Huige Li

Effects of Angiotensin II Infusion on the Expression and Function of NAD(P)H Oxidase and Components of Nitric Oxide/cGMP Signaling

Angiotensin II infusion causes endothelial dysfunction by increasing NAD(P)H oxidase-mediated vascular superoxide production. However, it remains to be elucidated how in vivo angiotensin II treatment may alter the expression of the gp91phox isoforms and the endothelial nitric oxide synthase (NOS III) and subsequent signaling events and whether, in addition to the NAD(P)H oxidase, NOS III contributes to vascular superoxide formation. We therefore studied the influence of in vivo angiotensin II treatment (7 days) in rats on endothelial function and on the expression of the NAD(P)H oxidase subunits p22phox, nox1, nox4, and gp91phox and NOS III. Further analysis included the expression of NO-downstream targets, the soluble guanylyl cyclase (sGC), the cGMP-dependent protein kinase I (cGK-I), and the expression and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) at Ser239 (P-VASP). Angiotensin II caused endothelial dysfunction and increased vascular superoxide. Likewise, we found an increase in vascular protein kinase C (PKC) activity, in the expression of nox1 (6- to 7-fold), gp91phox (3-fold), p22phox (3-fold), NOS III mRNA, and protein. NOS-inhibition with NG-nitro-l-arginine decreased superoxide in vessels from angiotensin II-treated animals, compatible with NOS-uncoupling. Vascular NO assessed with electron paramagnetic resonance was markedly reduced. Likewise, a decrease in sGC-expression and P-VASP levels was found. In vivo PKC-inhibition with chelerythrine reduced angiotensin II-induced superoxide production and markedly inhibited upregulation of NAD(P)H oxidase subunits. We therefore conclude that angiotensin II-induced increases in the activity and the expression of NAD(P)H oxidase are at least in part PKC-dependent. NADPH oxidase-induced superoxide production may trigger NOS III uncoupling, leading to impaired NO/cGMP signaling and to endothelial dysfunction in this animal model. The full text of this article is available at http://www.circresaha.org.

Resveratrol, a Polyphenolic Phytoalexin Present in Red Wine, Enhances Expression and Activity of Endothelial Nitric Oxide Synthase

Background—Estrogens can upregulate endothelial nitric oxide synthase (eNOS) in human endothelial cells by increasing eNOS promoter activity and enhancing the binding activity of the transcription factor Sp1. Resveratrol, a polyphenolic phytoalexin found in grapes and wine, has been reported to act as an agonist at the estrogen receptor. Therefore, we tested the effect of this putative phytoestrogen on eNOS expression in human endothelial cells. Methods and Results—Incubation of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells with resveratrol for 24 to 72 hours upregulated eNOS mRNA expression in a time- and concentration-dependent manner (up to 2.8-fold). eNOS protein expression and eNOS-derived NO production were also increased after long-term incubation with resveratrol. Resveratrol increased the activity of the eNOS promoter (3.5-kb fragment) in a concentration-dependent fashion, with the essential trans-stimulated sequence being located in the proximal 263 bp of the promoter sequence. In addition, eNOS mRNA was stabilized by resveratrol. The effect of resveratrol on eNOS expression was not modified by the estrogen receptor antagonists ICI 182780 and RU 58668. In electrophoretic mobility shift assays, nuclear extracts from resveratrol-incubated EA.hy 926 cells showed no enhanced binding activity of the eNOS promoter-relevant transcription factors Sp1, GATA, PEA3, YY1, or Elf-1. In addition to its long-term effects on eNOS expression, resveratrol also enhanced the production of bioactive NO in the short-term (after a 2-minute incubation). Conclusions—In concert with other effects, the stimulation of eNOS expression and activity may contribute to the cardiovascular protective effects attributed to resveratrol.

Nitric oxide in the pathogenesis of vascular disease

Nitric oxide (NO) is synthesized by at least three distinct isoforms of NO synthase (NOS). Their substrate and cofactor requirements are very similar. All three isoforms have some implications, physiological or pathophysiological, in the cardiovascular system. The endothelial NOS III is physiologically important for vascular homeostasis, keeping the vasculature dilated, protecting the intima from platelet aggregates and leukocyte adhesion, and preventing smooth muscle proliferation. Central and peripheral neuronal NOS I may also contribute to blood pressure regulation. Vascular disease associated with hypercholesterolaemia, diabetes, and hypertension is characterized by endothelial dysfunction and reduced endothelium‐mediated vasodilation. Oxidative stress and the inactivation of NO by superoxide anions play an important role in these disease states. Supplementation of the NOS substrate L‐arginine can improve endothelial dysfunction in animals and man. Also, the addition of the NOS cofactor (6R)‐5,6,7,8‐tetrahydrobiopterin improves endothelium‐mediated vasodilation in certain disease states. In cerebrovascular stroke, neuronal NOS I and cytokine‐inducible NOS II play a key role in neurodegeneration, whereas endothelial NOS III is important for maintaining cerebral blood flow and preventing neuronal injury. In sepsis, NOS II is induced in the vascular wall by bacterial endotoxin and/or cytokines. NOS II produces large amounts of NO, which is an important mediator of endotoxin‐induced arteriolar vasodilatation, hypotension, and shock. Copyright

Estrogens increase transcription of the human endothelial NO synthase gene analysis of the transcription factors involved

Estrogens have been found to reduce the incidence of cardiovascular disease that has been ascribed in part to an increased expression and/or activity of the vasoprotective endothelial NO synthase (NOS III). Some reports have shown that the level of expression of this constitutive enzyme can be upregulated by estrogens. The current study investigates the molecular mechanism of the NOS III upregulation in human endothelial EA.hy 926 cells. Incubation of EA.hy 926 cells with 17beta-estradiol or the more stable 17alpha-ethinyl estradiol enhanced NOS III mRNA and protein expression up to 1.8-fold, without changing the stability of the NOS III mRNA. There was no enhancement of NOS III mRNA after incubation of EA.hy 926 cells with testosterone, progesterone, or dihydrocortisol or when 17alpha-ethinyl estradiol was added together with the estrogen antagonist RU58668, indicating a specific estrogenic response. Nuclear run-on assays indicated that the increase in NOS III mRNA is the result of an estrogen-induced enhancement of NOS III gene transcription. In transient transfection experiments using a 1.6 kb human NOS III promoter fragment (which contains no bona fide estrogen-responsive element, ERE), basal promoter activity was enhanced 1.7-fold by 17alpha-ethinyl estradiol. In electrophoretic mobility shift assays, nuclear extracts from estrogen-incubated EA.hy 926 cells showed no enhanced binding activity either for the ERE-like motif in the human NOS III promoter or for transcription factor GATA. However, binding of transcription factor Sp1 (which is essential for the activity of the human NOS III promoter) was significantly enhanced by estrogens. These data suggest that the estrogen stimulation of the NOS III promoter could be mediated in part by an increased activity of transcription factor Sp1.

Vascular oxidative stress, nitric oxide and atherosclerosis

In the vascular wall, reactive oxygen species (ROS) are produced by several enzyme systems including NADPH oxidase, xanthine oxidase, uncoupled endothelial nitric oxide synthase (eNOS) and the mitochondrial electron transport chain. On the other hand, the vasculature is protected by antioxidant enzyme systems, including superoxide dismutases, catalase, glutathione peroxidases and paraoxonases, which detoxify ROS. Cardiovascular risk factors such as hypercholesterolemia, hypertension, and diabetes mellitus enhance ROS generation, resulting in oxidative stress. This leads to oxidative modification of lipoproteins and phospholipids, mechanisms that contribute to atherogenesis. In addition, oxidation of tetrahydrobiopterin may cause eNOS uncoupling and thus potentiation of oxidative stress and reduction of eNOS-derived NO, which is a protective principle in the vasculature. This review summarizes the latest advances in the role of ROS-producing enzymes, antioxidative enzymes as well as NO synthases in the initiation and development of atherosclerosis.

Physiological mechanisms regulating the expression of endothelial-type NO synthase.

Although endothelial nitric oxide synthase (eNOS) is a constitutively expressed enzyme, its expression is regulated by a number of biophysical, biochemical, and hormonal stimuli, both under physiological conditions and in pathology. This review summarizes the recent findings in this field. Shear stress, growth factors (such as transforming growth factor-beta, fibroblast growth factor, vascular endothelial growth factor, and platelet-derived growth factor), hormones (such as estrogens, insulin, angiotensin II, and endothelin 1), and other compounds (such as lysophosphatidylcholine) upregulate eNOS expression. On the other hand, the cytokine tumor necrosis factor-alpha and bacterial lipopolysaccharide downregulate the expression of this enzyme. The growth status of cells, the actin cytoskeleton, and NO itself are also important regulators of eNOS expression. Both transcriptional and posttranscriptional mechanisms are involved in the expressional regulation of eNOS. Different signaling pathways are involved in the regulation of eNOS promoter activity and eNOS mRNA stability. Changes in eNOS expression and activity under pathophysiological conditions and the pharmacological modulation of eNOS expression are subject of a subsequent brief review (part 2) to be published in the next issue of this journal.

Regulation of endothelial-type NO synthase expression in pathophysiology and in response to drugs.

In many types of cardiovascular pathophysiology such as hypercholesterolemia and atherosclerosis, diabetes, cigarette smoking, or hypertension (with its sequelae stroke and heart failure) the expression of endothelial NO synthase (eNOS) is altered. Both up- and downregulation of eNOS have been observed, depending on the underlying disease. When eNOS is upregulated, the upregulation is often futile and goes along with a reduction in bioactive NO. This is due to an increased production of superoxide generated by NAD(P)H oxidase and by an uncoupled eNOS. A number of drugs with favorable effects on cardiovascular disease upregulate eNOS expression. The resulting increase in vascular NO production may contribute to their beneficial effects. These compounds include statins, angiotensin-converting enzyme inhibitors, AT1 receptor antagonists, calcium channel blockers, and some antioxidants. Other drugs such as glucocorticoids, whose administration is associated with cardiovascular side effects, downregulate eNOS expression. Stills others such as the immunosuppressants cyclosporine A and FK506/tacrolimus or erythropoietin have inconsistent effects on eNOS. Thus regulation of eNOS expression and activity contributes to the overall action of several classes of drugs, and the development of compounds that specifically upregulate this protective enzyme appears as a desirable target for drug development.

Inhibitors of Histone Deacetylation Downregulate the Expression of Endothelial Nitric Oxide Synthase and Compromise Endothelial Cell Function in Vasorelaxation and Angiogenesis

Abstract— The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) inhibits hypoxia-stimulated angiogenesis. Endothelial nitric oxide synthase (eNOS)–derived NO is central to angiogenesis signaling in endothelial cells (ECs). We hypothesized that the HDAC-dependent regulation of angiogenesis may involve a modulatory effect on eNOS expression. The HDAC inhibitors TSA, butyric acid (BuA), and MS-275 time- and concentration-dependently suppressed eNOS protein levels to 41±2%, 46±12%, and 40±12% of control, respectively. In parallel, TSA and BuA also downregulated eNOS mRNA expression to 21±4% and 37±4% of control. TSA also attenuated the NO-dependent relaxation of porcine coronary arteries (P <0.0001, TSA 1 &mgr;mol/L) and prevented tube formation in a human angiogenesis assay. Although vascular endothelial growth factor substitution did not compensate for the inhibitory effect of TSA, exogenous NO reversed the inhibition of angiogenesis by TSA. To address the underlying signaling mechanism, we characterized the effect of TSA on eNOS gene transcription and mRNA half-life. Although TSA decreased both eNOS protein and mRNA levels, TSA paradoxically enhanced the activity of the eNOS promoter, and did not alter the eNOS transcription rate in nuclear run-on experiments, suggesting that TSA posttranscriptionally targets eNOS mRNA. These data indicate that HDAC-dependent mechanisms contribute to the regulation of eNOS expression in ECs.

Nebivolol Inhibits Superoxide Formation by NADPH Oxidase and Endothelial Dysfunction in Angiotensin II–Treated Rats

Nebivolol is a &bgr;1-receptor antagonist with vasodilator and antioxidant properties. Because the vascular NADPH oxidase is an important superoxide source, we studied the effect of nebivolol on endothelial function and NADPH oxidase activity and expression in the well-characterized model of angiotensin II–induced hypertension. Angiotensin II infusion (1 mg/kg per day for 7 days) caused endothelial dysfunction in male Wistar rats and increased vascular superoxide as detected by lucigenin-derived chemiluminescence, as well as dihydroethidine staining. Vascular NADPH oxidase activity, as well as expression at the mRNA and protein level, were markedly upregulated, as well as NOS III uncoupled, as evidenced by NO synthase III inhibitor experiments and dihydroethidine staining and by markedly decreased hemoglobin–NO concentrations. Treatment with the &bgr;-receptor blocker nebivolol but not metoprolol (10 mg/kg per day for each drug) normalized endothelial function, reduced superoxide formation, increased NO bioavailability, and inhibited upregulation of the activity and expression of the vascular NADPH oxidase, as well as membrane association of NADPH oxidase subunits (Rac1 and p67phox). In addition, NOS III uncoupling was prevented. In vitro treatment with nebivolol but not atenolol or metoprolol induced a dissociation of p67phox and Rac1, as well as an inhibition of NADPH oxidase activity assessed in heart membranes from angiotensin II–infused animals, as well as in homogenates of Nox1 and cytosolic subunit–transfected and phorbol ester–stimulated HEK293 cells. These findings indicate that nebivolol interferes with the assembly of NADPH oxidase. Thus, inhibitory effects of this &bgr;-blocker on vascular NADPH oxidase may explain, at least in part, its beneficial effect on endothelial function in angiotensin II–induced hypertension.

Therapeutic effect of enhancing endothelial nitric oxide synthase (eNOS) expression and preventing eNOS uncoupling

Nitric oxide (NO) produced by the endothelium is an important protective molecule in the vasculature. It is generated by the enzyme endothelial NO synthase (eNOS). Similar to all NOS isoforms, functional eNOS transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH), via the flavins flavin adenine dinucleotide and flavin mononucleotide in the carboxy‐terminal reductase domain, to the heme in the amino‐terminal oxygenase domain. Here, the substrate L‐arginine is oxidized to L‐citrulline and NO. Cardiovascular risk factors such as diabetes mellitus, hypertension, hypercholesterolaemia or cigarette smoking reduce bioactive NO. These risk factors lead to an enhanced production of reactive oxygen species (ROS) in the vessel wall. NADPH oxidases represent major sources of this ROS and have been found upregulated in the presence of cardiovascular risk factors. NADPH‐oxidase‐derived superoxide avidly reacts with eNOS‐derived NO to form peroxynitrite (ONOO‐). The essential NOS cofactor (6R‐)5,6,7,8‐tetrahydrobiopterin (BH4) is highly sensitive to oxidation by this ONOO‐. In BH4 deficiency, oxygen reduction uncouples from NO synthesis, thereby converting NOS to a superoxide‐producing enzyme. Among conventional drugs, compounds interfering with the renin‐angiotensin‐aldosterone system and statins can reduce vascular oxidative stress and increase bioactive NO. In recent years, we have identified a number of small molecules that have the potential to prevent eNOS uncoupling and, at the same time, enhance eNOS expression. These include the protein kinase C inhibitor midostaurin, the pentacyclic triterpenoids ursolic acid and betulinic acid, the eNOS enhancing compounds AVE9488 and AVE3085, and the polyphenolic phytoalexin trans‐resveratrol. Such compounds enhance NO production from eNOS also under pathophysiological conditions and may thus have therapeutic potential.