Akiko Fukatsu

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.

A Specific Role for eNOS-Derived Reactive Oxygen Species in Atherosclerosis Progression

Objective—When the availability of tetrahydrobiopterin (BH4) is deficient, endothelial nitric oxide synthase (eNOS) produces superoxide rather than NO (uncoupled eNOS). We have shown that the atherosclerotic lesion size was augmented in apolipoprotein E–deficient (ApoE-KO) mice overexpressing eNOS because of the enhanced superoxide production. In this study, we addressed the specific importance of uncoupled eNOS in atherosclerosis, and the potential mechanistic role for specific versus nonspecific antioxidant strategies in restoring eNOS coupling. Methods and Results—We crossed mice overexpressing eNOS in the endothelium (eNOS-Tg) with mice overexpressing GTP-cyclohydrolase I (GCH), the rate-limiting enzyme in BH4 synthesis, to generate ApoE-KO/eNOS-Tg/GCH-Tg mice. As a comparison, ApoE-KO/eNOS-Tg mice were treated with vitamin C. Atherosclerotic lesion formation was increased in ApoE-KO/eNOS-Tg mice compared with ApoE-KO mice. GCH overexpression in ApoE-KO/eNOS-Tg/GCH-Tg mice increased vascular BH4 levels and reduced plaque area. This reduction was associated with decreased superoxide production from uncoupled eNOS. Vitamin C treatment failed to reduce atherosclerotic lesion size in ApoE-KO/eNOS-Tg mice, despite reducing overall vascular superoxide production. Conclusion—In contrast to vitamin C treatment, augmenting BH4 levels in the endothelium by GCH overexpression reduced the accelerated atherosclerotic lesion formation in ApoE-KO/eNOS-Tg mice, associated with a reduction of superoxide production from uncoupled eNOS.

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.

Augmentation of Vascular Remodeling by Uncoupled Endothelial Nitric Oxide Synthase in a Mouse Model of Diabetes Mellitus

Objective—Diabetes mellitus is associated with increased oxidative stress, which induces oxidation of tetrahydrobiopterin (BH4) in vessel wall. Without enough BH4, eNOS is uncoupled to L-arginine and produces superoxide rather than NO. We examined the role of uncoupled eNOS in vascular remodeling in diabetes. Methods and Results—Diabetes mellitus was produced by streptozotocin in C57BL/6J mice. Under stable hyperglycemia, the common carotid artery was ligated, and neointimal formation was examined 4 weeks later. In diabetic mice, the neointimal area was dramatically augmented. This augmentation was associated with increased aortic superoxide formation, reduced aortic BH4/dihydrobiopterin (BH2) ratio, and decreased plasma nitrite and nitrate (NOx) levels compared with nondiabetic mice. Chronic BH4 treatment (10 mg/kg/d) reduced the neointimal area in association with suppressed superoxide production and inflammatory changes in vessels. BH4/BH2 ratio in vessel wall was preserved, and plasma NOx levels increased. Furthermore, in the presence of diabetes, overexpression of bovine eNOS resulted in augmentation of neointimal area, accompanied by increased superoxide production in the endothelium. Conclusions—In diabetes, increased oxidative stress by uncoupled NOSs, particularly eNOS, causes augmentation of vascular remodeling. These findings indicate restoration of eNOS coupling has an atheroprotective benefit in diabetes.