Hisako Matsui-Hirai

Endothelial cellular senescence is inhibited by nitric oxide: Implications in atherosclerosis associated with menopause and diabetes

Senescence may contribute to the pathogenesis of atherosclerosis. Although the bioavailability of nitric oxide (NO) is limited in senescence, the effect of NO on senescence and its relationship to cardiovascular risk factors have not been investigated fully. We studied these factors by investigating senescence-associated β-galactosidase (SA-β-gal) and human telomerase activity in human umbilical venous endothelial cells (HUVECs). Treatment with NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-aminoethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) and transfection with endothelial NO synthase (eNOS) into HUVECs each decreased the number of SA-β-gal positive cells and increased telomerase activity. The NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) abolished the effect of eNOS transfection. The physiological concentration of 17β-estradiol activated hTERT, decreased SA-β-gal-positive cells, and caused cell proliferation. However, ICI 182780, an estrogen receptor-specific antagonist, and l-NAME each inhibited these effects. Finally, we investigated the effect of NO bioavailability on high glucose-promoted cellular senescence of HUVECs. Inhibition by eNOS transfection of this cellular senescence under high glucose conditions was less pronounced. Treatment with l-arginine or l-citrulline of eNOS-transfected cells partially inhibited, and combination of l-arginine and l-citrulline with antioxidants strongly prevented, high glucose-induced cellular senescence. These data demonstrate that NO can prevent endothelial senescence, thereby contributing to the anti-senile action of estrogen. The ingestion of NO-boosting substances, including l-arginine, l-citrulline, and antioxidants, can delay endothelial senescence under high glucose. We suggest that the delay in endothelial senescence through NO and/or eNOS activation may have clinical utility in the treatment of atherosclerosis in the elderly.

Nitric oxide and endothelial cellular senescence

Cellular senescence is characterized by permanent exit from the cell cycle and the appearance of distinct morphological and functional changes associated with an impairment of cellular homeostasis. Many studies support the occurrence of vascular endothelial cell senescence in vivo, and the senescent phenotype of endothelial cells can be transformed from anti-atherosclerotic to pro-atherosclerotic. Thus, endothelial cell senescence promotes endothelial dysfunction and may contribute to the pathogenesis of age-associated vascular disorders. Emerging evidence suggests that increasing nitric oxide (NO) bioavailability or endothelial NO synthase (eNOS) activity activates telomerase and delays endothelial cell senescence. In this review, we discuss the potential mechanisms underlying the ability of NO to prevent endothelial cell senescence and describe the possible changes in the NO-mediated anti-senescence effect under pathophysiological conditions, including oxidative stress and hyperglycemia. Further understanding of the mechanisms underlying the anti-senescence effect of NO in endothelial cells will provide insights into the potential of eNOS-based anti-senescence therapy for age-associated vascular disorders.

NADPH oxidase inhibitor, apocynin, restores the impaired endothelial-dependent and -independent responses and scavenges superoxide anion in rats with type 2 diabetes complicated by NO dysfunction.

Objective:  We investigated the effect of apocynin, an NADPH oxidase inhibitor, in the impairment of vascular responses in Otsuka Long‐Evans Tokushima Fatty (OLETF) rats (type 2 diabetic rat model) with or without (w/wo) N‐nitro‐l‐arginine methyl ester treatment.

Sarpogrelate HCl, a selective 5-HT2A antagonist, retards the progression of atherosclerosis through a novel mechanism

Although sarpogrelate HCl is widely used for the prevention of arterial thrombosis, its effect on atherosclerosis is unknown. Accordingly, we here investigated the effects of sarpogrelate HCl on a rabbit model of atherosclerosis. Male rabbits were fed a 0.5% cholesterol diet (HCD) (Gp 1), HCD with vitamin E (Gp 2), HCD with vitamin E and sarpogrelate (Gp 3), or HCD with sarpogrelate alone (Gp 4) for 8 weeks. The atherosclerotic area was decreased by feeding of vitamin E and sarpogrelate (16.9+/-2.0% in Gp 1 vs. 8.2+/-2.0% in Gp 3). Tone-related basal NO release was higher in Gps 3 and 4. Acetylcholine-induced relaxation tended to be improved in Gp 3. The amount of eNOS mRNA was increased in Gp 4, and aortic cyclic GMP concentration showed the same tendency. O(2)(-) release tended to be decreased in Gps 2 and 3. The matrix metalloproteinase-1 (MMP-1)-positive area was decreased, and the percentage ratio of cell numbers of smooth muscle cells/macrophages in the plaque was increased in Gp 3. The results demonstrated that sarpogrelate HCl retards the progression of atherosclerosis in rabbits, and that this effect is enhanced by concomitant administration of vitamin E. Although upregulation of eNOS may play a role as one of the underlying mechanisms, our results suggest that an additional mechanism-possibly involving the antiproliferative effects of sarpogrelate HCl on smooth muscle cells and macrophages-may also play an important role.

Dose-Dependent Modulatory Effects of Insulin on Glucose-induced Endothelial Senescence in vitro and in vivo: A Relationship between Telomeres and Nitric Oxide

The elderly are prone to postprandial hyperglycemia that increases their cardiovascular risk. Although insulin therapy is necessary to treat diabetes, high plasma concentrations of insulin may cause the development of atherosclerosis and accelerate endothelial senescence. We assumed that high glucose causes stress-induced premature senescence and replicative senescence and examined the regulatory role of insulin in endothelial senescence and functions under different glucose conditions. Exposure of human endothelial cells to high glucose (22 mM) for 3 days increased senescence-associated-β-galactosidase activity, a senescence marker, and decreased telomerase activity, a replicative senescence marker. Physiological concentrations of insulin preserved telomere length and delayed endothelial senescence under high-glucose conditions. The effect of insulin under high-glucose conditions was associated with reduced reactive oxygen species and increased nitric oxide (NO). Small interfering RNA targeting endothelial NO synthase reduced the antisenescence effects of insulin. Physiological concentrations of insulin also reversed high glucose-induced increases in p53 and vascular cell adhesion molecule-1 and decreases in senescence marker protein-30. On the other hand, when insulin was given at any concentrations under normal glucose or at high concentrations under high glucose, its ability to promote cellular senescence was unrelated to endothelial NO. Finally, streptozotocin-induced diabetes showed more senescent cells in the aortic endothelium of aged rats compared with age-matched control and insulin-treated animals. Conclusively, the regulatory effects of insulin on endothelial senescence were modulated by the glucose environment. These data may help explain insulins complicated roles in atherosclerosis in the elderly.

17β-Estradiol inhibits NADPH oxidase activity through the regulation of p47phox mRNA and protein expression in THP-1 cells

In this report, we demonstrate that NADPH oxidase is activated by tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) in human monocytic cells (THP-1 cells) differentiated with phorbol ester (PMA) and that physiological concentration of 17beta-estradiol inhibits NADPH oxidase activity in THP-1 cells stimulated with TNF-alpha plus IFN-gamma. This effect is mediated by estrogen receptor based on estrogen receptor antagonist (ICI 182, 780) that diminishes inhibition by 17beta-estradiol. This inhibition is specific in 17beta-estradiol because 17alpha-estradiol, testosterone and progesterone do not inhibit NADPH oxidase activity. Activation of NADPH oxidase induced by TNF-alpha plus IFN-gamma is caused by up-regulation of p47(phox) (cytosolic component of NADPH oxidase) expression. 17beta-Estradiol prevents the up-regulation of p47(phox) mRNA and protein expression. This prevention of p47(phox) expression depends on the inhibition of NF-kappaB activation. Our results implicate that 17beta-estradiol has an anti-atherosclerotic effects through the improvement of nitric oxide (NO) bioavailability caused by the regulation of superoxide (O(2)(-)) production.

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.

Clinical factors such as B-type natriuretic peptide link to factor VII, endothelial NO synthase and estrogen receptor α polymorphism in elderly women

AIMS This study evaluated the presence of genetic mutations in relation to thrombosis or atherosclerosis in elderly women. MAIN METHODS This is an observational study of 93 Japanese women with a mean age of 80.9 years recruited from outpatient clinics of Nagoya University and its related hospitals. Ten single nucleotide polymorphisms (SNPs) were studied. Each gene studied acts in or is related to either blood coagulation (factor V Leiden, prothrombin G20210A, factor XIII Val34Leu, factor VII Arg353Gln, MTHFR C677T, beta-fibrinogen G-455A, PAI-1 4G/5G), metabolic syndrome-related pathways (PPARalpha Leu162Val), or endothelium/estrogen system (eNOS Glu298Asp, ERalpha IVS1-401). SNPs were analyzed for their relation to clinical values including lipids, B-type natriuretic peptide (BNP), fasting plasma glucose, tumor necrosis factor-alpha, interleukin-6, cyclic GMP, and nitric oxide metabolites. KEY FINDINGS Comparisons between the distributions of different genotypes and clinical values showed three relationships. First, factor VII Arg353Gln and HDL-cholesterol (HDL-C) were linked to Arg/Arg carriers at higher levels (P=.049). The HDL-C to LDL-cholesterol ratio supported this link (P=.027). Second, eNOS Glu298Asp and triglycerides were linked to Glu/Glu carriers at higher levels (P=.031). Third, ERalpha IVS1-401 and BNP were related to CC genotype at lower levels (P=.031). Additionally, the last two relations showed that genotype does not influence the demarcation line of biomarkers, but the plasma/serum levels of biomarkers instead. SIGNIFICANCE Correlations of factor VII Arg353Gln with HDL-C and eNOS Glu298Asp with triglycerides are new findings. Polymorphisms in the endothelium/estrogen system and the heart failure marker BNP are also correlated, with ERalpha IVS1-401 being the first identified marker. SNPs may be helpful for understanding the pathophysiology of atherosclerotic diseases in elderly women.