Biomedical subjects

Huige Li

Publications and source records attributed to Huige Li.

14 recordsLinked to original sources

PubMed2006

Nebivolol inhibits superoxide formation by NADPH oxidase and endothelial dysfunction in angiotensin II-treated rats.

Nebivolol is a beta(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 beta-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 p67(phox)). In addition, NOS III uncoupling was prevented. In vitro treatment with nebivolol but not atenolol or metoprolol induced a dissociation of p67(phox) 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 beta-blocker on vascular NADPH oxidase may explain, at least in part, its beneficial effect on endothelial function in angiotensin II-induced hypertension.

Acridines↗

PubMed2006

Reversal of endothelial nitric oxide synthase uncoupling and up-regulation of endothelial nitric oxide synthase expression lowers blood pressure in hypertensive rats.

OBJECTIVES: We sought to examine the hypothesis that a pharmacologic up-regulation of endothelial nitric oxide synthase (eNOS) combined with a reversal of eNOS uncoupling provides a protective effect against cardiovascular disease. BACKGROUND: Many cardiovascular diseases are associated with oxidant stress involving protein kinase C (PKC) and uncoupling of eNOS. METHODS: Messenger ribonucleic acid (mRNA) expression was analyzed with RNase protection assay or quantitative real-time polymerase chain reaction, vascular nitric oxide (NO) with spin trapping, and reactive oxygen species (ROS) with dihydroethidium fluorescence. RESULTS: Aortas of spontaneously hypertensive rats (SHR) showed an elevated production of ROS when compared with aortas of Wistar-Kyoto rats (WKY). The aortic expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits (Nox1, Nox2, Nox4, and p22phox) was higher in SHR compared with WKY. In SHR, aortic production of ROS was reduced by the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), indicating eNOS "uncoupling" in hypertension. Oral treatment with the PKC inhibitor midostaurin reduced aortic Nox1 expression, diminished ROS production, and reversed eNOS uncoupling in SHR. Aortic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) were significantly reduced in SHR compared with WKY. Midostaurin normalized BH4 levels in SHR. In both WKY and SHR, midostaurin increased aortic expression of eNOS mRNA and protein, stimulated bioactive NO production, and enhanced relaxation of the aorta to acetylcholine. Midostaurin lowered blood pressure in SHR and, to a lesser extent, in WKY; the compound did not change blood pressure in WKY made hypertensive with L-NAME. CONCLUSIONS: Pharmacologic interventions that combine eNOS up-regulation and reversal of eNOS uncoupling can markedly increase bioactive NO in the vasculature and produce beneficial hemodynamic effects such as a reduction of blood pressure.

Animals↗

PubMed2005

A blend of polyphenolic compounds explains the stimulatory effect of red wine on human endothelial NO synthase.

A high intake of polyphenolic compounds is likely to have beneficial effects on the cardiovascular system. Especially red wine is a rich source of polyphenols, and we have previously shown that French red wine upregulates eNOS, a protective enzyme in the cardiovascular system. The current study tested (poly)phenolic constituents of red wine for their ability to enhance eNOS expression (and the activity of a 3.5-kb human eNOS promoter) in human EA.hy 926 endothelial cells. Of the compounds tested, we found 3,4',5-trihydroxy-trans-stilbene (trans-resveratrol) to be the most efficacious stimulator of eNOS expression (and eNOS transcription), but this compound alone could not explain the total stimulatory effect of red wine. The flavanols catechin and epicatechin, the flavonols fisetin, myricetin, isoquercitrin and hyperoside, the anthocyanins delphinidin, malvidin, and paeonidin, gallic acid, and the hydroxycinnamic acids ferulic acid and sinapinic acid did not change eNOS expression or eNOS promoter activity in any substantial way. The flavonol quercetin inhibited eNOS expression (with no effect on eNOS promoter activity). Cinnamic acid was a rather potent enhancer of eNOS expression, however with an efficacy of only 170%. Surprisingly, it reduced eNOS promoter activity. The anthocyanins cyanidin, the hydroxycinnamic acids p-coumaric acid and caffeic acid, and the phenolic acids benzoic acid and vanillic acid also enhanced eNOS expression moderately (with no effect on eNOS promoter activity). Thus, the increase in eNOS in response to red wine involves several polyphenolic compounds with a major contribution from trans-resveratrol and lesser contributions from cinnamic and hydroxycinnamic acids, cyanidin, and some phenolic acids.

Anthocyanins↗

PubMed2005

Midostaurin upregulates eNOS gene expression and preserves eNOS function in the microcirculation of the mouse.

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is a powerful vasodilator and possesses vasoprotective effects. Therefore, augmentation of eNOS expression and -activity by pharmacological means could provide protection against cardiovascular disease. However, this concept has been questioned recently, because in several disease models, eNOS upregulation was associated with a dysfunctional enzyme (referred to as eNOS uncoupling). In contrast, the present study demonstrates that an eNOS gene expression-enhancing compound with additional protein kinase C (PKC) inhibitory properties can upregulate eNOS while preserving its enzymatic function. Apolipoprotein E-knockout mice were treated for 7 days with midostaurin (4'-N-benzoyl staurosporine, compound CGP 41251, 50-125 mg/kg/day), a PKC inhibitor previously shown to increase eNOS expression and NO production in cultured human endothelial cells. Midostaurin treatment enhanced eNOS mRNA expression (RNase protection assay) in mouse aorta, kidney, and heart in a dose-dependent fashion. In the dorsal skinfold microcirculation, midostaurin produced an arteriolar vasorelaxation (intravital microscopy), which could be prevented by the NOS inhibitor L-NAME, indicating that the upregulated eNOS remained functional. In organ chamber experiments, the aorta from midostaurin-treated mice showed an enhanced NO-mediated relaxation in response to acetylcholine. Accordingly, serum levels of nitrite/nitrate (NO-Analyzer) were increased, and the production of reactive oxygen species in the aorta (L-012 chemiluminescence) was reduced by midostaurin. Thus, in mice in vivo, midostaurin treatment results in enhanced expression of eNOS with preserved enzyme function and enhanced production of bioactive NO. Given the beneficial effects of endothelial-derived NO, vasoprotective and anti-atherosclerotic effects are likely to ensue.

Animals↗

PubMed2004

Flavonoids from artichoke (Cynara scolymus L.) up-regulate endothelial-type nitric-oxide synthase gene expression in human endothelial cells.

Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) represents an antithrombotic and anti-atherosclerotic principle in the vasculature. Hence, an enhanced expression of eNOS in response to pharmacological interventions could provide protection against cardiovascular diseases. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVECs), an artichoke leaf extract (ALE) increased the activity of the human eNOS promoter (determined by luciferase reporter gene assay). An organic subfraction from ALE was more potent in this respect than the crude extract, whereas an aqueous subfraction of ALE was without effect. ALE and the organic subfraction thereof also increased eNOS mRNA expression (measured by an RNase protection assay) and eNOS protein expression (determined by Western blot) both in EA.hy 926 cells and in native HUVECs. NO production (measured by NO-ozone chemiluminescence) was increased by both extracts. In organ chamber experiments, ex vivo incubation (18 h) of rat aortic rings with the organic subfraction of ALE enhanced the NO-mediated vasodilator response to acetylcholine, indicating that the up-regulated eNOS remained functional. Caffeoylquinic acids and flavonoids are two major groups of constituents of ALE. Interestingly, the flavonoids luteolin and cynaroside increased eNOS promoter activity and eNOS mRNA expression, whereas the caffeoylquinic acids cynarin and chlorogenic acid were without effect. Thus, in addition to the lipid-lowering and antioxidant properties of artichoke, an increase in eNOS gene transcription may also contribute to its beneficial cardiovascular profile. Artichoke flavonoids are likely to represent the active ingredients mediating eNOS up-regulation.

Animals↗

PubMed2004

Dexamethasone lacks effect on blood pressure in mice with a disrupted endothelial NO synthase gene.

Cushing's syndrome and systemic administration of glucocorticoids are associated with hypertension, but the underlying molecular mechanism is only partially understood. We have shown previously that dexamethasone downregulates the expression of the endothelial NO synthase (eNOS) gene in human endothelial cells and in the rat and that this may contribute to the blood pressure-raising effect of the steroid [Proc. Natl. Acad. Sci. USA 96 (1999) 13357]. In the current communication, we demonstrated that dexamethasone increased mean arterial blood pressure in wild-type C-57 Bl6 mice (eNOS+/+ mice), but had no effect on blood pressure in mice with a disrupted eNOS gene (eNOS-/- mice) derived from the same strain. The NOS inhibitor ethylisothiourea, used for control purposes, showed a hypertensive effect in eNOS+/+ mice, but no such effect in eNOS-/- mice. Serum NO2-/NO3- levels, an indicator of total body NO synthesis, decreased significantly when eNOS+/+ mice were treated with dexamethasone. eNOS-/- mice had lower serum NO2-/NO3- levels per se, which were not changed significantly by dexamethasone. Dexamethasone decreased the expression of eNOS in three major organs of the mouse investigated, namely the heart, the liver, and the kidney. We conclude that the expressional downregulation of eNOS and the ensuing reduction in vascular NO production contributes to the hypertension caused by glucocorticoids.

Animals↗

PubMed2003

Histamine upregulates gene expression of endothelial nitric oxide synthase in human vascular endothelial cells.

BACKGROUND: Histamine has a short-term, transient, stimulating effect on endothelial nitric oxide synthase (eNOS) activity; however, long-term effects on eNOS have not been described yet. In addition, the vascular effect of histamine seems to depend critically on eNOS functionality. Therefore, we studied the effects of histamine on eNOS gene expression and function. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA.hy 926 cells, histamine upregulated eNOS mRNA (RNase protection assay) and protein (electron microscopic immunocytochemistry) expression. The upregulation of eNOS could be prevented by mepyramine, a selective antagonist at the H1 receptor, but not by H2 and H3 receptor antagonists. Incubation of EA.hy 926 cells with histamine led to the activation of calcium/calmodulin-dependent protein kinase II (CaMK II; in vitro phosphorylation assay). The histamine-induced eNOS expression was completely prevented by KN-93, an inhibitor of CaMK II. Histamine increased the activity of a 1.6-kb human eNOS promoter fragment (luciferase reporter gene assay), an effect that was also blocked by mepyramine. Under normal conditions, eNOS upregulation by histamine resulted in increased nitric oxide production (measured by nitric oxide chemiluminescence and RFL-6 reporter cell assay). Under conditions of oxidative stress, however, the eNOS upregulated by histamine produced reactive oxygen species (CM-H2DCFDA oxidation-based fluorescence assay). CONCLUSIONS: Stimulation of the H1 receptor increases eNOS transcription in endothelial cells by a signaling pathway involving CaMK II. This eNOS upregulation may be protective under normal conditions, but it may become harmful under conditions of oxidative stress when eNOS produces reactive oxygen species at the expense of nitric oxide.

Calcium-Calmodulin-Dependent Protein Kinase Type 2↗

PubMed2003

Red wine increases the expression of human endothelial nitric oxide synthase: a mechanism that may contribute to its beneficial cardiovascular effects.

OBJECTIVES: The study tested the effect of red wine on endothelial-type nitric oxide synthase (eNOS) expression and eNOS activity in human endothelial cells. BACKGROUND: Endothelial-type nitric oxide (NO) synthase exerts vasoprotective effects. Moderate alcohol consumption has been associated with a reduction of cardiovascular disease, and red wine seems to offer more benefits than any other type of drink. However, the molecular basis of this protective effect is unclear. METHODS: Human endothelial cells were treated with red wine, and eNOS messenger ribonucleic acid (mRNA) expression was measured by RNase protection assay, eNOS protein expression by Western blotting, and eNOS activity by RFL-6 reporter cell assay. The eNOS promoter activity was analyzed in transfected endothelial cells; binding activities of relevant transcription factors were determined by electrophoretic mobility shift assay. RESULTS: Incubation of endothelial cells with red wines from France upregulated eNOS mRNA and protein expression. In contrast, red wines from Germany showed little or no effect on eNOS expression. No significant difference in eNOS mRNA expression could be detected between "en barrique" (matured in oak barrels) and "non-barrique" (matured in steel tanks)-produced French red wines. Endothelial cells treated with French red wines produced up to three times more bioactive NO than did control cells. French red wines increased the activity of the eNOS promoter, with the essential trans-stimulated sequence being located in the proximal 326 bp of the promoter sequence. The eNOS mRNA stability was also increased by red wine. CONCLUSIONS: The increase in eNOS expression and activity brought about by red wines from France (and probably other locations) may contribute to the beneficial effects of this beverage on the cardiovascular system.

Cardiovascular Diseases↗

PubMed2002

Inhibitors of histone deacetylation downregulate the expression of endothelial nitric oxide synthase and compromise endothelial cell function in vasorelaxation and angiogenesis.

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 micromol/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.

Animals↗

PubMed2002

Dual effect of ceramide on human endothelial cells: induction of oxidative stress and transcriptional upregulation of endothelial nitric oxide synthase.

BACKGROUND: Generation of the second-messenger molecule ceramide by stimulated sphingomyelinase activity has been implicated in the inflammatory processes contributing to the pathogenesis of atherosclerosis. However, reports of stimulatory effects of ceramide on endothelial NO production in animal models suggest antiatherosclerotic effects of the molecule. Therefore, we investigated long-term effects of ceramide on NO generation in human endothelial cells. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs) and in HUVEC-derived EA.hy 926 endothelial cells, C6-ceramide (N-hexanoyl-D-erythro-sphingosine) reduced the generation of bioactive NO (RFL-6 reporter-cell assay). At the same time, the signaling molecule increased endothelial NO synthase (eNOS) mRNA (RNase protection assay) and protein expression (Western blot). C6-ceramide stimulated eNOS transcription by a signaling mechanism involving protein phosphatase PP2A but did not modify the stability of the eNOS mRNA. Endothelial generation of reactive oxygen species (ROS) was increased by C6-ceramide [5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA) oxidation-based fluorescence assay], and this effect was partially reversed by the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). On the other hand, (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)) normalized in part the ceramide-induced reduction in bioactive NO. CONCLUSIONS: Ceramide produces oxidative stress in human endothelial cells, thereby reducing bioactive NO. The partial reversal of this reduction by BH(4) and the diminution of ROS generation by L-NAME suggest that ceramide promotes NADPH oxidase activity of eNOS, leading to ROS formation at the expense of NO synthesis. The ceramide-induced upregulation of eNOS gene transcription can be considered an ineffective compensatory mechanism. The decreased bioavailability of NO is likely to favor a proatherogenic role of ceramide.

Cell Line↗

PubMed2002

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.

Cells, Cultured↗

PubMed2002

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 gp91(phox) 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 p22(phox), nox1, nox4, and gp91(phox) 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), gp91(phox) (3-fold), p22(phox) (3-fold), NOS III mRNA, and protein. NOS-inhibition with N(G)-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.

Angiotensin II↗

PubMed2002

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.

Animals↗

PubMed2002

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.

Animals↗