Yasumasa Ikeda

Disruption of Nuclear Vitamin D Receptor Gene Causes Enhanced Thrombogenicity in Mice

Vitamin D metabolites influence the expression of various genes involved in calcium homeostasis, cell differentiation, and regulation of the immune system. Expression of these genes is mediated by the activation of the nuclear vitamin D receptor (VDR). Previous studies have shown that a hormonally active form of vitamin D, 1α,25-dihydroxyvitamin D3, exerts anticoagulant effects in cultured monocytic cells. To clarify whether activation of VDR plays any antithrombotic actions in vivo, hemostatic/thrombogenic systems were examined in normocalcemic VDR knock-out (KO) mice on a high calcium diet and compared with wild type and hypocalcemic VDRKO mice that were fed a regular diet. Platelet aggregation was enhanced significantly in normocalcemic VDRKO mice compared with wild type and hypocalcemic VDRKO mice. Aortic endothelial nitric-oxide (NO) synthase expression and urinary NOx excretions were reduced in hypocalcemic VDRKO mice, but not in normocalcemic VDRKO mice. Northern blot and RT-PCR analyses revealed that the gene expression of antithrombin in the liver as well as that of thrombomodulin in the aorta, liver and kidney was down-regulated in hypo- and normocalcemic VDRKO mice. Whereas tissue factor mRNA expression in the liver and kidney was up-regulated in VDRKO mice regardless of plasma calcium level. Furthermore, VDRKO mice manifested an exacerbated multi-organ thrombus formation after exogenous lipopolysaccharide injection regardless of the calcemic conditions. These results demonstrate that activation of nuclear VDR elicits antithrombotic effects in vivo, and suggest that the VDR system may play a physiological role in the maintenance of antithrombotic homeostasis.

Androgen Receptor Gene Knockout Male Mice Exhibit Impaired Cardiac Growth and Exacerbation of Angiotensin II-induced Cardiac Fibrosis

Androgen has anabolic effects on cardiac myocytes and has been shown to enhance left ventricular enlargement and function. However, the physiological and patho-physiological roles of androgen in cardiac growth and cardiac stress-induced remodeling remains unclear. We aimed to clarify whether the androgen-nuclear androgen receptor (AR) system contributes to the cardiac growth and angiotensin II (Ang II)-stimulated cardiac remodeling by using systemic AR-null male mice. AR knock-out (ARKO) male mice, at 25 weeks of age, and age-matched wild-type (WT) male mice were treated with or without Ang II stimulation (2.0 mg/kg/day) for 2 weeks. ARKO mice with or without Ang II stimulation showed a significant reduction in the heart-to-body weight ratio compared with those of WT mice. In addition, echocardiographic analysis demonstrated impairments of both the concentric hypertrophic response and left ventricular function in Ang II-stimulated ARKO mice. Western blot analysis of the myocardium revealed that activation of extracellular signal-regulated kinases (ERK) 1/2 and ERK5 by Ang II stimulation were lower in ARKO mice than those of WT mice. Ang II stimulation caused more prominent cardiac fibrosis in ARKO mice than in WT mice with enhanced expression of types I and III collagen and transforming growth factor-β1 genes and with increased Smad2 activation. These results suggest that, in male mice, the androgen-AR system participates in normal cardiac growth and modulates cardiac adaptive hypertrophy and fibrosis during the process of cardiac remodeling under hypertrophic stress.

Cardiac-specific Deletion of LKB1 Leads to Hypertrophy and Dysfunction

LKB1 encodes a serine/threonine kinase, which functions upstream of the AMP-activated protein kinase (AMPK) superfamily. To clarify the role of LKB1 in heart, we generated and characterized cardiac myocyte-specific LKB1 knock-out (KO) mice using α-myosin heavy chain-Cre deletor strain. LKB1-KO mice displayed biatrial enlargement with atrial fibrillation and cardiac dysfunction at 4 weeks of age. Left ventricular hypertrophy was observed in LKB1-KO mice at 12 weeks but not 4 weeks of age. Collagen I and III mRNA expression was elevated in atria at 4 weeks, and atrial fibrosis was seen at 12 weeks. LKB1-KO mice displayed cardiac dysfunction and atrial fibrillation and died within 6 months of age. Indicative of a prohypertrophic environment, the phosphorylation of AMPK and eEF2 was reduced, whereas mammalian target of rapamycin (mTOR) phosphorylation and p70S6 kinase phosphorylation were increased in both the atria and ventricles of LKB1-deficient mice. Consistent with vascular endothelial growth factor mRNA and protein levels being significantly reduced in LKB1-KO mice, these mice also exhibited a reduction in capillary density of both atria and ventricles. In cultured cardiac myocytes, LKB1 silencing induced hypertrophy, which was ameliorated by the expression of a constitutively active form AMPK or by treatment with the inhibitor of mTOR, rapamycin. These findings indicate that LKB1 signaling in cardiac myocytes is essential for normal development of the atria and ventricles. Cardiac hypertrophy and dysfunction in LKB1-deficient hearts are associated with alterations in AMPK and mTOR/p70S6 kinase/eEF2 signaling and with a reduction in vascular endothelial growth factor expression and vessel rarefaction.

Adiponectin deficiency: a model of pulmonary hypertension associated with pulmonary vascular disease

Adiponectin (APN) is an adipocyte-derived factor that exists at high concentrations in serum and has anti-inflammatory and systemic vascular-protective properties. In this study, we investigated the role of APN in pulmonary vascular homeostasis. We found that APN localizes to the luminal side of blood vessels in lung and acts in vitro to block TNF-alpha-induced E-selectin upregulation in pulmonary artery endothelial cells. Targeted deletion of the APN gene in mice leads to a vascular phenotype in lung characterized by E-selectin upregulation and age-dependent increases in perivascular inflammatory cell infiltration and pulmonary arterial pressures. Taken together, these findings demonstrate an important role for APN in lung vascular homeostasis and suggest that APN-deficient states may contribute to the pathogenesis of inflammatory pulmonary vascular disease and to the development of pulmonary hypertension.

Estrogen Regulates Hepcidin Expression via GPR30-BMP6-Dependent Signaling in Hepatocytes

Hepcidin, a liver-derived iron regulatory protein, plays a crucial role in iron metabolism. It is known that gender differences exist with respect to iron storage in the body; however, the effects of sex steroid hormones on iron metabolism are not completely understood. We focused on the effects of the female sex hormone estrogen on hepcidin expression. First, ovariectomized (OVX) and sham-operated mice were employed to investigate the effects of estrogen on hepcidin expression in an in vivo study. Hepcidin expression was decreased in the livers of OVX mice compared to the sham-operated mice. In OVX mice, bone morphologic protein-6 (BMP6), a regulator of hepcidin, was also found to be downregulated in the liver, whereas ferroportin (FPN), an iron export protein, was upregulated in the duodenum. Both serum and liver iron concentrations were elevated in OVX mice relative to their concentrations in sham-operated mice. In in vitro studies, 17β-estradiol (E2) increased the mRNA expression of hepcidin in HepG2 cells in a concentration-dependent manner. E2-induced hepatic hepcidin upregulation was not inhibited by ICI 182720, an inhibitor of the estrogen receptor; instead, hepcidin expression was increased by ICI 182720. E2 and ICI 182720 exhibit agonist actions with G-protein coupled receptor 30 (GPR30), the 7-transmembrane estrogen receptor. G1, a GPR30 agonist, upregulated hepcidin expression, and GPR30 siRNA treatment abolished E2-induced hepcidin expression. BMP6 expression induced by E2 was abolished by GPR30 silencing. Finally, both E2 and G1 supplementation restored reduced hepatic hepcidin and BMP6 expression and reversed the augmentation of duodenal FPN expression in the OVX mice. In contrast, serum hepcidin was elevated in OVX mice, which was reversed in these mice with E2 and G1. Thus, estrogen is involved in hepcidin expression via a GPR30-BMP6-dependent mechanism, providing new insight into the role of estrogen in iron metabolism.

Pitavastatin, an HMG-CoA Reductase Inhibitor, Exerts eNOS-Independent Protective Actions Against Angiotensin II–Induced Cardiovascular Remodeling and Renal Insufficiency

Angiotensin II (Ang II) plays a pivotal role in cardiovascular remodeling leading to hypertension, myocardial infarction, and stroke. Pitavastatin, an HMG-CoA reductase inihibitor, is known to have pleiotropic actions against the development of cardiovascular remodeling. The objectives of this study were to clarify the beneficial effects as well as the mechanism of action of pitavastatin against Ang II–induced organ damage. C57BL6/J mice at 10 weeks of age were infused with Ang II for 2 weeks and were simultaneously administered pitavastatin or a vehicle. Pitavastatin treatment improved Ang II–induced left ventricular hypertrophy and diastolic dysfunction and attenuated enhancement of cardiac fibrosis, cardiomyocyte hypertrophy, coronary perivascular fibrosis, and medial thickening. Ang II–induced oxidative stress, cardiac TGFβ-1 expression, and Smad 2/3 phosphorylation were all attenuated by pitavastatin treatment. Pitavastatin also reduced Ang II–induced cardiac remodeling and diastolic dysfunction in eNOS−/− mice as in wild-type mice. In eNOS−/− mice, the Ang II–induced cardiac oxidative stress and TGF-β–Smad 2/3 signaling pathway were enhanced, and pitavastatin treatment attenuated the enhanced oxidative stress and the signaling pathway. Moreover, pitavastatin treatment reduced the high mortality rate and improved renal insufficiency in Ang II–treated eNOS−/− mice, with suppression of glomerular oxidative stress and TGF-β-Smad 2/3 signaling pathway. In conclusion, pitavastatin exerts eNOS-independent protective actions against Ang II–induced cardiovascular remodeling and renal insufficiency through inhibition of the TGF-β-Smad 2/3 signaling pathway by suppression of oxidative stress.

Heparin Cofactor II Is a Novel Protective Factor Against Carotid Atherosclerosis in Elderly Individuals

Background—Thrombin plays a crucial role in atherothrombotic changes. Because heparin cofactor II (HCII) inhibits thrombin actions after binding to dermatan sulfate at injured arterial walls, HCII may negatively regulate thrombin actions in vascular walls. We hypothesized that plasma HCII activity is a preventive factor against atherosclerotic changes, especially in elderly individuals who already have atherosclerotic vascular injuries. Methods and Results—Maximum plaque thickness (MPT) in the carotid artery was measured by ultrasonography in 306 Japanese elderly individuals (154 men and 152 women; age, 40 to 91 years; 68.9±11.1 years, mean±SD). The relevance of cardiovascular risk factors including plasma HCII activity to the severity of MPT was statistically evaluated. Plasma HCII activity decreased with age. Simple linear regression analysis after adjustments for age and sex showed that lipoprotein(a), glycosylated hemoglobin A1c, and presence of diabetes mellitus significantly contributed to an increase in MPT values (r =0.119, P <0.05; r =0.196, P <0.001; and r =0.227, P <0.0001, respectively). In contrast, high-density lipoprotein (HDL) cholesterol and HCII activity were negatively correlated with MPT values (r =−0.117, P <0.05, and r =−0.202, P <0.0005, respectively). Multiple regression analysis revealed that plasma HCII activity and HDL cholesterol independently contributed to the suppression of MPT values and that the antiatherogenic contribution of HCII activity was stronger than that of HDL cholesterol (P <0.001 and P <0.05, respectively). Conclusions—These results suggest that HCII can be a novel and independent antiatherogenic factor. Moreover, HCII is a stronger predictive factor than HDL cholesterol against carotid atherosclerosis in elderly individuals.

Androgen-androgen receptor system protects against angiotensin II-induced vascular remodeling.

Age-related andropause promotes cardiovascular disease in males. Although we had previously reported that the androgen-androgen receptor (AR) system plays important roles in cardiac growth and remodeling, the systems involvement in vascular remodeling remains unclear. To clarify this role, 25-wk-old male AR knockout (ARKO) mice and littermate male wild-type (WT) mice were divided into two groups with and without angiotensin II (Ang II) administration (2.0 mg/kg . d) for 14 d, respectively. No morphological differences in the coronary artery and thoracic aorta were observed between the groups without Ang II. Ang II stimulation markedly increased medial thickness and perivascular fibrosis in ARKO mice, with enhanced TGF-beta1, collagen type I, and collagen type III gene expression in the aorta. Ang II stimulation also prominently increased superoxide production, lipid peroxidation, and gene expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components in ARKO mice compared with WT mice. In addition, phosphorylation of c-Jun N-terminal kinase (JNK) and phosphorylated (Smad2/3) was remarkably enhanced in Ang II-treated ARKO mice compared with Ang II-treated WT mice. Notably, daily urinary nitric oxide (NO) metabolites excretion as a marker of NO bioavailability, aortic endothelial NO synthase expression and phosphorylation, and Akt phosphorylation were significantly reduced in ARKO mice compared with WT mice, regardless of Ang II stimulation. In conclusion, the androgen-AR system is required for the preservation of NO bioavailability through Akt-endothelial NO synthase system activation and exerts protective effects against Ang II-induced vascular remodeling by regulating oxidative stress, c-Jun N-terminal kinase (JNK) signaling, and the TGF-beta-phosphorylated Smad pathway.

Endothelial Nitric Oxide Synthase–Independent Protective Action of Statin Against Angiotensin II–Induced Atrial Remodeling via Reduced Oxidant Injury

Activation of the renin-angiotensin system exacerbates atrial remodeling, leading to atrial fibrillation and thrombosis, especially in a condition with decreased NO bioavailability. Recently, it has been reported that statins reduce the incidence of atrial fibrillation through attenuation of atrial remodeling; however, the mechanisms have not been completely elucidated. Therefore, we aimed to clarify the beneficial effect of statin on atrial remodeling in condition with reduced NO bioavailability. Endothelial NO synthase−/− mice were sham operated or infused with angiotensin II (Ang II) via an osmotic minipump for 2 weeks, and Ang II–infused mice were divided into 3 treatment groups: pitavastatin, Tempol (a free radical scavenger), or vehicle. Echocardiography and electrocardiography showed that Ang II infusion caused left atrial enlargement and a high incidence of atrial fibrillation, whereas pitavastatin and Tempol prevented these abnormalities. In histological analysis, Ang II–induced atrial interstitial fibrosis, perivascular fibrosis, and cardiomyocyte hypertrophy were all attenuated by pitavastatin and Tempol. Immunohistochemical staining showed that Ang II downregulated thrombomodulin and tissue factor pathway inhibitor and upregulated tissue factor and plasminogen activator inhibitor 1 in the left atrium and that pitavastatin and Tempol corrected the thrombogenic condition. Moreover, pitavastatin and Tempol reduced Ang II–induced atrial superoxide production and atrial transforming growth factor-&bgr;1 expression and Smad 2/3 phosphorylation. Atrial rac1-GTPase activity, known to activate NADPH oxidase, was attenuated by pitavastatin but not by Tempol. In conclusion, pitavastatin exerts endothelial NO synthase–independent protective actions against Ang II–induced atrial remodeling and atrial fibrillation with enhanced thrombogenicity through suppression of oxidant injury.

Cyclooxygenase-2 induction by adiponectin is regulated by a sphingosine kinase-1 dependent mechanism in cardiac myocytes

The adipose‐derived plasma protein, adiponectin (APN), has various protective effects on cardiovascular diseases. In this study, we show that endogenous APN is required for full cyclooxygenase‐2 (COX‐2) induction by ischemia‐reperfusion injury in the heart in vivo. In rat neonatal cardiac myocytes, APN‐induced COX‐2 expression was reduced by treatment with a sphingosine kinase‐1 (SphK‐1) inhibitor or siRNA targeting SphK‐1. Treatment with a sphingosine‐1‐phosphate (S1P) receptor antagonist also diminished COX‐2 expression in response to APN stimulation. These findings suggest that APN is a physiological regulator of COX‐2 signaling in the heart and that this regulation occurs in part via a SphK‐1–S1P receptor dependent mechanism in cardiac myocytes.