Nox4 NADPH Oxidase Mediates Peroxynitrite-dependent Uncoupling of Endothelial Nitric-oxide Synthase and Fibronectin Expression in Response to Angiotensin II: ROLE OF MITOCHONDRIAL REACTIVE OXYGEN SPECIES*

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces endothelial nitric-oxide synthase (eNOS) uncoupling with enhanced generation of reactive oxygen species (ROS) and decreased production of NO. Ang II promotes a rapid increase in 3-nitrotyrosine formation, and uric acid attenuates Ang II-induced decrease in NO bioavailability, demonstrating that peroxynitrite mediates the effects of Ang II on eNOS dysfunction. Ang II rapidly up-regulates Nox4 protein. Inhibition of Nox4 abolishes the increase in ROS and peroxynitrite generation as well as eNOS uncoupling triggered by Ang II, indicating that Nox4 is upstream of eNOS. This pathway contributes to Ang II-mediated fibronectin accumulation in MCs. Ang II also elicits an increase in mitochondrial abundance of Nox4 protein, and the oxidase contributes to ROS production in mitochondria. Overexpression of mitochondrial manganese superoxide dismutase prevents the stimulatory effects of Ang II on mitochondrial ROS production, loss of NO availability, and MC fibronectin accumulation, whereas manganese superoxide dismutase depletion increases mitochondrial ROS, NO deficiency, and fibronectin synthesis basally and in cells exposed to Ang II. This work provides the first evidence that uncoupled eNOS is responsible for Ang II-induced MC fibronectin accumulation and identifies Nox4 and mitochondrial ROS as mediators of eNOS dysfunction. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal fibrosis.     

Involvement of the mitogen-activated protein kinase cascade in peroxynitrite-mediated arachidonic acid release in vascular smooth muscle cells

Eicosanoid production is reduced when the nitric oxide (NO·) pathway is inhibited or when the inducible NO synthase gene is deleted, indicating that the NO· and arachidonic acid pathways are linked. We hypothesized that peroxynitrite, formed by the reaction of NO· and superoxide anion, may cause signaling events leading to arachidonic acid release and subsequent eicosanoid generation. Western blot analysis of rat arterial smooth muscle cells demonstrated that peroxynitrite (100–500 µM) and 3-morpholinosydnonimine (SIN-1; 200 µM) stimulate phosphorylation of extracellular signal-regulated kinase (ERK), p38, and cytosolic phospholipase A₂ (cPLA₂). We found that peroxynitrite-induced arachidonic acid release was completely abrogated by the mitogen-activated protein/ERK kinase (MEK) inhibitor U0126 and by calcium chelators. With the p38 inhibitor SB-20219, we demonstrated that peroxynitrite-induced p38 phosphorylation led to minor arachidonic acid release, whereas U0126 completely blocked p38 phosphorylation. Addition of arachidonic acid caused p38 phosphorylation, suggesting that arachidonic acid or its metabolites are responsible for p38 activation. KN-93, a specific inhibitor of Ca²⁺/calmodulin-dependent kinase II (CaMKII), revealed no role for this kinase in peroxynitrite-induced arachidonic acid release in our cell system. Together, these results show that in response to peroxynitrite the cell initiates the MEK/ERK cascade leading to cPLA₂ activation and arachidonic acid release. Thus studies investigating the role of the NO· pathway on eicosanoid production must consider the contribution of signaling pathways initiated by reactive nitrogen species. These findings may provide evidence for a new role of peroxynitrite as an important reactive nitrogen species in vascular disease. reactive nitrogen species; prostaglandin H₂ synthase; extracellular signal-regulated kinase; p38; cytosolic phospholipase A₂     

Sestrin 2 and AMPK Connect Hyperglycemia to Nox4-Dependent Endothelial Nitric Oxide Synthase Uncoupling and Matrix Protein Expression

Mesangial matrix accumulation is an early feature of glomerular pathology in diabetes. Oxidative stress plays a critical role in hyperglycemia-induced glomerular injury. Here, we demonstrate that, in glomerular mesangial cells (MCs), endothelial nitric oxide synthase (eNOS) is uncoupled upon exposure to high glucose (HG), with enhanced generation of reactive oxygen species (ROS) and decreased production of nitric oxide. Peroxynitrite mediates the effects of HG on eNOS dysfunction. HG upregulates Nox4 protein, and inhibition of Nox4 abrogates the increase in ROS and peroxynitrite generation, as well as the eNOS uncoupling triggered by HG, demonstrating that Nox4 functions upstream from eNOS. Importantly, this pathway contributes to HG-induced MC fibronectin accumulation. Nox4-mediated eNOS dysfunction was confirmed in glomeruli of a rat model of type 1 diabetes. Sestrin 2-dependent AMP-activated protein kinase (AMPK) activation attenuates HG-induced MC fibronectin synthesis through blockade of Nox4-dependent ROS and peroxynitrite generation, with subsequent eNOS uncoupling. We also find that HG negatively regulates sestrin 2 and AMPK, thereby promoting Nox4-mediated eNOS dysfunction and increased fibronectin. These data identify a protective function for sestrin 2/AMPK and potential targets for intervention to prevent fibrotic injury in diabetes.     

Interactions of peroxynitrite,tetrahydrobiopterin, ascorbic acid,and thiols: implications for uncoupling endothelial nitric-oxide synthase

Tetrahydrobiopterin (BH4) serves as a critical co-factor for the endothelial nitric-oxide synthase (eNOS). A deficiency of BH4 results in eNOS uncoupling, which is associated with increased superoxide and decreased NO* production. BH4 has been suggested to be a target for oxidation by peroxynitrite (ONOO-), and ascorbate has been shown to preserve BH4 levels and enhance endothelial NO* production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO- with BH4 was studied using electron spin resonance and the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine. ONOO- reacted with BH4 6-10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO- and BH4 was the trihydrobiopterin radical (BH3.), which was reduced back to BH4 by ascorbate, whereas thiols were not efficient in recycling of BH4. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells (BAECs) by measuring superoxide and NO* using spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine and the NO*-spin trap iron-diethyldithiocarbamate. Bolus ONOO-, the ONOO- donor 3-morpholinosydnonimine, and an inhibitor of BH4 synthesis (2,4-diamino-6-hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO* production. Exogenous BH4 supplementation restored endothelial NO* production. Treatment of BAECs with both BH4 and ascorbate prior to ONOO- prevented uncoupling of eNOS by ONOO-. This study demonstrates that endothelial BH4 is a crucial target for oxidation by ONOO- and that the BH4 reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO- uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH4 from oxidation but recycles BH3. radical back to BH4.     

eNOS-β-Actin Interaction Contributes to Increased Peroxynitrite Formation during Hyperoxia in Pulmonary Artery Endothelial Cells and Mouse Lungs

Oxygen toxicity is the most severe side effect of oxygen therapy in neonates and adults. Pulmonary damage of oxygen toxicity is related to the overproduction of reactive oxygen species (ROS). In the present study, we investigated the effect of hyperoxia on the production of peroxynitrite in pulmonary artery endothelial cells (PAEC) and mouse lungs. Incubation of PAEC under hyperoxia (95% O₂) for 24 h resulted in an increase in peroxynitrite formation. Uric acid, a peroxynitrite scavenger, prevented hyperoxia-induced increase in peroxynitrite. The increase in peroxynitrite formation is accompanied by increases in nitric oxide (NO) release and endothelial NO synthase (eNOS) activity. We have previously reported that association of eNOS with β-actin increases eNOS activity and NO production in lung endothelial cells. To study whether eNOS-β-actin association contributes to increased peroxynitrite production, eNOS-β-actin interaction were inhibited by reducing β-actin availability or by using a synthetic peptide (P326TAT) containing a sequence corresponding to the actin binding site on eNOS. We found that disruption of eNOS-β-actin interaction prevented hyperoxia-induced increases in eNOS-β-actin association, eNOS activity, NO and peroxynitrite production, and protein tyrosine nitration. Hyperoxia failed to induce the increases in eNOS activity, NO and peroxynitrite formation in COS-7 cells transfected with plasmids containing eNOS mutant cDNA in which amino acids leucine and tryptophan were replaced with alanine in the actin binding site on eNOS. Exposure of mice to hyperoxia resulted in significant increases in eNOS-β-actin association, eNOS activity, and protein tyrosine nitration in the lungs. Our data indicate that increased association of eNOS with β-actin in PAEC contributes to hyperoxia-induced increase in the production of peroxynitrite which may cause nitrosative stress in pulmonary vasculature.     

Nitric oxide activates p21ras and leads to the inhibition of endothelial NO synthase by protein nitration

Recent data has indicated that exogenous nitric oxide (NO) has the ability to decrease endogenous NO production by inhibiting the enzyme responsible for its generation, NO synthase (NOS). Our previous studies have indicated that increased generation of reactive oxygen species (ROS) play an important role in the inhibitory event. However, the mechanisms for these effects remain unclear. Previous studies have suggested that NO can activate p21ras. Thus, the objective of this study was to determine whether NO-mediated activation of p21ras is involved in the inhibitory process, and to further elucidate the involvement of ROS. Using primary cultures of ovine pulmonary arterial endothelial cells we demonstrated that the NO donor SpermineNONOate, increased p21ras activity by 2.3-fold compared to untreated cells, and that the farnesyl-transferase inhibitor, alpha-hydroxyfarnesylphosphonic acid, reduced p21ras activity and significantly reduced inhibition of eNOS. The overexpression of p21ras increased, while the overexpression of an NO unresponsive mutant of p21ras (p21ras C118S) reduced, the inhibition of eNOS by NO. Further, we identified an increase in the level of superoxide and peroxynitrite in endothelial cells exposed to NO that was reduced by p21ras C118S transient transfection. Conversely, levels of superoxide and peroxynitrite could be increased by the over expression of wild type p21ras. Similarly, eNOS nitration induced by NO exposure was reduced by p21ras C118S transient transfection, and increased by the overexpression of wild-type p21ras. Finally, results also demonstrated that eNOS itself was a significant producer of superoxide, and that this appeared to be related to a p21ras-dependent increase in phosphorylation of Ser1177. Our results implicate a signaling pathway involving p21ras activation, superoxide generation, and peroxynitrite formation as being important in the NO-mediated inhibition of eNOS.     

Early determinants of H2O2-induced endothelial dysfunction

Reactive oxygen species (ROS) can stimulate nitric oxide (NO(*)) production from the endothelium by transient activation of endothelial nitric oxide synthase (eNOS). With continued or repeated exposure, NO(*) production is reduced, however. We investigated the early determinants of this decrease in NO(*) production. Following an initial H(2)O(2) exposure, endothelial cells responded by increasing NO(*) production measured electrochemically. NO(*) concentrations peaked by 10 min with a slow reduction over 30 min. The decrease in NO(*) at 30 min was associated with a 2.7-fold increase in O(2)(*-) production (p < 0.05) and a 14-fold reduction of the eNOS cofactor, tetrahydrobiopterin (BH(4), p < 0.05). Used as a probe for endothelial dysfunction, the integrated NO(*) production over 30 min upon repeated H(2)O(2) exposure was attenuated by 2.1-fold (p = 0.03). Endothelial dysfunction could be prevented by BH(4) cofactor supplementation, by scavenging O(2)(*-) or peroxynitrite (ONOO(-)), or by inhibiting the NADPH oxidase. Hydroxyl radical (()OH) scavenging did not have an effect. In summary, early H(2)O(2)-induced endothelial dysfunction was associated with a decreased BH(4) level and increased O(2)(*-) production. Dysfunction required O(2)(*-), ONOO(-), or a functional NADPH oxidase. Repeated activation of the NADPH oxidase by ROS may act as a feed forward system to promote endothelial dysfunction.     

Adiponectin improves endothelial function in hyperlipidemic rats by reducing oxidative/nitrative stress and differential regulation of eNOS/iNOS activity

Plasma adiponectin level is significantly reduced in patients with metabolic syndrome, and vascular dysfunction is an important pathological event in these patients. However, whether adiponectin may protect endothelial cells and attenuate endothelial dysfunction caused by metabolic disorders remains largely unknown. Adult rats were fed with a regular or a high-fat diet for 14 wk. The aorta was isolated, and vascular segments were incubated with vehicle or the globular domain of adiponectin (gAd; 2 mug/ml) for 4 h. The effect of gAd on endothelial function, nitric oxide (NO) and superoxide production, nitrotyrosine formation, gp91(phox) expression, and endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) activity/expression was determined. Severe endothelial dysfunction (maximal vasorelaxation in response to ACh: 70.3 +/- 3.3 vs. 95.2 +/- 2.5% in control, P < 0.01) was observed in hyperlipidemic aortic segments, and treatment with gAd significantly improved endothelial function (P < 0.01). Paradoxically, total NO production was significantly increased in hyperlipidemic vessels, and treatment with gAd slightly reduced, rather than increased, total NO production in these vessels. Treatment with gAd reduced (-78%, P < 0.01) superoxide production and peroxynitrite formation in hyperlipidemic vascular segments. Moreover, a moderate attenuation (-30%, P < 0.05) in gp91(phox) and iNOS overexpression in hyperlipidemic vessels was observed after gAd incubation. Treatment with gAd had no effect on eNOS expression but significantly increased eNOS phosphorylation (P < 0.01). Most noticeably, treatment with gAd significantly enhanced eNOS (+83%) but reduced iNOS (-70%, P < 0.01) activity in hyperlipidemic vessels. Collectively, these results demonstrated that adiponectin protects the endothelium against hyperlipidemic injury by multiple mechanisms, including promoting eNOS activity, inhibiting iNOS activity, preserving bioactive NO, and attenuating oxidative/nitrative stress.     

3-Morpholinosyndnonimine inhibits 5-hydroxytryptamine-induced phosphorylation of nitric oxide synthase in endothelial cells.

5-Hydroxytryptamine (5-HT) is a vasoactive substance that is taken up by endothelial cells to activate endothelial nitrite oxide synthase (eNOS). The activation of eNOS results in the production of nitric oxide (NO), which is responsible for vasodilation of blood vessels. NO also interacts with superoxide anion (O2*-) to form peroxynitrite (ONOO-), a potent oxidant that has been shown to induce vascular endothelial dysfunction. We examined the ability of 3-morpholinosyndnonimine (SIN-1), an ONOO- generator, to inhibit 5-HT-induced phosphorylation of eNOS in cultured bovine aortic endothelial cells (BAECs). We observed that 5-HT phosphorylates Ser1179 eNOS in a time- and concentration-dependent manner. Maximum phosphorylation occurred at 30 sec using a concentration of 1.0 microM 5-HT. BAECs treated with SIN-1 (1-1000 microM) for 30 min showed no significant increase in eNOS phosphorylation. However, 5-HT-induced eNOS phosphorylation was inhibited in cells treated with various concentrations of SIN-1 for 30 min and stimulated with 5-HT. These data suggest that an increase in ONOO- as a result of an increase in the production of O2*-, may feedback to inhibit 5-HT-induced eNOS phosphorylation at Ser1179 and therefore, contribute to endothelial dysfunction associated with cardiovascular diseases.     

20-hydroxyeicosatetraenoic acid causes endothelial dysfunction via eNOS uncoupling

Nitric oxide (NO), generated from L-arginine by endothelial nitric oxide synthase (eNOS), is a key endothelial-derived factor whose bioavailability is essential to the normal function of the endothelium. Endothelium dysfunction is characterized by loss of NO bioavailability because of either reduced formation or accelerated degradation of NO. We have recently reported that overexpression of vascular cytochrome P-450 (CYP) 4A in rats caused hypertension and endothelial dysfunction driven by increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a major vasoconstrictor eicosanoid in the microcirculation. To further explore cellular mechanisms underlying CYP4A-20-HETE-driven endothelial dysfunction, the interactions between 20-HETE and the eNOS-NO system were examined in vitro. Addition of 20-HETE to endothelial cells at concentrations as low as 1 nM reduced calcium ionophore-stimulated NO release by 50%. This reduction was associated with a significant increase in superoxide production. The increase in superoxide in response to 20-HETE was prevented by N(G)-nitro-L-arginine methyl ester, suggesting that uncoupled eNOS is a source of this superoxide. The response to 20-HETE was specific in that 19-HETE did not affect NO or superoxide production, and, in fact, the response to 20-HETE could be competitively antagonized by 19(R)-HETE. 20-HETE had no effect on phosphorylation of eNOS protein at serine-1179 or threonine-497 following addition of calcium ionophore; however, 20-HETE inhibited association of eNOS with 90-kDa heat shock protein (HSP90). In vivo, impaired acetylcholine-induced relaxation in arteries overexpressing CYP4A was associated with a marked reduction in the levels of phosphorylated vasodilator-stimulated phosphoprotein, an indicator of bioactive NO, that was reversed by inhibition of 20-HETE synthesis or action. Because association of HSP90 with eNOS is critical for eNOS activation and coupled enzyme activity, inhibition of this association by 20-HETE may underlie the mechanism, at least in part, by which increased CYP4A expression and activity cause endothelial dysfunction.     

Cigarette smoke and LDL cooperate in reducing nitric oxide bioavailability in endothelial cells via effects on both eNOS and NADPH oxidase

The ubiquitous free radical nitric oxide (NO) plays an important role in many biological processes, including the regulation of both vascular tone and inflammatory response; however, its role in the effects of cigarette smoke exposure on atherosclerosis remains unclear. Our aim was to study the mechanisms of NO regulation in endothelial cells in response to cigarette smoke exposure in vitro. Using human umbilical vein endothelial cells (HUVEC), we have demonstrated that combining non-toxic concentrations of cigarette smoke bubbled through PBS (smoke-bubbled PBS [sbPBS]) with native LDL (nLDL) significantly reduces the amount of bioavailable NO. The effect is comparable to that seen with oxidized LDL (oxLDL), but has not been seen with sbPBS or nLDL alone. Mechanistic investigations showed that the combination of sbPBS+nLDL did not reduce the amount of endothelial nitric oxide synthase (eNOS), but did inhibit its enzymatic activity. Concomitantly, both sbPBS+nLDL and oxLDL significantly increased the production of reactive oxygen species (ROS) in the form of superoxide anions (()O(2)(-)) and peroxynitrite (ONOO(-)) in HUVEC. Selective inhibition of NADPH oxidase prevented this response. Incubation of sbPBS+nLDL revealed the formation of 7-ketocholesterol (7-KC) and 7-hydroxycholesterol, which are indicators for oxidative modification of LDL. This could explain the reported increase in circulatory levels of oxLDL in smokers. Our results suggest that reduction of functional NO in response to a combination of sbPBS+nLDL is secondary to both reduction of eNOS activity and stimulation of NADPH oxidase activity. Because sbPBS alone showed no effect on eNOS activity or ROS formation, nLDL should be included in cigarette-smoke-related mechanistic in vitro experiments on endothelial cells to be more reflective of the clinical situation.     

Ceramide-induced activation of NADPH oxidase and endothelial dysfunction in small coronary arteries

We tested the hypothesis that ceramide induces endothelial dysfunction in small coronary arteries via NADPH oxidase-mediated superoxide and resulting peroxynitrite formation. With the use of dihydroethidium as a superoxide indicator, C(2)-ceramide was found to increase superoxide production in the endothelial cells of small coronary arteries, which was inhibited by the NADPH oxidase inhibitors N-vanillylnonanamide, apocynin, and diphenylene iodonium. NADPH oxidase expression was confirmed in endothelial cells, as indicated by the immunoblotting of its subunits gp91(phox) and p47(phox). C(2)-ceramide increased NADPH oxidase activity by 52%, which was blocked by NADPH oxidase inhibitors but not by inhibitors of NO synthase, xanthine oxidase, and mitochondrial electron transport chain enzymes. By Western blot analysis, ceramide-induced NADPH oxidase activation was found to be associated with the translocation of p47(phox) to the membrane. In isolated and pressurized small coronary arteries, N-vanillylnonanamide, apocynin, or uric acid, a peroxynitrite scavenger, largely restored the inhibitory effects of ceramide on bradykinin- and A-23187-induced vasorelaxation. With the use of nitrotyrosine as a marker, C(2)-ceramide was found to increase peroxynitrite in small coronary arteries, which could be blocked by uric acid. We conclude that NADPH oxidase-mediated superoxide production and subsequent peroxynitrite formation mediate ceramide-induced endothelial dysfunction in small coronary arteries.     

The role of mitochondrial dysfunction and neuronal nitric oxide in animal models of neurodegenerative diseases

Excitotoxicity, mitochondrial dysfunction and free radical induced oxidative damage have been implicated in the pathogenesis of several different neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease. Much of the interest in the association of neurodegeneration with mitochondrial dysfunction and oxidative damage emerged from animal studies using mitochondrial toxins. Within mitochondria 1-methyl-4-phenylpyridinium (MPP⁺), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), acts to inhibit NADH-coenzyme Q reductase (complex I) of the electron transport chain. MPTP produces Parkinsonism in humans, primates, and mice. Similarly, lesions produced by the reversible inhibitor of succinate dehydrogenase (complex II), malonate, and the irreversible inhibitor, 3-nitropropionic acid (3-NP), closely resemble the histologic, neurochemical and clinical features of HD in both rats and non-human primates. The interruption of oxidative phosphorylation results in decreased levels of ATP. A consequence is partial neuronal depolarization and secondary activation of voltage-dependent NMDA receptors, which may result in excitotoxic neuronal cell death (secondary excitotoxicity). The increase in intracellular Ca²⁺ concentration leads to an actiation of Ca²⁺ dependent enzymes, including the constitutive neuronal nitric oxide synthase (cnNOS) which produces NO·. NO· may react with the superoxide anion to form peroxynitrite. We show that systemic administration of 7-nitroindazole (7-NI), a relatively specific inhibitor of cnNOS in vivo. attenuates lesions produced by striatal malonate injections or systemic treatment with 3-NP or MPTP. Furthermore 7-NI attenuated increases in lactate production and hydroxyl radical and 3-nitrotyrosine generation in vivo, which may be a consequence of peroxynitrite formation. Our results suggest that neuronal nitric oxide synthase inhibitors may be useful in the treatment of neurologic diseases in which excitotoxic mechanisms play a role. (Mol Cell Biochem 174: 193–197, 1997)     

Endothelial Nitric Oxide Synthase and Superoxide Mediate Hemodynamic Initiation of Intracranial Aneurysms

Background

Hemodynamic insults at arterial bifurcations are believed to play a critical role in initiating intracranial aneurysms. Recent studies in a rabbit model indicate that aneurysmal damage initiates under specific wall shear stress conditions when smooth muscle cells (SMCs) become pro-inflammatory and produce matrix metalloproteinases (MMPs). The mechanisms leading to SMC activation and MMP production during hemodynamic aneurysm initiation are unknown. The goal is to determine if nitric oxide and/or superoxide induce SMC changes, MMP production and aneurysmal remodeling following hemodynamic insult.

Methods

Bilateral common carotid artery ligation was performed on rabbits (n = 19, plus 5 sham operations) to induce aneurysmal damage at the basilar terminus. Ligated animals were treated with the nitric oxide synthase (NOS) inhibitor LNAME (n = 7) or the superoxide scavenger TEMPOL (n = 5) and compared to untreated animals (n = 7). Aneurysm development was assessed histologically 5 days after ligation. Changes in NOS isoforms, peroxynitrite, reactive oxygen species (ROS), MMP-2, MMP-9, and smooth muscle α-actin were analyzed by immunohistochemistry.

Results

LNAME attenuated ligation-induced IEL loss, media thinning and bulge formation. In untreated animals, immunofluorescence showed increased endothelial NOS (eNOS) after ligation, but no change in inducible or neuronal NOS. Furthermore, during aneurysm initiation ROS increased in the media, but not the intima, and there was no change in peroxynitrite. In LNAME-treated animals, ROS production did not change. Together, this suggests that eNOS is important for aneurysm initiation but not by producing superoxide. TEMPOL treatment reduced aneurysm development, indicating that the increased medial superoxide is also necessary for aneurysm initiation. LNAME and TEMPOL treatment in ligated animals restored α-actin and decreased MMPs, suggesting that eNOS and superoxide both lead to SMC de-differentiation and MMP production.

Conclusion

Aneurysm-inducing hemodynamics lead to increased eNOS and superoxide, which both affect SMC phenotype, increasing MMP production and aneurysmal damage.     

Reactive oxygen species as cardiovascular mediators: Lessons from endothelial-specific protein overexpression mouse models

The term reactive oxygen species (ROS) summarizes several small chemical compounds such as superoxide, peroxynitrite, hydrogen peroxide and nitric oxide. The stoichiometry of the chemical reactions underlying generation and metabolism is subject of tight enzymatic regulation resulting in well balanced steady-state concentrations throughout the healthy body. ROS are short-lived and usually active at the site of production only, e.g. in vascular endothelial cells. Although an increase of vascular ROS-production is considered an important pathogenic factor in cardiovascular diseases, there is evidence for physiological or even beneficial effects as well. We have generated several transgenic mice using the Tie-2 promotor which expresses an enzyme of interest specifically in vascular endothelial cells. Here, we review some results obtained with mice carrying a Tie-2-driven overexpression of catalase or endothelial nitric oxide synthase (eNOS). Tie-2-catalase mice have a strongly reduced steady-state concentration of vascular hydrogen peroxide and show profound hypotension that is not dependent on the bioavailability of endothelial nitric oxide but is completely reversible by treatment with the catalase inhibitor aminotriazole. A similar hypotension was observed in transgenic mice with an endothelial-specific overexpression of eNOS but this hypotension is entirely dependent on vascular eNOS activity. These observations suggest a tonic effect of hydrogen peroxide on vascular smooth muscle. Further studies suggested that hydrogen peroxide promotes the exercise-induced increase of vascular eNOS expression and inhibits the release of endothelial progenitor cells induced by exercise training. In summary, our data support the concept of a dual role of ROS in the vascular system.     

Evidence against tetrahydrobiopterin depletion of vascular tissue exposed to nitric oxide/superoxide or nitroglycerin

Several cardiovascular disorders, including atherosclerosis and tolerance to the antianginal drug nitroglycerin (GTN), may be associated with the generation of superoxide anions, which react with nitric oxide (NO) to yield peroxynitrite. According to a widely held view, oxidation of tetrahydrobiopterin (BH₄) by peroxynitrite causes uncoupling of endothelial NO synthase (eNOS), resulting in reduced NO bioavailability and endothelial dysfunction under conditions of oxidative stress. In this study we determined the levels of reduced biopterins and endothelial function in cultured cells exposed to peroxynitrite and GTN as well as in blood vessels isolated from GTN-tolerant guinea pigs and rats. BH₄ was rapidly oxidized by peroxynitrite and 3-morpholino sydnonimine (SIN-1) in buffer, but this was prevented by glutathione and not observed in endothelial cells exposed to SIN-1 or GTN. Prolonged treatment of the cells with 0.1 mM GTN caused slow N^G-nitro-l-arginine-sensitive formation of reactive oxygen species without affecting eNOS activity. Endothelial function and BH₄/BH₂ levels were identical in blood vessels of control and GTN-tolerant animals. Our results suggest that peroxynitrite-triggered BH₄ oxidation does not occur in endothelial cells or GTN-exposed blood vessels. GTN seems to trigger minor eNOS uncoupling that is unrelated to BH₄ depletion and without observable consequence on eNOS function.     

Protective effects of honokiol against oxidized LDL-induced cytotoxicity and adhesion molecule expression in endothelial cells

Honokiol, a compound extracted from Chinese medicinal herb Magnolia officinalis, has several biological effects. However, its protective effects against endothelial injury remain unclarified. In this study, we examined whether honokiol prevented oxidized low-density lipoprotein (oxLDL)-induced vascular endothelial dysfunction. Incubation of oxLDL with honokiol (2.5-20 microM) inhibited copper-induced oxidative modification as demonstrated by diene formation, thiobarbituric acid reactive substances (TBARS) assay and electrophoretic mobility assay. Expression of adhesion molecules (ICAM, VCAM and E-selectin) and endothelial NO synthase (eNOS) affected by oxLDL was investigated by flow cytometry and Western blot. We also measured the production of reactive oxygen species (ROS) using the fluorescent probe 2',7'-dichlorofluorescein acetoxymethyl ester (DCF-AM). Furthermore, several apoptotic phenomena including increased cytosolic calcium, alteration of mitochondrial membrane potential, cytochrome c release and activation of caspase-3 were also investigated. Apoptotic cell death was characterized by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain. The results showed that honokiol prevented the copper-induced oxidative modification of LDL. Honokiol also ameliorated the oxLDL-diminished eNOS protein expression and reduced the oxLDL-induced adhesion molecules and the adherence of THP-1 cells to HUVECs. Furthermore, honokiol attenuated the oxLDL-induced cytotoxicity, apoptotic features, ROS generation, intracellular calcium accumulation and the subsequent mitochondrial membrane potential collapse, cytochrome c release and activation of caspase-3. Our results suggest that honokiol may have clinical implications in the prevention of atherosclerotic vascular disease.     

Indoxyl sulfate potentiates endothelial dysfunction via reciprocal role for reactive oxygen species and RhoA/ROCK signaling in 5/6 nephrectomized rats

Accumulative indoxyl sulfate (IS) retained in chronic kidney disease (CKD) can potentiate vascular endothelial dysfunction, and herein, we aim at elucidating the underlying mechanisms from the perspective of possible association between reactive oxygen species (ROS) and RhoA/ROCK pathway. IS-treated nephrectomized rats are administered with antioxidants including NADPH oxidase inhibitor apocynin, SOD analog tempol, and mitochondrion-targeted SOD mimetic mito-TEMPO to scavenge ROS, or ROCK inhibitor fasudil to obstruct RhoA/ROCK pathway. First, we find in response to IS stimulation, antioxidants treatments suppress increased aortic ROCK activity and expression levels. Additionally, ROCK blockade prevent IS-induced increased NADPH oxidase expression (mainly p22phox and p47phox), mitochondrial and intracellular ROS (superoxide and hydrogen peroxide) generation, and decreased Cu/Zn-SOD expression in thoracic aortas. Apocynin, mito-TEMPO, and tempol also reverse these markers of oxidative stress. These results suggest that IS induces excessive ROS production and ROCK activation involving a circuitous relationship in which ROS activate ROCK and ROCK promotes ROS overproduction. Finally, ROS and ROCK depletion attenuate IS-induced decrease in nitric oxide (NO) production and eNOS expression levels, and alleviate impaired vasomotor responses including increased vasocontraction to phenylephrine and decreased vasorelaxation to acetylcholine, thereby preventing cardiovascular complications accompanied by CKD. Taken together, excessive ROS derived from NADPH oxidase and mitochondria coordinate with RhoA/ROCK activation in a form of positive reciprocal relationship to induce endothelial dysfunction through disturbing endothelium-dependent NO signaling upon IS stimulation in CKD status.     

Cell oxidant stress delivery and cell dysfunction onset in type 2 diabetes

Most known pathways of diabetic complications involve oxidative stress. The mitochondria electron transport chain is a significant source of reactive oxygen species (ROS) in insulin secretory cells, insulin peripheral sensitive cells and endothelial cells. Elevated intracellular glucose level induces tricarboxylic acid cycle electron donor overproduction and mitochondrial proton gradient increase leading to an increase in electron transporter lifetime. Subsequently, the electrons leaked combine with respiratory oxygen (O₂) resulting in superoxide anion (^•O₂⁻) production. Advanced glycation end products derive ROS via interaction with their receptors. Elevated diacylglycerol and ROS activate the protein kinase C pathway which, in turn, activates NADPH oxidases. A vicious circle of pathway derived ROS installs. Pathologic pathways induced ROS are activated and persistent though glycemia returns to normal due to hyperglycemia memory. Endothelial nitric oxide synthase may produce both superoxide anion (^•O₂⁻) and nitric oxide (NO) leading to peroxynitrite (^•ONOO⁻) generation. Homocysteine is also implicated in oxidative stress pathogenesis. In this paper we have highlighted the pathologic mechanisms of ROS on atherosclerosis, renal dysfunction, retina dysfunction and nerve dysfunction in type 2 diabetes. Cell oxidant stress delivery have pivotal role in cell dysfunction onset and progression of angiopathies but an early introduction of good glycemic control may protect cells more efficiently than antioxidants.     

Janus-faced role of endothelial NO synthase in vascular disease: uncoupling of oxygen reduction from NO synthesis and its pharmacological reversal

Endothelial NO synthase (eNOS) is the predominant enzyme responsible for vascular NO synthesis. A functional eNOS transfers electrons from NADPH to its heme center, where L-arginine is oxidized to L-citrulline and NO. Common conditions predisposing to atherosclerosis, such as hypertension, hypercholesterolemia, diabetes mellitus and smoking, are associated with enhanced production of reactive oxygen species (ROS) and reduced amounts of bioactive NO in the vessel wall. NADPH oxidases represent major sources of ROS in cardiovascular pathophysiology. NADPH oxidase-derived superoxide avidly interacts with eNOS-derived NO to form peroxynitrite (ONOO(-)), which oxidizes the essential NOS cofactor (6R-)5,6,7,8-tetrahydrobiopterin (BH(4)). As a consequence, oxygen reduction uncouples from NO synthesis, thereby rendering NOS to a superoxide-producing pro-atherosclerotic enzyme. Supplementation with BH(4) corrects eNOS dysfunction in several animal models and in patients. Administration of high local doses of the antioxidant L-ascorbic acid (vitamin C) improves endothelial function, whereas large-scale clinical trials do not support a strong role for oral vitamin C and/or E in reducing cardiovascular disease. Statins, angiotensin-converting enzyme inhibitors and AT1 receptor blockers have the potential of reducing vascular oxidative stress. Finally, novel approaches are being tested to block pathways leading to oxidative stress (e.g. protein kinase C) or to upregulate antioxidant enzymes.