Free Radical Generators Cause Changes in Endothelial and Inducible Nitric Oxide Synthases and Endothelin-1 Immunoreactivity in Endothelial Cells from Hyperlipidemic Rabbits

doi:10.1006/mgme.1997.2664

Molecular Genetics and Metabolism

Volume 63, Issue 3, March 1998, Pages 191-197

https://doi.org/10.1006/mgme.1997.2664 Get rights and content

Abstract

Reactive oxygen species (ROS) play an important role in the damage of vascular endothelium during atherogenesis and impaired endothelium-dependent vasorelaxation. We have studied the effect of two ROS generators (H₂O₂and menadione) and one of the most potent antioxidants (morin) on the double immunofluorescent staining of endothelial cells (EC) from both Watanabe Heritable Hyperlipidemic (WHHL) and New Zealand White (NZW) rabbits in primary cultures using antibodies against endothelin-1 (ET-1), endothelial (eNOS), and inducible nitric oxide synthase (iNOS). In aortic EC from normal rabbits, ROS decreased the immunoreactivity of eNOS and ET-1 and this effect was significantly reversed by morin. In atherosclerotic rabbits, ROS had the same effect on the immunoreactivity of eNOS and ET-1 but also induced the expression of iNOS immunoreactivity. In general, the cells from WHHL rabbits were less sensitive to the protective effects of morin and more sensitive to the effects of ROS. It thus appears that the protective effect of morin may be due to neutralization of ROS and may be considered for the treatment of early stages of atherosclerosis, before macroscopic lesions have occurred.

References (38)

G Aliev et al.
Changes in vessel ultrastructure during ischemia and reperfusion of rabbit hindlimb: Implications for therapeutical interventions
Microvasc Res
(1993)
JF Seccombe et al.
Oxygen radical-mediated vascular injury selectively inhibits receptor-dependent release of nitric oxide from canine coronary arteries
J Thor Cardiovasc Surg
(1994)
B Tesfamariam
Free radicals in diabetic endothelial cell dysfunction
Free Rad Biol Med
(1994)
TW Wu et al.
Morin: A wood pigment that protects three types of human cells in the cardiovascular system against oxyradical damage
Biochem Pharmacol
(1994)
A Chinellato et al.
Receptor-mediated pathways of endothelium activity in experimental atherosclerosis
Pharmacol Res
(1995)
A Loesch et al.
Colocalization of endothelin, vasopressin and serotonin in cultured endothelial cells of rabbit aorta
Peptides
(1991)
M Yanagisawa et al.
A novel potent vasoconstrictor peptide produced by vascular endothelial cells
Nature
(1988)
RF Furchgott
Studies on relaxation of rabbit aorta by sodium nitrite: the basis for the proposal that the acid-activable inhibitory factor from bovine retractor penis is inorganic nitrite and the endothelium-derived relaxing factor is nitric oxide
Vasodilatation: Vascular Smooth Muscle, Peptides, Autonomic Nerves, and Endothelium
(1988)
RM Palmer et al.
Vascular endothelial cells synthesize nitric oxide from L-arginine
Nature
(1988)
S Moncada et al.
Nitric oxide: Physiology, pathophysiology and pharmacology
Pharmacol Rev
(1991)

RM Rapoport et al.

Agonist-induced endothelium-dependent relaxation in rat thoracic aorta may be mediated through cGMP

Circ Res

(1983)

PS Tsao et al.

Enhanced endothelial adhesiveness in hypercholesterolemia is attenuated by L-arginine

Circulation

(1994)

WM Radomski et al.

Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium

Lancet

(1987)

UC Garg et al.

Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosino monophosphate inhibits mitogenesis and proliferation of cultured rat vascular smooth muscle cells

J Clin Invest

(1989)

R Ross

The pathogenesis of atherosclerosis: A perspective for the 1990s

Nature

(1993)

U Förstermann et al.

Selective attenuation of endothelium-mediated vasodilatation in atherosclerotic human coronary arteries

Circ Res

(1988)

GJ Dusting

Nitric oxide in cardiovascular disorders

J Vasc Res

(1995)

RL Minor et al.

Diet-induced atherosclerosis increases the release of nitrogen oxide from rabbit aorta

J Clin Invest

(1990)

Cited by (38)

A tailed primers protocol to identify the association of eNOS gene variable number of tandem repeats polymorphism with ischemic stroke in Chinese Han population by capillary electrophoresis
2013, Gene
Citation Excerpt :
Because 4a/4a did not appear in some populations, 4a/4a vs 4b/4b analysis was not included in the meta-analysis. It has been well recognized that eNOS may have possible therapeutic potential in the treatment of inflammation related disorders (Aliev et al., 1998; Hen et al., 2007; Isordia-Salas et al., 2010; Jaramillo et al., 2008; Van-Assche et al., 2011). Based upon its biological plausibility for involvement in atherosclerosis, association with inflammatory diseases, and its potential influence on NO production (Antoniades et al., 2007), we chose eNOS as candidate gene.
Endothelial nitric oxide synthase (eNOS) plays an important role in mediating endothelium-dependent vasodilatation and antithrombotic action and is thus involved in the development of ischemic stroke (IS). Controversial results regarding the association of eNOS gene variable number of tandem repeats (VNTR) polymorphism with IS have been reported by conventional PCR-polyacrylamide gel electrophoresis methods. We aimed to identify any common association of eNOS gene VNTR polymorphism with IS in Chinese Han population by capillary electrophoresis (CE). The VNTR polymorphism of 27 bp within the eNOS intron-4 was determined by CE with specially designed tailed primers in Chinese Han patients with IS (n = 457) and matched elderly controls without IS (n = 457). Significant differences in BMI, WHR, hypertension, diabetes, smoking, TG, HDL, LDL, LDL, and FBG were observed between cases and controls. The distributions of eNOS VNTR polymorphism were not significantly associated with IS after adjustment for cardiovascular risk factors (OR = 1.18, 95% CI: 0.82–1.69). This finding was consistent with the further meta-analysis in Asians. The meta-analysis in Americans demonstrated that 4a/4b + 4a/4a genotype was significantly associated with IS risk with an OR of 1.54 (95% CI, 1.09–2.17) compared with the 4b/4b genotype. Our data suggests that BMI, WHR, hypertension, diabetes, smoking, TG, LDL, and FBG may increase the risk of IS. However, eNOS VNTR polymorphism may be not an independent major contributor for IS in Chinese Han population. The VNTR polymorphism might be associated with IS in Americans based on meta-analysis.
Morin inhibits interleukin-1β-induced nitric oxide and prostaglandin E <inf>2</inf> production in human chondrocytes
2012, International Immunopharmacology
Citation Excerpt :
Their data were supported by Harasstani et al., who found the inhibition of NO production by morin in LPS-induced RAW 264.7 cells [20]. Similar results were also demonstrated by Lee and Aliev et al. [30,31]. PGE2 is also an inflammatory factor which is elevated by COX-2.
It is well known that the inflammatory cytokines play important roles in osteoarthritis (OA). In the present study, we investigated the anti-inflammatory properties of morin in chondrocytes. The nitric oxide (NO) production was determined by Griess method, the prostaglandin E2 (PGE₂) production was detected by Enzyme-linked immunosorbent assay (ELISA). The expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were investigated by quantitative real-time PCR and western blot. In addition, western blotting and immunofluorescence staining were performed to investigate the protein level of inhibitor of nuclear factor-κB (IκB-α) and the translocation of nuclear factor kappa B (NF-κB). For the in vivo study, morin was administered by intra-articular injection in rats, and the gene expression of iNOS and COX-2 was assessed. We showed that morin inhibited the production of NO and PGE₂ as well as the expression of iNOS and COX-2 in interleukin-1-beta (IL-1β)-induced chondrocytes. In addition, morin suppressed the degradation of IκB-α as well as the translocation of NF-κB. In vivo study, morin exerted anti-inflammatory properties in an IL-1β-induced rat OA model. Our data suggest that morin possess potential value in the treatment of OA.
Free cholesterol accumulation impairs antioxidant activities and aggravates apoptotic cell death in menadione-induced oxidative injury
2011, Archives of Biochemistry and Biophysics
Although the relationship between hypercholesterolemia and oxidative stress has been extensively investigated, direct evidence regarding to the roles of cholesterol accumulation in the generations of reactive oxygen species (ROS) and apoptotic cell death under oxidative stress is lack. In this study, we investigated productions of superoxide anions ( $O_{2}^{-}$ ) and nitric oxide (NO), and apoptotic cell death in wild type Chinese hamster ovary (CHO) cells and cholesterol accumulated CHO cells genetically and chemically. Oxidative stress was induced by menadione challenge. The results revealed that abundance of free cholesterol (FC) promoted menadione-induced $O_{2}^{-}$ and NO productions. FC accumulation down-regulated eNOS expression but up-regulated NADPH oxidases, and inhibited the activities of superoxide dismutase (SOD) and catalase. Treatment of menadione increased the expressions of iNOS and qp91 phox, enhanced the activities of SOD and catalase in the wild-type CHO cells but inhibited the activity of glutathione peroxidase in the cholesterol accumulated CHO cells. Moreover, FC abundance promoted apoptotic cell death in these cells. Taken together, those results suggest that free cholesterol accumulation aggravates menadione-induced oxidative stress and exacerbates apoptotic cell death.
Characterization of CoCl<inf>2</inf>-induced reactive oxygen species (ROS): Inductions of neurite outgrowth and endothelin-2/vasoactive intestinal contractor in PC12 cells by CoCl<inf>2</inf> are ROS dependent, but those by MnCl<inf>2</inf> are not
2007, Neuroscience Letters
CoCl₂ and MnCl₂ are hypoxic mimetic agents. We previously found that expression of ET-2/VIC, one of hypoxia-related factors, and the induction of neurite outgrowth in PC12 cells through ROS induced by CoCl₂. MnCl₂ also are known to induce neurite outgrowth in PC12 cells. However, it is unclear whether the mechanism of the effect induced by these metals is same. In the present study, we evaluated biological effects induced by MnCl₂ and compared with those induced by CoCl₂. Furthermore, we analyzed sources of CoCl₂-induced ROS generation. MnCl₂ up-regulated ET-2/VIC gene expression and ET-2/VIC peptide production as CoCl₂ did, but not affect ET-1 gene expression, in the neurite outgrowth of PC12 cells. NAC did not at all inhibit the effects induced by MnCl₂. Furthermore, addition of MnCl₂ to the culture medium did not generate ROS as CoCl₂ did. These results indicate that ET-2/VIC expression is a common pathway in neurite outgrowth induced by CoCl₂ and MnCl₂, but the effects induced by CoCl₂ are ROS dependent, whereas the effects induced by MnCl₂ are ROS independent. Taken together, the mechanism for the effects by CoCl₂ was different from that by MnCl₂. The ROS, were not decomposed by catalase or SOD, were rapidly generated by reaction of CoCl₂ mainly with components of HS rather than with FBS or DMEM. Some ROS generated by reaction of CoCl₂ with components of HS may participate in the observed neurite outgrowth of PC12 cells.
Mechanisms underlying dysfunction of carotid arteries in genetically hyperlipidemic rabbits
2006, Nitric Oxide - Biology and Chemistry
In the present study we compared the vascular reactivity and integrity of the nitric oxide (NO)–cyclic 3′,5′-guanosine monophopsphate (cGMP) pathway in carotid arteries of hyper- and normolipidemic rabbits. Vasodilation to acetylcholine, nitroglycerin, and sodium nitroprusside was desensitized in hyperlipidemia, but the nitroprusside-induced relaxation was normalized by an NO synthase inhibitor in endothelium-intact and -denuded vessels. Hyperlipidemic carotid arteries exhibited increased basal NO (detected by EPR spin-trapping) and reactive oxygen species formation (detected by chemiluminescence), whereas acetylcholine-induced NO formation was nearly abolished. Hyperlipidemia increased NADPH-dependent superoxide formation in carotid membranes, and carotid cryosections stained with the fluorescent dye dihydroethidium revealed increased endothelial and medial reactive oxygen species formation. Hyperlipidemia elicited macrophage invasion into the carotid wall, as detected by a dot-immunoblot. The basal activity of cGMP-dependent proteinkinase, the nitroprusside-stimulated activity of soluble guanylyl cyclase, and its protein expression were decreased by hyperlipidemia. The cGMP phosphodiesterase activity was marginally increased by hyperlipidemia, such that the ratio of cGMP-forming vs. -degrading capacity was decreased by 2-fold. Hyperlipidemia triggers infiltration of macrophages into the carotid wall and endothelial as well as smooth muscle superoxide formation. Consequently, relaxation of the carotid arteries are impaired due to smooth muscle and endothelial dysfunction.
Protective effect of ebselen on cytotoxicity induced by cholestane-3 beta, 5 alpha, 6 beta-triol in ECV-304 cells
2006, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
The protective effect of ebselen, with documented glutathione peroxidase-like activity and antioxidative and anti-inflammatory properties, on the cytotoxicity induced by oxysterol was investigated in ECV-304 cells with cholestane-3β, 5α, 6β-triol (3-triol), one of the most toxic oxysterols. 3-triol exhibited significant cytotoxicity to ECV-304 cells in dose- and time-dependent manners. Pre-incubations with ebselen at different concentrations for 4 h effectively inhibited the decreases of the cell viability and the intracellular thiols level induced by 3-triol; suppressed the 3-triol-caused increases of the GPx and NOS activities, the LDH leakage and MDA formation. The inhibition of ebselen to the generation of intracellular ROS induced by 3-triol was monitored by luminol-, lucigenin-derived chemiluminescence and DCFH-DA-derived fluorescence assays. Our results suggest that ebselen inhibited 3-triol-induced enhancement of intracellular ROS level and the cytotoxicity of 3-triol is contributed to, for the most part, an enhanced formation of intracellular O₂^·−; nevertheless, the mitochondria were not the main source of intercellular O₂^·− contributed to the cytotoxicity of 3-triol. Ebselen lost its high protection against 3-triol-induced injuries in the presence of GSH probably due to the formation of the ebselen-GSH adduct. In conclusion, our investigations provide new utility for ebselen as a prospective antiatherosclerotic in both clinical and non-clinical situations.

View all citing articles on Scopus

^☆: Vanhoutte, P

¹: Current address: Department of Cell Biology and Atherosclerosis, The Lerner Research Institute, Cleveland Clinic Foundation, NC10 9500 Euclid Avenue, Cleveland, OH 44195.

²: To whom correspondence should be addressed at Autonomic Neuroscience Institute, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, U.K. Fax: (+44) 171 830 2949. E-mail:[email protected].

View full text

Regular ArticleFree Radical Generators Cause Changes in Endothelial and Inducible Nitric Oxide Synthases and Endothelin-1 Immunoreactivity in Endothelial Cells from Hyperlipidemic Rabbits☆

Abstract

Microvasc Res

J Thor Cardiovasc Surg

Free Rad Biol Med

Biochem Pharmacol

Pharmacol Res

Peptides

A novel potent vasoconstrictor peptide produced by vascular endothelial cells

Nature

Studies on relaxation of rabbit aorta by sodium nitrite: the basis for the proposal that the acid-activable inhibitory factor from bovine retractor penis is inorganic nitrite and the endothelium-derived relaxing factor is nitric oxide

Vasodilatation: Vascular Smooth Muscle, Peptides, Autonomic Nerves, and Endothelium

Vascular endothelial cells synthesize nitric oxide from L-arginine

Nature

Nitric oxide: Physiology, pathophysiology and pharmacology

Pharmacol Rev

Agonist-induced endothelium-dependent relaxation in rat thoracic aorta may be mediated through cGMP

Circ Res

Enhanced endothelial adhesiveness in hypercholesterolemia is attenuated by L-arginine

Circulation

Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium

Lancet

Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosino monophosphate inhibits mitogenesis and proliferation of cultured rat vascular smooth muscle cells

J Clin Invest

The pathogenesis of atherosclerosis: A perspective for the 1990s

Nature

Selective attenuation of endothelium-mediated vasodilatation in atherosclerotic human coronary arteries

Circ Res

Nitric oxide in cardiovascular disorders

J Vasc Res

Diet-induced atherosclerosis increases the release of nitrogen oxide from rabbit aorta

J Clin Invest

Regular Article
Free Radical Generators Cause Changes in Endothelial and Inducible Nitric Oxide Synthases and Endothelin-1 Immunoreactivity in Endothelial Cells from Hyperlipidemic Rabbits☆