. Polyphenols prevent the enhanced release of microparticles, macrovascular inflammation and oxidative stress, and microvascular endothelial dysfunction independently of blood pressure, shear stress and mineralocorticoid receptor activation in a model of hyperaldosteronism.     

Role of Dimethylarginine Dimethylaminohydrolases in the Regulation of Endothelial Nitric Oxide Production

Reduced NO is a hallmark of endothelial dysfunction, and among the mechanisms for impaired NO synthesis is the accumulation of the endogenous nitric-oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Free ADMA is actively metabolized by the intracellular enzyme dimethylarginine dimethylaminohydrolase (DDAH), which catalyzes the conversion of ADMA to citrulline. Decreased DDAH expression/activity is evident in disease states associated with endothelial dysfunction and is believed to be the mechanism responsible for increased methylarginines and subsequent ADMA-mediated endothelial nitric-oxide synthase impairment. Two isoforms of DDAH have been identified; however, it is presently unclear which is responsible for endothelial ADMA metabolism and NO regulation. The current study investigated the effects of both DDAH-1 and DDAH-2 in the regulation of methylarginines and endothelial NO generation. Results demonstrated that overexpression of DDAH-1 and DDAH-2 increased endothelial NO by 24 and 18%, respectively. Moreover, small interfering RNA-mediated down-regulation of DDAH-1 and DDAH-2 reduced NO bioavailability by 27 and 57%, respectively. The reduction in NO production following DDAH-1 gene silencing was associated with a 48% reduction in l-Arg/ADMA and was partially restored with l-Arg supplementation. In contrast, l-Arg/ADMA was unchanged in the DDAH-2-silenced cells, and l-Arg supplementation had no effect on NO. These results clearly demonstrate that DDAH-1 and DDAH-2 manifest their effects through different mechanisms, the former of which is largely ADMA-dependent and the latter ADMA-independent. Overall, the present study demonstrates an important regulatory role for DDAH in the maintenance of endothelial function and identifies this pathway as a potential target for treating diseases associated with decreased NO bioavailability.     

Hypersensitivity to thromboxane receptor mediated cerebral vasomotion and CBF oscillations during acute NO-deficiency in rats

Background

Low frequency (4–12 cpm) spontaneous fluctuations of the cerebrovascular tone (vasomotion) and oscillations of the cerebral blood flow (CBF) have been reported in diseases associated with endothelial dysfunction. Since endothelium-derived nitric oxide (NO) suppresses constitutively the release and vascular effects of thromboxane A₂ (TXA₂), NO-deficiency is often associated with activation of thromboxane receptors (TP). In the present study we hypothesized that in the absence of NO, overactivation of the TP-receptor mediated cerebrovascular signaling pathway contributes to the development of vasomotion and CBF oscillations.

Methodology/Principal Findings

Effects of pharmacological modulation of TP-receptor activation and its downstream signaling pathway have been investigated on CBF oscillations (measured by laser-Doppler flowmetry in anesthetized rats) and vasomotion (measured by isometric tension recording in isolated rat middle cerebral arteries, MCAs) both under physiological conditions and after acute inhibition of NO synthesis. Administration of the TP-receptor agonist U-46619 (1 µg/kg iv.) to control animals failed to induce any changes of the systemic or cerebral circulatory parameters. Inhibition of the NO synthesis by nitro-L-arginine methyl esther (L-NAME, 100 mg/kg iv.) resulted in increased mean arterial blood pressure and a decreased CBF accompanied by appearance of CBF-oscillations with a dominant frequency of 148±2 mHz. U-46619 significantly augmented the CBF-oscillations induced by L-NAME while inhibition of endogenous TXA₂ synthesis by ozagrel (10 mg/kg iv.) attenuated it. In isolated MCAs U-46619 in a concentration of 100 nM, which induced weak and stable contraction under physiological conditions, evoked sustained vasomotion in the absence of NO, which effect could be completely reversed by inhibition of Rho-kinase by 10 µM Y-27632.

Conclusion/Significance

These results suggest that hypersensitivity of the TP-receptor – Rho-kinase signaling pathway contributes to the development of low frequency cerebral vasomotion which may propagate to vasospasm in pathophysiological states associated with NO-deficiency.     

Chronic deficiency of nitric oxide affects hypoxia inducible factor-1α (HIF-1α) stability and migration in human endothelial cells

Background

Endothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated.

Results

Endothelial nitric oxide synthase (eNOS) was chronically inhibited either by N ^G-Nitro-l-arginine methyl ester (l-NAME) treatment or its expression was down-regulated by RNA interference. After long-term l-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1α (HIF-1α). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O₂ consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1α accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells.

Conclusion

Based on our results, we propose that basal release of NO may act as a negative controller of HIF-1α levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction.     

Therapeutic dose of nebivolol, a nitric oxide-releasing β-blocker, reduces atherosclerosis in cholesterol-fed rabbits

Nitric oxide (NO) exerts a plethora of vascular beneficial effects. The NO-releasing β-blocker nebivolol is a racemic mixture of d/l-enantiomers that displays negative inotropic as well as direct vasodilating activity. The in vivo antiatherogenic activity of therapeutic doses of the β-blocker with antioxidant properties carvedilol (12.5 mg/day) or nebivolol (5 mg/day) was tested in cholesterol-fed rabbits. Animals received a 1% cholesterol-rich diet alone (controls) or mixed with drugs (treated animals) for 8 weeks. While it did not affect hyperlipidemia, nebivolol inhibited the development of atherosclerosis, expressed as computer-assisted imaging analysis of aortic area covered by lesions (23.3 ± 4.1% in treated vs 38.2 ± 6.4% in control animals, p < 0.01). Differently, in our experimental condition of therapeutic drug doses, this antiatherogenic effect did not reach statistical significance in rabbits treated with carvedilol (32.5 ± 5.1% aortic area covered by lesions, p = NS vs controls). Plasma nitrates increased in rabbits treated with nebivolol while both β-blockers reduced LDL oxidation. Moreover, nebivolol induced a consistent increase of endothelial reactivity and aortic eNOS expression compared with control animals (p < 0.05) and those receiving carvedilol (p < 0.05). Since NO may exert beneficial effects in atherosclerosis, a NO-dependent mechanism could explain this data. These observations suggest that the NO-releasing β-blocker, nebivolol, might represent an effective pharmacological approach for preventing atherosclerotic lesion progression.     

Probiotics (VSL#3) Prevent Endothelial Dysfunction in Rats with Portal Hypertension: Role of the Angiotensin System

Aims

Portal hypertension characterized by generalized vasodilatation with endothelial dysfunction affecting nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) has been suggested to involve bacterial translocation and/or the angiotensin system. The possibility that ingestion of probiotics prevents endothelial dysfunction in rats following common bile duct ligation (CBDL) was evaluated.

Methods

Rats received either control drinking water or the probiotic VSL#3 solution (50 billion bacteria.kg body wt⁻¹.day⁻¹) for 7 weeks. After 3 weeks, rats underwent surgery with either resection of the common bile duct or sham surgery. The reactivity of mesenteric artery rings was assessed in organ chambers, expression of proteins by immunofluorescence and Western blot analysis, oxidative stress using dihydroethidium, and plasma pro-inflammatory cytokine levels by flow cytometry.

Results

Both NO- and EDH-mediated relaxations to acetylcholine were reduced in the CBDL group compared to the sham group, and associated with a reduced expression of Cx37, Cx40, Cx43, IK_Ca and SK_Ca and an increased expression of endothelial NO synthase (eNOS). In aortic sections, increased expression of NADPH oxidase subunits, angiotensin converting enzyme, AT1 receptors and angiotensin II, and formation of ROS and peroxynitrite were observed. VSL#3 prevented the deleterious effect of CBDL on EDH-mediated relaxations, vascular expression of connexins, IK_Ca, SK_Ca and eNOS, oxidative stress, and the angiotensin system. VSL#3 prevented the CBDL-induced increased plasma TNF-α, IL-1α and MCP-1 levels.

Conclusions

These findings indicate that VSL#3 ingestion prevents endothelial dysfunction in the mesenteric artery of CBDL rats, and this effect is associated with an improved vascular oxidative stress most likely by reducing bacterial translocation and the local angiotensin system.     

Tetrahydrocurcumin Protects against Cadmium-Induced Hypertension,Raised Arterial Stiffness and Vascular Remodeling in Mice

Background

Cadmium (Cd) is a nonessential heavy metal, causing oxidative damage to various tissues and associated with hypertension. Tetrahydrocurcumin (THU), a major metabolite of curcumin, has been demonstrated to be an antioxidant, anti-diabetic, anti-hypertensive and anti-inflammatory agent. In this study, we investigated the protective effect of THU against Cd-induced hypertension, raised arterial stiffness and vascular remodeling in mice.

Methods

Male ICR mice received CdCl₂ (100 mg/l) via drinking water for 8 weeks. THU was administered intragastrically at dose of 50 or 100 mg/kg/day concurrently with Cd treatment.

Results

Administration of CdCl₂ significantly increased arterial blood pressure, blunted vascular responses to vasoactive agents, increased aortic stiffness, and induced hypertrophic aortic wall remodeling by increasing number of smooth muscle cells and collagen deposition, decreasing elastin, and increasing matrix metalloproteinase (MMP)-2 and MMP-9 levels in the aortic medial wall. Supplementation with THU significantly decreased blood pressure, improved vascular responsiveness, and reversed the structural and mechanical alterations of the aortas, including collagen and elastin deposition. The reduction on the adverse response of Cd treatment was associated with upregulated eNOS and downregulated iNOS protein expressions, increased nitrate/nitrite level, alleviated oxidative stress and enhanced antioxidant glutathione. Moreover, THU also reduced the accumulation of Cd in the blood and tissues.

Conclusions

Our results suggest that THU ameliorates cadmium-induced hypertension, vascular dysfunction, and arterial stiffness in mice through enhancing NO bioavailability, attenuating oxidative stress, improving vascular remodeling and decreasing Cd accumulation in other tissues. THU has a beneficial effect in moderating the vascular alterations associated with Cd exposure.     

Sonic Hedgehog Carried by Microparticles Corrects Angiotensin II-Induced Hypertension and Endothelial Dysfunction in Mice

Microparticles are small fragments of the plasma membrane generated after cell stimulation. We recently showed that Sonic hedgehog (Shh) is present in microparticles generated from activated/apoptotic human T lymphocytes and corrects endothelial injury through nitric oxide (NO) release. This study investigates whether microparticles bearing Shh correct angiotensin II-induced hypertension and endothelial dysfunction in mice. Male Swiss mice were implanted with osmotic minipumps delivering angiotensin II (0.5 mg/kg/day) or NaCl (0.9%). Systolic blood pressure and heart rate were measured daily during 21 days. After 7 day of minipump implantation, mice received i.v. injections of microparticles (10 µg/ml) or i.p. Shh receptor antagonist cyclopamine (10 mg/kg/2 days) during one week. Angiotensin II induced a significant rise in systolic blood pressure without affecting heart rate. Microparticles reversed angiotensin II-induced hypertension, and cyclopamine prevented the effects of microparticles. Microparticles completely corrected the impairment of acetylcholine- and flow-induced relaxation in vessels from angiotensin II-infused mice. The improvement of endothelial function induced by microparticles was completely prevented by cyclopamine treatment. Moreover, microparticles alone did not modify NO and O₂ ^{. -} production in aorta, but significantly increased NO and reduced O₂ ^{. -} productions in aorta from angiotensin II-treated mice, and these effects were blocked by cyclopamine. Altogether, these results show that microparticles bearing Shh correct angiotensin II-induced hypertension and endothelial dysfunction in aorta through a mechanism associated with Shh-induced NO production and reduction of oxidative stress. These microparticles may represent a new therapeutic approach in cardiovascular diseases associated with decreased NO production.     

Thrombin Has Biphasic Effects on the Nitric Oxide-cGMP Pathway in Endothelial Cells and Contributes to Experimental Pulmonary Hypertension

Background

A potential role for coagulation factors in pulmonary arterial hypertension has been recently described, but the mechanism of action is currently not known. Here, we investigated the interactions between thrombin and the nitric oxide-cGMP pathway in pulmonary endothelial cells and experimental pulmonary hypertension.

Principal Findings

Chronic treatment with the selective thrombin inhibitor melagatran (0.9 mg/kg daily via implanted minipumps) reduced right ventricular hypertrophy in the rat monocrotaline model of experimental pulmonary hypertension. In vitro, thrombin was found to have biphasic effects on key regulators of the nitric oxide-cGMP pathway in endothelial cells (HUVECs). Acute thrombin stimulation led to increased expression of the cGMP-elevating factors endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) subunits, leading to increased cGMP levels. By contrast, prolonged exposition of pulmonary endothelial cells to thrombin revealed a characteristic pattern of differential expression of the key regulators of the nitric oxide-cGMP pathway, in which specifically the factors contributing to cGMP elevation (eNOS and sGC) were reduced and the cGMP-hydrolyzing PDE5 was elevated (qPCR and Western blot). In line with the differential expression of key regulators of the nitric oxide-cGMP pathway, a reduction of cGMP by prolonged thrombin stimulation was found. The effects of prolonged thrombin exposure were confirmed in endothelial cells of pulmonary origin (HPAECs and HPMECs). Similar effects could be induced by activation of protease-activated receptor-1 (PAR-1).

Conclusion

These findings suggest a link between thrombin generation and cGMP depletion in lung endothelial cells through negative regulation of the nitric oxide-cGMP pathway, possibly mediated via PAR-1, which could be of relevance in pulmonary arterial hypertension.     

Tetrahydrobiopterin attenuates homocysteine induced endothelial dysfunction

Homocysteine is an independent risk factor for atherosclerotic vascular disease. It impairs endothelial function via increasing superoxide production and quenching nitric oxide (NO) release. Tetrahydrobiopterin (BH₄) is a critical cofactor that couples nitric oxide synthase and facilitates the production of nitric oxide (vs. superoxide anions). In the first study, the effects of hyperhomocysteinemia (0.1 mM, 3 h) on endothelium-dependent vasorelaxation to ACh and A23187 were examined in isolated segments of rat aortae in the presence or absence of BH₄ (0.1 mM). In the second study, the effects of hyperhomocysteinemia (24 h) on nitric oxide production and superoxide release (using lucigenin chemiluminescence) were studied in human umbilical vein endothelial cells in the absence or presence of BH₄ (10 M). Homocysteine incubation impaired receptor-dependent and -independent endothelial function to ACh and A23187. This effect was attenuated by BH₄. Furthermore, homocysteine exposure increased superoxide production and impaired agonist-stimulated nitric oxide release. These effects were attenuated by BH₄ (p < 0.05). Hyperhomocysteinemia impairs endothelial function, in part due to a diminished bioavailability of BH₄ with resultant uncoupling of nitric oxide synthase. BH₄ may represent an important target for strategies aimed at improving endothelial dysfunction secondary to hyperhomocysteinemia.     

Nebivolol improves diastolic dysfunction and myocardial remodeling through reductions in oxidative stress in the transgenic (mRen2) rat

Angiotensin II contributes to myocardial tissue remodeling and interstitial fibrosis through NADPH oxidase-mediated generation of oxidative stress in the progression of heart failure. Recent data have suggested that nebivolol, a third-generation β-blocker, improves diastolic dysfunction by targeting nitric oxide (NO) and metabolic pathways that decrease interstitial fibrosis. We sought to determine if targeting NO would improve diastolic function in a model of tissue renin-angiotensin system overactivation. We used the transgenic (TG) (mRen2)27 rat, which overexpresses the murine renin transgene and manifests insulin resistance and left ventricular dysfunction. We treated 6- to 7-wk-old TG (mRen2)27 rats and age-matched Sprague-Dawley control rats with nebivolol (10 mg·kg(-1)·day(-1)) or placebo via osmotic minipumps for a period of 21 days. Compared with Sprague-Dawley control rats, TG (mRen2)27 rats displayed a prolonged diastolic relaxation time and reduced initial filling rate associated with increased interstitial fibrosis and left ventricular hypertrophy. These findings were temporally related to increased NADPH oxidase activity and subunits p47(phox) and Rac1 and increased total ROS and peroxynitrite formation in parallel with reductions in the antioxidant heme oxygenase as well as the phosphorylation/activation of endothelial NO synthase and PKB/Akt. Treatment with nebivolol restored diastolic function and interstitial fibrosis through increases in the phosphorylation of 5'-AMP-activated protein kinase, Akt, and endothelial NO synthase and reductions in oxidant stress. These results support that targeting NO with nebivolol treatment improves diastolic dysfunction through reducing myocardial oxidative stress by enhancing 5'-AMP-activated protein kinase and Akt activation of NO biosynthesis.     

Inhibition of eNOS Partially Blunts the Beneficial Effects of Nebivolol on Angiotensin II-Induced Signaling in H9c2 Cardiomyoblasts

We have recently illustrated that nebivolol can inhibit angiotensin II (Ang II)-mediated signaling in cardiomyoblasts; however, to date, the detailed mechanism for the beneficial effects of nebivolol has not been studied. Here, we investigated whether the inhibition of NO bioavailability by blocking eNOS (endothelial nitric oxide synthase) using L-NG-nitroarginine methyl ester (L-NAME) would attenuate nebivolol-mediated favorable effects on Ang II-evoked signaling in H9c2 cardiomyoblasts. Our data reveal that the nebivolol-mediated antagonistic effects on Ang II-induced oxidative stress were retreated by concurrent pretreatment with L-NAME and nebivolol. Similarly, the expressions of pro-inflammatory markers TNF-α and iNOS stimulated by Ang II were not decreased with the combination of nebivolol plus L-NAME. In contrast, the nebivolol-induced reduction in the Ang II-triggered mTORC1 pathway and the mRNA levels of hypertrophic markers ANP, BNP, and β-MHC were not reversed with the addition of L-NAME to nebivolol. In compliance with these data, the inhibition of eNOS by L-N⁵-(1-Iminoethyl) ornithine (LNIO) and its upstream regulator AMP-activated kinase (AMPK) with compound C in the presence of nebivolol showed effects similar to those of the L-NAME plus nebivolol combination on Ang II-mediated signaling. Pretreatment with either compound C plus nebivolol or LNIO plus nebivolol showed similar effects to those of the L-NAME plus nebivolol combination on Ang II-mediated signaling. In conclusion, our data indicate that the rise in NO bioavailability caused by nebivolol via the stimulation of AMPK/eNOS signaling is key for its anti-inflammatory and antioxidant properties but not for its antihypertrophic response upon Ang II stimulation.     

Is NO the king? Pathophysiological benefit with uncertain clinical impact

NO is the "hero" molecule of the last few decades. It is a ubiquitous and omnipotent radical with both hemodynamic and antiproliferative effects within the cardiovascular system. NO is an important counterregulatory factor for vasoconstrictors and growth promoting substances. Endothelial dysfunction with decreased NO production is related to many cardiovascular disorders, such as coronary artery disease, heart failure and hypertension. Despite the important role of NO within the circulation, there is only limited evidence in the form of large clinical trials that NO delivery can reduce cardiovascular morbidity and mortality. Thus, NO donors are not in the first line therapy in ischemic heart disease, heart failure or arterial hypertension and NO delivery is recommended only in particular clinical situations, when a well established treatment is contraindicated or has an insufficient effect. It is concluded that the insufficient NO production is the principal disorder in endothelial dysfunction, which is related to cardiovascular pathology with deteriorated prognosis, but the impact of therapeutically increased NO bioactivity on the morbidity and mortality is inferior to well established treatment with ACE-inhibitors, AT(1) receptor blockers, beta-blockers, statins and certain antihypertensive drugs. There is little doubt that NO is king in the circulation, but kings seldom decide the battles.     

The effect of methylmercury (CH3HgCl) on the production of endothelium-derived relaxing factor (EDRF) by cultured human umbilical vascular endothelial cells based on its anti-aggregatory effect on human platelets

The effect of methylmercury (CH₃HgCl) on the production of endothelium-derived relaxing factor (EDRF) by cultured human umbilical vascular endothelial cells (HUVECs) based on its antiaggregatory effect on human platelets was examined. HUVECs were harvested from umbilical veins by collagenase treatment. The platelet aggregation test was performed with cuvettes lined with HUVECs. Platelet aggregation induced by 0.05 units thrombin/ml was inhibited in the presence of HUVECs. This HUVEC-dependent anti-platelet aggregatory effect was enhanced by the addition of bradykinin (10 nmol/L), which stimulates the production of EDRF. Indomethacin (IND, 1 mol/L) reduced the HUVEC-dependent anti-platelet aggregatory effect. The effect ofN ^G-monomethyl-L-arginine (L-NMMA, 100 mol/L), an inhibitor of nitric oxide synthase (NOS) in endothelial cells, on HUVECs pretreated with IND showed almost complete platelet aggregation similar to results without HUVECs. The anti-platelet aggregatory effect of HUVECs pretreated with IND seemed to depend mainly on EDRF. Methylmercury (MeHg) (20–50 mol/L) induced dose-dependent platelet aggregation in cuvettes, without HUVECs. Methylmercury (30 mol/L) induced less platelet aggregation in the presence of HUVECs than in their absence. The degree of inhibitory effect by HUVECs on MeHg-induced platelet aggregation was reduced dose-dependently (30–50 mol/L MeHg). Methylmercury-induced platelet aggregation at 50 mol/L MeHg with or without HUVECs was similar. These findings suggest that this simple new experimental system is useful for assessing the production of EDRF by HUVECs, and show that MeHg inhibits the production of EDRF by HUVECs, which may be involved in the etiology of cardiovascular diseases such as hypertension and arteriosclerosis.Abbreviations BK bradykinin - EDRF endothelium-derived relaxing factor - HUVECs human umbilical vascular endothelial cells - IND indomethacin - L-NMMA N ^G-monomethyl-L-arginine - MeHg methylmercury