Haruaki Kubota

Increasing dihydrobiopterin causes dysfunction of endothelial nitric oxide synthase in rats in vivo

An elevation of oxidized forms of tetrahydrobiopterin (BH(4)), especially dihydrobiopterin (BH(2)), has been reported in the setting of oxidative stress, such as arteriosclerotic/atherosclerotic disorders, where endothelial nitric oxide synthase (eNOS) is dysfunctional, but the role of BH(2) in the regulation of eNOS activity in vivo remains to be evaluated. This study was designed to clarify whether increasing BH(2) concentration causes endothelial dysfunction in rats. To increase vascular BH(2) levels, the BH(2) precursor sepiapterin (SEP) was intravenously given after the administration of the specific dihydrofolate reductase inhibitor methotrexate (MTX) to block intracellular conversion of BH(2) to BH(4). MTX/SEP treatment did not significantly affect aortic BH(4) levels compared with control treatment. However, MTX/SEP treatment markedly augmented aortic BH(2) levels (291.1 ± 29.2 vs. 33.4 ± 6.4 pmol/g, P < 0.01) in association with moderate hypertension. Treatment with MTX alone did not significantly alter blood pressure or BH(4) levels but decreased the BH(4)-to-BH(2) ratio. Treatment with MTX/SEP, but not with MTX alone, impaired ACh-induced vasodilator and depressor responses compared with the control treatment (both P < 0.05) and also aggravated ACh-induced endothelium-dependent relaxations (P < 0.05) of isolated aortas without affecting sodium nitroprusside-induced endothelium-independent relaxations. Importantly, MTX/SEP treatment significantly enhanced aortic superoxide production, which was diminished by NOS inhibitor treatment, and the impaired ACh-induced relaxations were reversed with SOD (P < 0.05), suggesting the involvement of eNOS uncoupling. These results indicate, for the first time, that increasing BH(2) causes eNOS dysfunction in vivo even in the absence of BH(4) deficiency, demonstrating a novel insight into the regulation of endothelial function.

Effect of caffeine contained in a cup of coffee on microvascular function in healthy subjects.

Recent epidemiological studies have demonstrated that coffee drinking is associated with reduced mortality of cardiovascular disease. However, its precise mechanisms remain to be clarified. In this study, we examined whether single ingestion of caffeine contained in a cup of coffee improves microvascular function in healthy subjects. A double-blind, placebo-controlled, crossover study was performed in 27 healthy volunteers. A cup of either caffeinated or decaffeinated coffee was drunk by the subjects, and reactive hyperemia of finger blood flow was assessed by laser Doppler flowmetry. In an interval of more than 2 days, the same experimental protocol was repeated with another coffee in a crossover manner. Caffeinated coffee intake slightly but significantly elevated blood pressure and decreased finger blood flow as compared with decaffeinated coffee intake. There was no significant difference in heart rate between caffeinated and decaffeinated coffee intake. Importantly, caffeinated coffee intake significantly enhanced post-occlusive reactive hyperemia of finger blood flow, an index of microvascular endothelial function, compared with decaffeinated coffee intake. These results provide the first evidence that caffeine contained in a cup of coffee enhances microvascular function in healthy individuals.

Development of an experimentally useful model of acute myocardial infarction: 2/3 nephrectomized triple nitric oxide synthases-deficient mouse

We investigated the effect of subtotal nephrectomy on the incidence of acute myocardial infarction (AMI) in mice deficient in all three nitric oxide synthases (NOSs). Two-thirds nephrectomy (NX) was performed on male triple NOSs(-/-) mice. The 2/3NX caused sudden cardiac death due to AMI in the triple NOSs(-/-) mice as early as 4months after the surgery. The 2/3NX triple NOSs(-/-) mice exhibited electrocardiographic ST-segment elevation, reduced heart rate variability, echocardiographic regional wall motion abnormality, and accelerated coronary arteriosclerotic lesion formation. Cardiovascular risk factors (hypertension, hypercholesterolemia, and hyperglycemia), an increased number of circulating bone marrow-derived vascular smooth muscle cell (VSMC) progenitor cells (a pro-arteriosclerotic factor), and cardiac up-regulation of stromal cell-derived factor (SDF)-1α (a chemotactic factor of the progenitor cells) were noted in the 2/3NX triple NOSs(-/-) mice and were associated with significant increases in plasma angiotensin II levels (a marker of renin-angiotensin system activation) and urinary 8-isoprostane levels (a marker of oxidative stress). Importantly, combined treatment with a clinical dosage of an angiotensin II type 1 receptor blocker, irbesartan, and a calcium channel antagonist, amlodipine, markedly prevented coronary arteriosclerotic lesion formation and the incidence of AMI and improved the prognosis of those mice, along with ameliorating all those pro-arteriosclerotic parameters. The 2/3NX triple NOSs(-/-) mouse is a new experimentally useful model of AMI. Renin-angiotensin system activation, oxidative stress, cardiovascular risk factors, and SDF-1α-induced recruitment of bone marrow-derived VSMC progenitor cells appear to be involved in the pathogenesis of AMI in this model.