Navin Kumar Thakur

Dehydroepiandrosterone retards atherosclerosis formation through its conversion to estrogen: the possible role of nitric oxide.

Dehydroepiandrosterone (DHEA) is speculated to have an antiatherosclerotic effect, although the mechanism of action remains unclear. The objective of the current study was to determine whether the antiatherosclerotic effect of DHEA is related to its conversion to estrogen and to define the role of nitric oxide (NO) in the antiatherosclerotic effect of DHEA. Forty-eight oophorectomized rabbits were divided into 5 groups and fed the following diets for 10 weeks: group 1, a regular rabbit diet plus 1% cholesterol (a high-cholesterol diet [HCD]); group 2, an HCD plus 0.3% DHEA; group 3, an HCD plus 0.3% DHEA and fadrozole (2.0 mg x kg(-1) x d(-1)), a specific aromatase inhibitor; group 4, an HCD plus 17beta-estradiol (20 microg x kg(-1) x d(-1)); and group 5, a regular diet. Atherosclerotic lesions, lipid deposition in aortic vessels, and basal and stimulated NO release were measured in the aforementioned groups of rabbits. NO release was measured by using an NO-selective electrode as well as by measuring vascular responses and the plasma NO metabolites nitrite and nitrate. The plasma total cholesterol level was increased, but there were no significant differences in lipid profile in the 4 groups of rabbits that were fed the HCD. The area occupied by atherosclerosis in the thoracic aorta was diminished by approximately 60% in the DHEA-treated rabbits (group 2) compared with the HCD group of rabbits (group 1); there was a corresponding 80% decrease in the estradiol group (group 4) but only a 30% decrease in the DHEA plus fadrozole group (group 3). In the aortas of rabbits from groups 1 and 3, the acetylcholine-induced and tone-related basal NO-mediated relaxations were diminished compared with those of the controls (group 5). However, these relaxations were restored in the aortas of group 2 and 4 rabbits, and an increase in NO release was observed in groups 2 and 4 compared with groups 1 and 3, as measured by an NO-selective electrode. Injection of neither solvent (20% ethanol/distilled water) nor fadrozole significantly affected the atherosclerotic area or the NO-related responses described above. We conclude that approximately 50% of the total antiatherosclerotic effect of DHEA was achieved through the conversion of DHEA to estrogen. NO may also play a role in the antiatherosclerotic effect of DHEA and 17beta-estradiol.

Physiological Concentration of 17β-Estradiol Retards the Progression of Severe Atherosclerosis Induced by a High-Cholesterol Diet Plus Balloon Catheter Injury Role of NO

The molecular mechanisms of the antiatherosclerotic effects of estrogen are not yet known. We evaluated the effects of 17beta-estradiol (E(2)) on high cholesterol diet- (HCD; standard diet and 1% cholesterol) and balloon injury-induced atherosclerosis in female New Zealand White rabbits. The abdominal aortas of 40 oophorectomized (Groups 1 through 5) and 8 nonoophorectomized (Group 6) rabbits were injured by balloon catheter, and the animals were then divided into the following groups and treated for 10 weeks: Group 1, standard diet; Group 2, standard diet plus a moderate dose of E(2) (100 microg x kg(-1) x d(-1)); Group 3, HCD; Group 4, HCD plus a moderate dose of E(2); Group 5, HCD plus a low dose of E(2) (20 microg x kg(-1) x d(-1)); and Group 6, HCD in nonoophorectomized rabbits. After the treatment phase, plasma E(2) was increased up to 282.2+/-45.5 pg/mL in Group 2, 263.0+/-41.5 pg/mL in Group 4, 87. 9+/-18.8 pg/mL in Group 5, and 45.6+/-7.3 pg/mL in Group 6. HCD-mediated increases in plasma lipid levels were not changed by E(2) treatment, whereas E(2) decreased the aortic intimal thickening in Group 2 animals compared with those in Group 1 and reduced atherosclerosis in the thoracic and abdominal aortas of Group 4, 5, and 6 rabbits compared with those in Group 3. E(2) restored the impaired abdominal aortic endothelium-dependent relaxation of balloon-injured and HCD-supplemented rabbits, and E(2) increased basal nitric oxide (NO) release. The basal NO-releasing effect showed a significant, inverse relation with the severity of atherosclerosis. Plasma E(2) concentration also showed a significant, inverse relation with atherosclerotic area. In conclusion, physiological concentrations of E(2) can retard the progression of severe atherosclerosis and stabilize atheromas induced by HCD and balloon injury. The retardation may be partially mediated by endothelial NO function in vessels treated with E(2).

Endothelium-dependent relaxation of rabbit atherosclerotic aorta was not restored by control of hyperlipidemia: the possible role of peroxynitrite (ONOO−)

We determined the role of ONOO(-) in nitric oxide (NO) mediated vascular response in atherosclerosis and regression following removal of dietary cholesterol. The effect of ONOO(-) on NO-mediated vascular responses was examined in vitro. Basal and stimulated NO release was estimated by an NO-selective electrode as well as vascular response and the plasma NO metabolites. An immunohistochemical study was also carried out. Responses were compared in normal controls, atherosclerotic rabbits fed 1% cholesterol diet for 6 or 9 weeks (atherosclerotic group) and animals fed a normal diet for 6-36 weeks after the high cholesterol diet for 6 or 9 weeks (regression group). ONOO(-) impaired the basal and acetylcholine-stimulated NO release, but did not affect endothelium-independent relaxation. After 15 weeks on a normal diet, the acetylcholine-stimulated and basal NO-mediated relaxation, which was diminished in the aorta induced by 6 weeks high cholesterol diet, became restored. However, the vascular response in the 9 weeks high cholesterol diet group did not return to normal after 36 weeks on a normal diet. iNOS was observed in atherosclerotic plaques in atherosclerotic and regression groups along with ONOO(-) in the 9 weeks high cholesterol diet group, but not in the 6 weeks group. Conclusively, ONOO(-) can play a role in impairment of NO-mediated vascular response during the regression of dietary cholesterol-induced atherosclerosis, not in the initiation of atherosclerosis.

Inhibition of inducible nitric oxide synthase gene expression by indomethacin or ibuprofen in β-amyloid protein-stimulated J774 cells

Recent studies show that a mononuclear phagocyte lineage, including microglia, plays a possible role in the pathogenesis of Alzheimers disease through nitric oxide (NO)-mediated neurotoxicity. Epidemiological studies show that nonsteroidal anti-inflammatory drugs (NSAIDs) have a protective effect against Alzheimers disease. Based on these observations, it has been hypothesized that an anti-Alzheimers disease effect of NSAIDs could result from the inhibition of NO synthesis. We report here that indomethacin or ibuprofen dose-dependently reduce beta-amyloid protein and interferon-gamma-induced NO production, accompanied by an inhibition of inducible nitric oxide synthase mRNA expression in J774 cells, a murine macrophage cell line. Aspirin, however, does not produce such an effect, suggesting that the cyclooxygenases pathway is not involved in the inhibitory effects of NSAIDs on beta-amyloid protein and interferon-gamma-induced NO production in J774 cells.

Anti-atherosclerotic Effect of β-blocker with Nitric Oxide–releasing Action on the Severe Atherosclerosis

It is not completely understood whether nitric oxide donors and &bgr;-adrenoceptor antagonists have anti-atherosclerotic effects. The anti-atherosclerotic effects of &bgr;-adrenergic receptor antagonists and nitric oxide donors on severe atherosclerosis induced by cholesterol and inhibition of nitric oxide synthesis were determined. Six groups of New Zealand white male rabbits were treated for 10 weeks, under the following regimens: group I: high-cholesterol diet (HCD) (standard diet plus 0.5% cholesterol); group II: HCD plus N G -nitro- l -arginine methyl ester ( l -NAME), an inhibitor of nitric oxide synthase; group III: HCD plus l -NAME and isosorbide dinitrate; group IV: HCD plus l -NAME and nitroglycerin; group V: HCD plus l -NAME and nipradilol (&bgr;-blocker with nitric oxide–releasing action); and group VI: HCD plus l -NAME and atenolol (&bgr;-blocker). Serum lipid levels did not differ among the six groups. Blood pressure and heart rates were slightly decreased in groups V and VI. The atherosclerotic area and aortic cholesterol increased in l -NAME-treated animals but not in animals in group V. The endothelium-dependent relaxations and basal nitric oxide release were impaired in the l -NAME treatment group, though not in group V, in comparison with those in group I. cGMP in the aorta was increased in groups III, IV, and V as compared with that in group II. Endothelial nitric oxide synthase mRNA was decreased in the aortae of l -NAME-treated rabbits and increased in aortae in group V, in comparison with that in group I. Conclusively, nipradilol, &bgr;-blocker with nitric oxide–releasing action, in contrast to the other &bgr;-blockers and nitric oxide donors, showed a successful anti-atherosclerotic effect through the restoration of nitric oxide bioavailability and possible interaction with oxygen radicals.

Estrogen and Nitric Oxide-Possible Mechanism of Anti-atherosclerotic Effect of Estrogen via Isoforms of Nitric Oxide Synthase

Although the atheroprotective effect of estrogen is well known, the mechanism is not completely understood. We investigated the role of nitric oxide in the atheroprotective effects of estrogen. The basal and stimulated NO response of arteries from rabbits were studied using Nω-monomethyl-L-arginine (L-NMMA), acetylcholine (ACh), and other compounds. The aorta of female rabbits released a greater amount of basal NO than did that of oophorectomized females and male rabbits. The greater basal release of NO in female rabbits was decreased in atherosclerotic vessels in animals fed a high cholesterol diet. We investigated the effect of estrogen on endothelial, neuronal and inducible nitric oxide synthase, (eNOS, nNOS, and iNOS) respectively. Preincubation with a physiological concentration of 17β-estradiol (10−12 to 10−8 M) over 8 hours significantly enhanced activity and protein concentration of eNOS in endothelial cells of cultured human umblical vein (HUVEC) and of bovine aortas (BAEC). 17β-estradiol also enhanced the release of NO from endothelial cells as measured by NO2−/NO3−, metabolites of NO. These effects were identified via a estrogen receptor-mediated system. 17β-estradiol (10−10 to 10−8 M) also enhanced the activity of partially purified nNOS in the cytosolic fraction of rabbit cerebellums by DEAE column-chromatography. Estrogen enhanced the fluorescence of dansyl-calmodulin. This may explain one mechanism of short term action of estrogen. The effect of estrogen on iNOS was contrast to those on eNOS or nNOS. When J774 cells, a murine macrophage cell line, were incubated with interferon-γ and lipopolysaccharide, iNOS was induced and a large amount of NO was released. Pre- or co-incubation of 17β-estradiol inhibited the induction of iNOS protein and NO release by a receptor mediated system. These results may offer one mechanism for the anti-atherosclerotic effect of 17β-estradiol.