Mitsuru Ohishi

Association of Hypoadiponectinemia With Impaired Vasoreactivity

Abstract—Endothelial dysfunction is a crucial feature in the evolution of atherosclerosis. Adiponectin is an adipocyte-specific plasma protein with antiatherogenic and antidiabetic properties. In the present study, we investigated the relation between adiponectin and endothelium-dependent vasodilation. We analyzed endothelial function in 202 hypertensive patients, including those who were not taking any medication. Forearm blood flow was measured by strain-gauge plethysmography. Plasma adiponectin level was highly correlated with the vasodilator response to reactive hyperemia in the total (r =0.257, P <0.001) and no-medication (r =0.296, P =0.026) groups but not with nitroglycerin-induced hyperemia, indicating that adiponectin affected endothelium-dependent vasodilation. Multiple regression analysis of data from all hypertensive patients revealed that plasma adiponectin level was independently correlated with the vasodilator response to reactive hyperemia. Vascular reactivity was also analyzed in aortic rings from adiponectin-knockout (KO) and wild-type (WT) mice. Adiponectin-KO mice showed obesity, hyperglycemia, and hypertension compared with WT mice after 4 weeks on an atherogenic diet. Endothelium-dependent vasodilation in response to acetylcholine was significantly reduced in adiponectin-KO mice compared with WT mice, although no significant difference was observed in endothelium-independent vasodilation in response to sodium nitroprusside. Our observations suggest that hypoadiponectinemia is associated with impaired endothelium-dependent vasorelaxation and that the measurement of plasma adiponectin level might be helpful as a marker of endothelial dysfunction.

Comparison between carotid-femoral and brachial-ankle pulse wave velocity as measures of arterial stiffness

Background Arterial stiffness is an important risk factor for cardiovascular disease. Carotid-femoral pulse wave velocity (cfPWV) is the most recognized and established index of arterial stiffness. An emerging automatic measure of PWV primarily used in the Asian countries is brachial-ankle PWV (baPWV). Method To systematically compare these two methodologies, we conducted a multicenter study involving a total of 2287 patients. Results There was a significant positive relation between baPWV and cfPWV (r = 0.73). Average baPWV was approximately 20% higher than cfPWV. Both cfPWV and baPWV were significantly and positively associated with age (r = 0.56 and 0.64), systolic blood pressure (r = 0.49 and 0.61), and the Framingham risk score (r = 0.48 and 0.63). The areas under the receiver operating curves (ROCs) of PWV to predict the presence of both stroke and coronary artery disease were comparable between cfPWV and baPWV. Conclusion Collectively, these results indicate that cfPWV and baPWV are indices of arterial stiffness that exhibit similar extent of associations with cardiovascular disease risk factors and clinical events.

Deletion of Angiotensin-Converting Enzyme 2 Accelerates Pressure Overload-Induced Cardiac Dysfunction by Increasing Local Angiotensin II

Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that cleaves angiotensin II to angiotensin 1-7. Recently, it was reported that mice lacking ACE2 (ACE2−/y mice) exhibited reduced cardiac contractility. Because mechanical pressure overload activates the cardiac renin–angiotensin system, we used ACE2−/y mice to analyze the role of ACE2 in the response to pressure overload. Twelve-week-old ACE2−/y mice and wild-type (WT) mice received transverse aortic constriction (TAC) or sham operation. Sham-operated ACE2−/y mice exhibited normal cardiac function and had morphologically normal hearts. In response to TAC, ACE2−/y mice developed cardiac hypertrophy and dilatation. Furthermore, their hearts displayed decreased cardiac contractility and increased fetal cardiac gene induction, compared with WT mice. In response to chronic pressure overload, ACE2−/y mice developed pulmonary congestion and increased incidence of cardiac death compared with WT mice. On a biochemical level, cardiac angiotensin II concentration and activity of mitogen-activated protein (MAP) kinases were markedly increased in ACE2−/y mice in response to TAC. Administration of candesartan, an AT1 subtype angiotensin receptor blocker, attenuated the hypertrophic response and suppressed the activation of MAP kinases in ACE2−/y mice. Activation of MAP kinases in response to angiotensin II was greater in cardiomyocytes isolated from ACE2−/y mice than in those isolated from WT mice. ACE2 plays an important role in dampening the hypertrophic response to pressure overload mediated by angiotensin II. Disruption of this regulatory function may accelerate cardiac hypertrophy and shorten the transition period from compensated hypertrophy to cardiac failure.

Upregulation of renin-angiotensin system during differentiation of monocytes to macrophages.

BACKGROUND We have demonstrated that accumulated macrophages in human coronary arteries strongly express angiotensin converting enzyme in accordance with the development of atheromatous plaques. However, there are few reports on the regulation of the renin-angiotensin system in macrophages and in monocytes as their source. OBJECTIVE To examine whether the renin-angiotensin system is upregulated during the differentiation of monocytes to macrophages, and whether it is further regulated by angiotensin II and cytokines. MATERIALS AND METHODS We used a human leukemia cell line, THP-1, for monocytes. Differentiated THP-1, induced by adding phorbol 12-myristate 13-acetate for 24 h, were used as macrophages. Expression of messenger RNA of the renin-angiotensin system components was measured by quantitative reverse-transcriptase polymerase chain reaction. Angiotensin converting enzyme activity and subtype-specific angiotensin-binding sites of cultured cells, and angiotensin II production in the culture medium were measured. RESULTS Macrophages expressed all components of the renin-angiotensin system except chymase. Cellular angiotensin converting enzyme activity and angiotensin II in the medium were increased 3.2- and 4.5-fold during differentiation, respectively. Expression of angiotensin II type 1 (AT1) and type 2 (AT2) receptors was increased 6.2-and 6.4-fold during differentiation, and was sustained for 7 days. Incubation with angiotensin II for 24 h caused downregulation of both AT1 and AT2 receptor messenger RNA, but the expression levels were still more than threefold higher compared with monocytes. The density of binding sites of AT1 and AT2 receptors in macrophages was 0.26 +/- 0.02 and 0.15 +/- 0.01 fmol/10(6) cells, respectively. CONCLUSION The renin-angiotensin system is markedly activated during monocyte/macrophage differentiation, and may participate in the development of atherosclerosis.

Endogenous Drp1 mediates mitochondrial autophagy and protects the heart against energy stress

Rationale: Both fusion and fission contribute to mitochondrial quality control. How unopposed fusion affects survival of cardiomyocytes and left ventricular function in the heart is poorly understood. Objective: We investigated the role of dynamin-related protein 1 (Drp1), a GTPase that mediates mitochondrial fission, in mediating mitochondrial autophagy, ventricular function, and stress resistance in the heart. Methods and Results: Drp1 downregulation induced mitochondrial elongation, accumulation of damaged mitochondria, and increased apoptosis in cardiomyocytes at baseline. Drp1 downregulation also suppressed autophagosome formation and autophagic flux at baseline and in response to glucose deprivation in cardiomyocytes. The lack of lysosomal translocation of mitochondrially targeted Keima indicates that Drp1 downregulation suppressed mitochondrial autophagy. Mitochondrial elongation and accumulation of damaged mitochondria were also observed in tamoxifen-inducible cardiac-specific Drp1 knockout mice. After Drp1 downregulation, cardiac-specific Drp1 knockout mice developed left ventricular dysfunction, preceded by mitochondrial dysfunction, and died within 13 weeks. Autophagic flux is significantly suppressed in cardiac-specific Drp1 knockout mice. Although left ventricular function in cardiac-specific Drp1 heterozygous knockout mice was normal at 12 weeks of age, left ventricular function decreased more severely after 48 hours of fasting, and the infarct size/area at risk after ischemia/reperfusion was significantly greater in cardiac-specific Drp1 heterozygous knockout than in control mice. Conclusions: Disruption of Drp1 induces mitochondrial elongation, inhibits mitochondrial autophagy, and causes mitochondrial dysfunction, thereby promoting cardiac dysfunction and increased susceptibility to ischemia/reperfusion.

Genetic variants at the 9p21 locus contribute to atherosclerosis through modulation of ANRIL and CDKN2A/B

UNLABELLED Genome-wide association studies (GWAS) have identified genetic variants contributing to the risk of cardiovascular disease (CVD) at the chromosome 9p21 locus. The CVD-associated region is adjacent to the two cyclin dependent kinase inhibitors (CDKN)2A and 2B and the last exons of the non-coding RNA, ANRIL. It is still not clear which of or how these transcripts are involved in the pathogenesis of atherosclerosis. OBJECTIVE We assessed the hypothesis that 9p21 locus polymorphisms influence the expression of the transcripts in the region (ANRIL, CDKN2A/B) and that these transcripts contribute to atherogenesis through the modulation of proliferation in VSMC. METHODS We genotyped 18 SNPs (r(2)<0.8 and MAF>0.05) across the region of interest: CDKN2A/B and ANRIL, encompassing the CVD-associated region. RNA and DNA were extracted from the blood of 57 volunteers (69-72 years old). Carotid ultrasound was performed in 56 subjects. CDKN2A/B and ANRIL (exons 1-2 and 17-18) expression was measured employing RT-PCR. Gene expression and cell growth were evaluated in cultured VSMC after the siRNA-mediated knock-down of ANRIL. RESULTS The risk alleles for atherosclerosis-related phenotypes were consistently associated with a lower expression of ANRIL when evaluating exons 1-2. Common carotid artery stenosis was associated with a significantly lower (P<0.01) expression of ANRIL (exons 1-2). ANRIL knock-down in VSMC caused significant variation in expression of CDKN2A/B (P<0.05) and reduction of cell growth (P<0.05) in vitro. CONCLUSION Disease-associated SNPs at the 9p21 locus predominantly affect the expression of ANRIL. Overall, our results suggest that several CVD-associated SNPs in the 9p21 locus affect the expression of ANRIL, which, in turn modulate cell growth, possibly via CDKN2A/B regulation.

Klotho suppresses TNF-α-induced expression of adhesion molecules in the endothelium and attenuates NF-κB activation

Klotho is a senescence suppressor protein that, when overexpressed, extends the lifespan of mice. Klotho-disrupted mice exhibit atherosclerosis and endothelial dysfunction, which led us to investigate the effect of the Klotho protein on vascular inflammation, particularly adhesion molecule expression. In this study, human umbilical vein endothelial cells (HUVECs) were preincubated with Klotho protein and then exposed to tumor necrosis factor-α (TNF-α) or vehicle. Reverse transcription-PCR and Western blot analyses revealed that Klotho suppressed TNF-α-induced expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). NF-κB activation, IκB phosphorylation induced by TNF-α were also attenuated by Klotho protein administration. The inhibition of eNOS phosphorylation by TNF-α was reversed by Klotho. Furthermore, Klotho inhibited TNF-α-induced monocyte adhesion to HUVECs and suppressed adhesion molecule expression in an organ culture of the rat aorta. These results suggest that Klotho suppresses TNF-α-induced expression of adhesion molecules and NF-κB activation. Klotho may have a role in the modulation of endothelial inflammation.

Enhanced Predictability of Myocardial Infarction in Japanese by Combined Genotype Analysis

To explore the genes responsible for myocardial infarction and restenosis after percutaneous transluminal coronary angioplasty, we performed association studies of the polymorphisms of the angiotensinogen and angiotensin-converting enzyme (ACE) genes. In the first study, normotensive myocardial infarction patients (n = 103) and control subjects (n = 103), who were matched for established risk factors with the myocardial infarction patients, were randomly selected. The angiotensinogen-TT genotype (T indicates threonine instead of methionine at position 235) was more frequent in the myocardial infarction group than in the control group (P < .05). The ACE-DD genotype (D indicates a deletion polymorphism in intron 16) was also more frequent in the myocardial infarction group (P < .0001). The odds ratio estimated by the combined analysis of the angiotensinogen-TT and ACE-DD genotypes (11.2) was markedly increased compared with that estimated separately from the angiotensinogen-TT (1.75) or ACE-DD (4.43) genotype. In the second study, we investigated 91 consecutive patients with acute myocardial infarction who underwent successful direct angioplasty. Combined analysis showed that the angiotensinogen-TT genotype did not enhance the predictability of myocardial infarction from the ACE-DD genotype. In conclusion, the angiotensinogen-TT genotype is a predictor for myocardial infarction, as well as the ACE-DD genotype, and the combined analysis of the angiotensinogen-TT and ACE-DD genotypes further enhanced the predictability of myocardial infarction in Japanese, suggesting its future clinical usefulness.

Mapping tissue angiotensin-converting enzyme and angiotensin AT1, AT2 and AT4 receptors.

Background The renin–angiotensin system (RAS) functions as both a circulating endocrine system and a tissue paracrine/autocrine system. As a circulating peptide, angiotensin II (Ang II) plays a prominent role in blood-pressure control and body fluid and electrolyte balance by acting on the AT1 receptor in the brain and peripheral tissues. As a paracrine/autocrine peptide, locally formed Ang II also plays additional roles in tissues involving the regulation of regional haemodynamics, cell growth and remodelling, and neurotransmitter release. Evidence is emerging that Ang II is not the only active peptide of the RAS, and other Ang II fragments may also have important biological activities. Objectives To provide a morphological basis for understanding novel actions of angiotensin-converting enzyme (ACE), Ang II and related peptides in tissues, this article will review the localization of ACE and AT1, AT2 and AT4 receptors in the central nervous system, blood vessels and kidney. Results and conclusion Autoradiographic mapping of the major components of the RAS has proved a valuable strategy to reveal, or suggest, cellular sites of novel actions for Ang II and related peptides in tissues. First, colocalization of ACE and AT1 receptors in the substantia nigra, the caudate nucleus and putamen of human and rat brain, which contain the dopamine-synthesizing neurons, suggests that the central RAS may be important in modulating central dopamine release. Secondly, the distribution of AT4 receptors with a striking association with cholinergic neurons, motor and sensory nuclei in the brain reveals that Ang IV may modulate central motor and sensory activities and memory. Thirdly, the occurrence of high levels of ACE and AT1 and/or AT2 receptors in the adventitia of blood vessels suggests important paracrine roles of the vascular RAS. Finally, the identification of abundant AT1 receptor and elucidation of its roles in the renomedullary interstitial cells of the kidney may provide a new impetus to study further the role of Ang II in the regulation of renal medullary function and blood pressure. Overall, circulating and locally produced Ang II and related peptides may exert a remarkable range of actions in the brain, kidney and cardiovascular system through multiple angiotensin receptors.

ANRIL: Molecular Mechanisms and Implications in Human Health

ANRIL is a recently discovered long non-coding RNA encoded in the chromosome 9p21 region. This locus is a hotspot for disease-associated polymorphisms, and it has been consistently associated with cardiovascular disease, and more recently with several cancers, diabetes, glaucoma, endometriosis among other conditions. ANRIL has been shown to regulate its neighbor tumor suppressors CDKN2A/B by epigenetic mechanisms and thereby regulate cell proliferation and senescence. However, the clear role of ANRIL in the pathogenesis of these conditions is yet to be understood. Here, we review the recent findings on ANRIL molecular characterization and function, with a particular focus on its implications in human disease.