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1.  Angiotensin II Receptor Blocker Ameliorates Stress-Induced Adipose Tissue Inflammation and Insulin Resistance 
PLoS ONE  2014;9(12):e116163.
A strong causal link exists between psychological stress and insulin resistance as well with hypertension. Meanwhile, stress-related responses play critical roles in glucose metabolism in hypertensive patients. As clinical trials suggest that angiotensin-receptor blocker delays the onset of diabetes in hypertensive patients, we investigated the effects of irbesartan on stress-induced adipose tissue inflammation and insulin resistance. C57BL/6J mice were subjected to 2-week intermittent restraint stress and orally treated with vehicle, 3 and 10 mg/kg/day irbesartan. The plasma concentrations of lipid and proinflammatory cytokines [Monocyte Chemoattractant Protein-1 (MCP-1), tumor necrosis factor-α, and interleukin-6] were assessed with enzyme-linked immunosorbent assay. Monocyte/macrophage accumulation in inguinal white adipose tissue (WAT) was observed with CD11b-positive cell counts and mRNA expressions of CD68 and F4/80 using immunohistochemistry and RT-PCR methods respectively. The mRNA levels of angiotensinogen, proinflammatory cytokines shown above, and adiponectin in WAT were also assessed with RT-PCR method. Glucose metabolism was assessed by glucose tolerance tests (GTTs) and insulin tolerance tests, and mRNA expression of insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) in WAT. Restraint stress increased monocyte accumulation, plasma free fatty acids, expression of angiotensinogen and proinflammatory cytokines including MCP-1, and reduced adiponectin. Irbesartan reduced stress-induced monocyte accumulation in WAT in a dose dependent manner. Irbesartan treatment also suppressed induction of adipose angiotensinogen and proinflammatory cytokines in WAT and blood, and reversed changes in adiponectin expression. Notably, irbesartan suppressed stress-induced reduction in adipose tissue weight and free fatty acid release, and improved insulin tolerance with restoration of IRS-1 and GLUT4 mRNA expressions in WAT. The results indicate that irbesartan improves stress-induced adipose tissue inflammation and insulin resistance. Our results suggests that irbesartan treatment exerts additive benefits for glucose metabolism in hypertensive patients with mental stress.
doi:10.1371/journal.pone.0116163
PMCID: PMC4281136  PMID: 25551221
2.  Non-invasive assessment of functionally significant coronary stenoses through mathematical analysis of spectral ECG components 
Open Heart  2014;1(1):e000144.
Objectives
The aim of this study was to evaluate the accuracy of the Multifunction CardioGram (MCG) in detecting the presence of functionally significant coronary ischaemia.
Methods and results
This prospective study evaluated the accuracy of the MCG, a new ECG analysis device used to diagnose ischaemic coronary artery disease (CAD). A consecutive 112 participants suspected to have CAD who were scheduled for elective coronary angiography (CAG) from October 2012 to December 2013 were examined. Their predictive values of relevant ischaemia were measured by MCG, standard ECG and Framingham Risk Score (FRS) and compared. Five levels of ischaemia based on CAG findings adjusted by fractional flow reserve (FFR) values and three levels of MCG score of high, borderline or low were used. The MCG (OR=2.67 (1.60 to 4.44), p<0.001) was the only test significantly associated with ischaemia level. The FFR values for individual MCG scores with low, borderline and high were 0.77 (0.70 to 0.86), 0.78 (0.71 to 0.82) and 0.69 (0.65 to 0.77), respectively, p=0.042. A high MCG score had a specificity of 90.4% (87.0% to 93.9%) in model 1 adjusted by FFR≤0.8 threshold and of 87.0% (83.2% to 90.8%) in model 2 adjusted by FFR≤0.75 threshold, and a negative predictive value of 82.5% (78.3% to 86.7%) in model 1 and of 83.8% (79.6% to 87.9%) in model 2 for the prediction of severe ischaemia.
Conclusions
The MCG showed high specificity with a high negative predictive value, suggesting that the MCG could be used not only to identify functionally significant ischaemia but to reduce unnecessary CAGs.
Trial registration number
UMIN ID: 000009992.
doi:10.1136/openhrt-2014-000144
PMCID: PMC4248141  PMID: 25469309
3.  Indoxyl Sulfate-Induced Activation of (Pro)renin Receptor Promotes Cell Proliferation and Tissue Factor Expression in Vascular Smooth Muscle Cells 
PLoS ONE  2014;9(10):e109268.
Chronic kidney disease (CKD) is associated with an increased risk of cardiovascular disease (CVD). (Pro)renin receptor (PRR) is activated in the kidney of CKD. The present study aimed to determine the role of indoxyl sulfate (IS), a uremic toxin, in PRR activation in rat aorta and human aortic smooth muscle cells (HASMCs). We examined the expression of PRR and renin/prorenin in rat aorta using immunohistochemistry. Both CKD rats and IS-administrated rats showed elevated expression of PRR and renin/prorenin in aorta compared with normal rats. IS upregulated the expression of PRR and prorenin in HASMCs. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed IS-induced expression of PRR and prorenin in HASMCs. Knock down of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR) and nuclear factor-κB p65 (NF-κB p65) with small interfering RNAs inhibited IS-induced expression of PRR and prorenin in HASMCs. Knock down of PRR inhibited cell proliferation and tissue factor expression induced by not only prorenin but also IS in HASMCs.
Conclusion
IS stimulates aortic expression of PRR and renin/prorenin through OAT3-mediated uptake, production of reactive oxygen species, and activation of AhR and NF-κB p65 in vascular smooth muscle cells. IS-induced activation of PRR promotes cell proliferation and tissue factor expression in vascular smooth muscle cells.
doi:10.1371/journal.pone.0109268
PMCID: PMC4208748  PMID: 25343458
4.  Hydrogen Sulfide Attenuates Cardiac Dysfunction Following Heart Failure via Induction of Angiogenesis 
Circulation. Heart failure  2013;6(5):10.1161/CIRCHEARTFAILURE.113.000299.
Background
Hydrogen sulfide (H2S) has been shown to induce angiogenesis in in vitro models and to promote vessel growth in the setting of hind-limb ischemia. The goal of the present study was to determine the therapeutic potential of a stable, long-acting H2S donor, diallyl trisulfide (DATS), in a model of pressure-overload heart failure and to assess the effects of chronic H2S therapy on myocardial vascular density and angiogenesis.
Methods and Results
Transverse aortic constriction (TAC) was performed in mice (C57BL/6J, 8-10 weeks of age). Mice received either vehicle or DATS (200 μg/kg) starting 24 hours after TAC and were followed for 12 weeks using echocardiography. H2S therapy with DATS improved left ventricular remodeling and preserved LV function in the setting of TAC. H2S therapy also increased the expression of the pro-angiogenic factor, vascular endothelial cell growth factor, while decreasing the angiogenesis inhibitor, angiostatin. Further studies revealed that H2S therapy increased the expression of the proliferation marker, Ki67, as well as increased the phosphorylation of endothelial nitric oxide synthase and increased the bioavailability of nitric oxide. Importantly, these changes were associated with an increase in vascular density within the H2S-treated hearts.
Conclusions
These results suggest that H2S therapy attenuates LV remodeling and dysfunction in the setting of heart failure by creating a pro-angiogenic environment for the growth of new vessels.
doi:10.1161/CIRCHEARTFAILURE.113.000299
PMCID: PMC3819451  PMID: 23811964
H2S donor; endothelial nitric oxide synthase; nitric oxide; angiogenesis; diallyl trisulfide
5.  Adiponectin Ameliorates Endotoxin-Induced Acute Cardiac Injury 
BioMed Research International  2014;2014:382035.
Background. Obesity is a risk factor for cardiovascular disease. Increasing evidence suggests that reduced levels of the adipocyte-derived plasma protein adiponectin are associated with an increased cardiovascular risk. Here, we examined the effects of adiponectin on lipopolysaccharide- (LPS-) induced acute cardiac injury in vivo. Methods and Results. A single dose of LPS (10 mg/kg) was intraperitoneally injected into wild-type (WT) and adiponectin-knockout (APN-KO) mice. Following LPS administration, APN-KO mice had exacerbation of left ventricular (LV) systolic dysfunction compared with WT mice. Administration of LPS to WT and APN-KO mice led to an increased expression of inflammatory cytokines including TNF-α and IL-6 in the heart, but the magnitude of this induction was greater in APN-KO mice compared to WT mice. Systemic delivery of an adenoviral vector expressing adiponectin (Ad-APN) improved LPS-induced LV dysfunction in APN-KO mice, and this effect was accompanied by the reduced expression of TNF-α and IL-6 in the heart. Administration of etanercept, a soluble TNF receptor abolished the reduced LV contractile function in response to LPS in APN-KO mice. Conclusion. These results suggest that adiponectin protects against LPS-induced acute cardiac injury by suppressing cardiac inflammatory responses, and could represent a potential therapeutic target in sepsis-associated myocardial dysfunction.
doi:10.1155/2014/382035
PMCID: PMC4142376  PMID: 25180179
6.  Association of Circulating C1q/TNF-Related Protein 1 Levels with Coronary Artery Disease in Men 
PLoS ONE  2014;9(6):e99846.
Objective
Obesity is a major risk factor for cardiovascular disease. Recent evidence demonstrates that dysregulation of fat-derived hormones, also known as adipokines, is linked with the pathogenesis of obesity-related disorders including coronary artery disease (CAD). Here, we investigated whether circulating level of an adipokine C1q/TNF-related protein (CTRP) 1 is associated with the prevalence of CAD.
Methods and Results
Consecutive 76 male CAD patients were enrolled from inpatients that underwent coronary angiography. Sixty four healthy male subjects served as controls. Plasma CTRP1 concentration was determined by enzyme-linked immunosorbent assay. CTRP1 levels were correlated positively with systolic blood pressure (BP) and triglyceride levels, and negatively with HDL cholesterol levels in all subjects. Plasma levels of CTRP1 were significantly higher in CAD patients than in control subjects (CAD: 443.3±18.6 ng/ml, control: 307.8±21.5 ng/ml, p<0.001). Multiple logistic regression analysis with body mass index, systolic BP, glucose, total cholesterol, HDL cholesterol, triglyceride, adiponectin and CTRP1 revealed that CTRP1 levels, together with systolic BP and HDL cholesterol, correlated with CAD.
Conclusions
Our data indicate the close association of high CTRP1 levels with CAD prevalence, suggesting that CTRP1 represents a novel biomarker for CAD.
doi:10.1371/journal.pone.0099846
PMCID: PMC4063765  PMID: 24945145
7.  Arterial Baroreflex Dysfunction Impairs Ischemia‐Induced Angiogenesis 
Background
Endothelium‐derived acetylcholine (eACh) plays an important role in the regulation of vascular actions in response to hypoxia, whereas arterial baroreflex (ABR) dysfunction impairs the eACh system. We investigated the effects of ABR dysfunction on ischemia‐induced angiogenesis in animal models of hindlimb ischemia with a special focus on eACh/nicotinic ACh receptor (nAChR) signaling activation.
Methods and Results
Male Sprague‐Dawley rats were randomly assigned to 1 of 3 groups that received (1) sham operation (control group), (2) sinoaortic denervation (SAD)‐induced ABR dysfunction (SAD group), or (3) SAD rats on diet with an acetylcholinesterase inhibitor pyridostigmine (30 mg/kg per day, SAD+Pyr group). After 4 weeks of the SAD intervention, unilateral limb ischemia was surgically induced in all animals. At postoperative day 14, SAD rats exhibited impaired angiogenic action (skin temperature and capillary density) and decreased angiogenic factor expressions (vascular endothelial growth factor [VEGF] and hypoxic inducible factor [HIF]‐1α) in ischemic muscles. These changes were restored by acetylcholinesterase inhibition. Rats with ABR dysfunction had lower eACh levels than did control rats, and this effect was recovered in SAD+Pyr rats. In α7‐nAChR knockout mice, pyridostigmine improved ischemia‐induced angiogenic responses and increased the levels of VEGF and HIF‐1α. Moreover, nicotinic receptor blocker inhibited VEGF expression and VEGF receptor 2 phosphorylation (p‐VEGFR2) induced by ACh analog.
Conclusions
Thus, ABR dysfunction appears to impair ischemia‐induced angiogenesis through the reduction of eACh/α7‐nAChR‐dependent and ‐independent HIF‐1α/VEGF‐VEGFR2 signaling activation.
doi:10.1161/JAHA.114.000804
PMCID: PMC4309071  PMID: 24820655
acetylcholinesterase inhibitor; angiogenesis; arterial baroreflex; non‐neural cholinergic system; peripheral vascular disease
8.  H2S Protects Against Pressure Overload Induced Heart Failure via Upregulation of Endothelial Nitric Oxide Synthase (eNOS) 
Circulation  2013;127(10):1116-1127.
Introduction
Cystathionine gamma-lyase (CSE) produces H2S via enzymatic conversion of L-cysteine and plays a critical role in cardiovascular homeostasis. We investigated the effects of genetic modulation of CSE and exogenous H2S therapy in the setting of pressure overload-induced heart failure.
Methods and Results
Transverse aortic constriction (TAC) was performed in wild-type (WT), CSE knockout (KO), and cardiac specific CSE transgenic (CS-CSE Tg) mice. In addition, C57BL/6J or CSE KO mice received a novel–H2S donor (SG-1002). Mice were followed for 12 weeks using echocardiography. We observed a >60% reduction in myocardial and circulating H2S levels following TAC. CSE KO mice exhibited cardiac dilatation and dysfunction significantly greater than WT mice following TAC and CS-CSE Tg mice maintained cardiac structure and function following TAC. H2S therapy with SG-1002 resulted in cardioprotection during TAC via upregulation of the VEGF-Akt-eNOS-nitric oxide-cGMP pathway with preserved mitochondrial function, attenuated oxidative stress, and increased myocardial vascular density.
Conclusions
Our results demonstrate that H2S levels are decreased in mice in the setting of heart failure. Moreover, CSE plays a critical role in the preservation of cardiac function in heart failure and oral H2S therapy prevents the transition from compensated to decompensated heart failure in part via upregulation of endothelial nitric oxide synthase (eNOS) and increased NO bioavailability.
doi:10.1161/CIRCULATIONAHA.112.000855
PMCID: PMC3670187  PMID: 23393010
nitric oxide (NO); cyclic guanosine monophosphate (cGMP); vascular endothelial growth factor (VEGF); left ventricular function; hypertrophy
9.  Recovery of Flow-Mediated Vasodilatation after Repetitive Measurements Is Involved in Early Vascular Impairment: Comparison with Indices of Vascular Tone 
PLoS ONE  2014;9(1):e83977.
In repetitive measurements of flow-mediated dilatation (FMD), the duration of the interval between measurements remains controversial. In this pilot study, we conducted three sequential measurements of low–flow-mediated constriction (L-FMC), FMD and flow-mediated total dilation (FMTD; L-FMC+ FMD) at baseline and intervals of 15 and 60 min in 30 healthy males. FMD15, L-FMC15, and FMTD15 were significantly lower than the respective first measurements, but all indices showed full recovery at 60 min in all subjects. The baseline diameter was slightly increased at 15 min and restored at 60 min, but the maximum diameter, and the baseline and reactive flow velocity unchanged. We examined the relationship between recovery rate of FMTD at 15 min (FMTD-R) and cardio-ankle vascular index (CAVI). Univariate analysis showed moderate correlation between FMTD-R, and CAVI and L-FMC0. Patients were divided according to FMTD-R value; the low-FMTD-R group [below the median value (−26.2%)] included a significantly higher proportion of smokers and higher CAVI values than the high-FMTD-R group. The reproducibility of FMTD and FMTD-R was evaluated in another group of 25 healthy subjects. The range of variation across measurements was 1.1% for FMTD and 4.6% for FMTD-R; with intraclass correlation coefficients of 0.93 and 0.95, respectively. The present study demonstrated blunted recovery of FMD within 15 min, suggesting the need for selection of a more adequate interval between measurements to avoid underestimation of FMD in subsequent measurements. The findings demonstrated the reproducibility of FMTD-R and FMTD measurements, and that FMTD-R might be involved in arterial stiffness and early vascular impairment in the healthy subjects.
doi:10.1371/journal.pone.0083977
PMCID: PMC3879271  PMID: 24392103
10.  Regulation of Apelin and Its Receptor Expression in Adipose Tissues of Obesity Rats with Hypertension and Cultured 3T3-L1 Adipocytes 
Experimental Animals  2014;63(2):257-267.
The apelin/APJ system has been implicated in obesity-related hypertension. We investigated the mechanism responsible for the pathogenesis of obesity-related hypertension with a special focus on the crosstalk between AngII/its type 1 receptor (AT1R) signaling and apelin/APJ expression. Sprague-Dawley rats fed a high-fat (obesity-related hypertension, OH) or normal-fat diet (NF) for 15 weeks were randomly assigned to one of two groups and administered vehicle or perindopril for 4 weeks. Compared to the NF rats, the OH rats showed lower levels of plasma apelin and apelin/APJ mRNAs of perirenal adipose tissues, and these changes were restored by perindopril. Administration of the AT1R antagonist olmesartan resulted in the restoration of the reduction of apelin and APJ expressions induced by AngII for 48 h in 3T3-L1 adipocytes. Among several inhibitors for extracellular signal-regulated kinases 1/2 (ERK1/2) PD98059, p38 mitogen-activated protein kinase (p38MAPK) SB203580 and phosphatidylinositol 3-kinase (PI3K) LY294002, the latter showed an additive effect on AngII-mediated inhibitory effects. In addition, the levels of p-Akt, p-ERK and p38MAPK proteins were decreased by long-term treatment with AngII (120 min), and these changes were restored by Olmesartan. Apelin/APJ appears to be impaired in obesity-related hypertension. The AngII inhibition-mediated beneficial effects are likely attributable, at least in part, to restoration of p38/ERK-dependent apelin/APJ expression in diet-induced obesity-related hypertension.
doi:10.1538/expanim.63.257
PMCID: PMC4160987  PMID: 24770651
angiotensin II; apelin; hypertension; obesity; 3T3-L1 adipocytes
11.  Transcriptional Regulation of an Insulin-Sensitizing Adipokine Adipolin/CTRP12 in Adipocytes by Krüppel-Like Factor 15 
PLoS ONE  2013;8(12):e83183.
Obese states characterized by chronic inflammation are closely linked to the development of metabolic dysfunction. We identified adipolin/CTRP12 as an insulin-sensitizing and anti-inflammatory adipokine. Although obese conditions down-regulate adipolin expression, its molecular mechanism is largely unknown. Here we show that the transcriptional regulator Krüppel-like factor (KLF) 15 is involved in the regulation of adipolin expression in adipocytes. White adipose tissue from diet-induced obese (DIO) mice showed decreased expression of KLF9 and KLF15 among several KLFs, which was accompanied by reduced expression of adipolin. In cultured 3T3L1 adipocytes, treatment with TNFα significantly reduced the mRNA levels of KLF9, KLF15 and adipolin. Adenovirus-mediated overexpression of KLF15 but not KLF9 reversed TNFα-induced reduction of adipolin expression in adipocytes. Conversely, gene targeting ablation of KLF15 attenuated adipolin expression in adipocytes. Expression of KLF15 but not KLF9 enhanced the promoter activity of adipolin in HEK293 cells. Pretreatment of 3T3L1 adipocytes with the JNK inhibitor SP600125, but not p38 MAPK inhibitor SB203580 blocked the inhibitory effects of TNFα on adipolin and KLF15 expression. These data suggest that adipose inflammation under conditions of obesity suppresses adipolin expression via JNK-dependent down-regulation of KLF15 in adipocytes.
doi:10.1371/journal.pone.0083183
PMCID: PMC3865152  PMID: 24358263
12.  Mechanisms With Clinical Implications for Atrial Fibrillation–Associated Remodeling: Cathepsin K Expression, Regulation, and Therapeutic Target and Biomarker 
Background
The cysteine protease cathepsin K (CatK) has been implicated in the pathogenesis of cardiovascular disease. We sought to determine the link between atrial fibrillation (AF) and plasma CatK levels and to investigate the expression of and therapeutic target for CatK in vivo and in vitro.
Methods and Results
Plasma CatK and extracellular matrix protein peptides (intact procollagen type I of N‐terminal propeptide; carboxyl‐terminal telopeptide of type I collagen [ICTP]) were measured in 209 consecutive patients with AF (paroxysmal AF, 146; persistent AF, 63) and 112 control subjects. In addition, the regulation of CatK expression was investigated in vivo and vitro. Patients with AF had higher plasma CatK and ICTP levels than did control subjects. Patients with persistent AF had higher levels of plasma CatK and ICTP than did patients with paroxysmal AF. CatK was correlated with ICTP concentration and left atrial diameter in all subjects. In rabbits, superoxide production, CatK activity, fibrosis, and the levels of atrial tissue angiotensin II, angiotensin type 1 receptor, gp91phox, phospho‐p38 mitogen‐activated protein kinase, and CatK were greater in those with tachypacing‐induced AF than in controls, and these changes were reversed with angiotensin type 1 receptor antagonist. Olmesartan and mitogen‐activated protein kinase inhibitor decreased the CatK expression induced by angiotensin II in rat neonatal myocytes.
Conclusions
These data indicated that increased plasma CatK levels are linked with the presence of AF. Angiotensin type 1 receptor antagonist appears to be effective in alleviating atrial fibrosis in a rabbit AF model, partly reducing angiotensin type 1 receptor‐p38mitogen‐activated protein kinase‐dependent and ‐independent CatK activation, thus preventing AF.
doi:10.1161/JAHA.113.000503
PMCID: PMC3886768  PMID: 24342995
angiotensin type 1 receptor; atrial fibrillation; cathepsin K; extracellular matrix; mitogen‐activated protein kinase
13.  A combination of anatomical and functional evaluations improves the prediction of cardiac event in patients with coronary artery bypass 
BMJ Open  2013;3(11):e003474.
Objective
To study the usefulness of combined risk stratification of coronary CT angiography (CTA) and myocardial perfusion imaging (MPI) in patients with previous coronary-artery-bypass grafting (CABG).
Design
A retrospective, observational, single centre study.
Setting and patients
204 patients (84.3% men, mean age 68.7±7.6) undergoing CTA and MPI.
Main outcome measures
CTA defined unprotected coronary territories (UCT; 0, 1, 2 or 3) by evaluating the number of significant stenoses which were defined as the left main trunk ≥50% diameter stenosis, other native vessel stenosis ≥70% or graft stenosis ≥70%. Using a cut-off value with receiver-operating characteristics analysis, all patients were divided into four groups: group A (UCT=0, summed stress score (SSS)<4), group B (UCT≥1, SSS<4), group C (UCT=0, SSS≥4) and group D (UCT≥1, SSS≥4).
Results
Cardiac events, as a composite end point including cardiac death, non-fatal myocardial infarction, unstable angina requiring revascularisation and heart-failure hospitalisation, were observed in 27 patients for a median follow-up of 27.5 months. The annual event rates were 1.1%, 2%, 5.7% and 12.9% of patients in groups A, B, C and D, respectively (log rank p value <0.0001). Adding UCT or SSS to a model with significant clinical factors including left ventricular ejection fraction, time since CABG and Euro SCORE II improved the prediction of events, while adding UCT and SSS to the model improved it greatly with increasing C-index, net reclassification improvement and integrated discrimination improvement.
Conclusions
The combination of anatomical and functional evaluations non-invasively enhances the predictive accuracy of cardiac events in patients with CABG.
doi:10.1136/bmjopen-2013-003474
PMCID: PMC3831107  PMID: 24220113
14.  Pitavastatin-induced angiogenesis and arteriogenesis is mediated by Notch1 in a murine hindlimb ischemia model without induction of VEGF 
Notch signaling is reported to regulate angiogenesis, interacting with vascular endothelial growth factor (VEGF) signaling. HMG CoA reductase inhibitors (statins) also alter Notch signaling in vascular cells, but the mechanism and involvement of Notch and VEGF signaling in statin-mediated angiogenesis remain unclear. Here, we examined how statins activate the endothelial Notch1, and promote angiogenesis and arteriogenesis. We examined blood flow recovery after hindlimb ischemia in wild-type (WT) and Notch1 mutant mice treated with or without pitavastatin (3 mg/kg/day, p.o.). Although VEGF induction was not altered in ischemic limbs, pitavastatin promoted blood flow recovery in ischemic limbs in control mice but not in Notch1 mutant mice. Furthermore, pitavastatin induced endothelial ephrinB2 downstream of Notch1 and increased the density of both capillaries and arterioles in the ischemic limbs of WT but not of Notch1 mutant mice. Pitavastatin (100 nmol/l) rapidly activated γ-secretase and Notch1 in human umbilical vein endothelial cells without VEGF induction, which was suppressed by pharmacological inhibition and knockdown of Akt. Pitavastatin also augmented endothelial proliferation and tube formation on Matrigel, which were suppressed by either γ-secretase inhibition or knockdown of Notch1. Pitavastatin-induced microvascular sprouting was also impaired in Notch1 mutant aortic explants. Taken together, pitavastatin activates Notch1 through Akt-dependent stimulation of γ-secretase in endothelial cells, and thereby increases vasculogenesis without VEGF induction.
doi:10.1038/labinvest.2011.5
PMCID: PMC3807100  PMID: 21301413
Akt; angio/arteriogenesis; endothelial function; Notch signal pathway; statins
15.  Adiponectin‐Mediated Modulation of Lymphatic Vessel Formation and Lymphedema 
Background
Obesity is linked with an increased risk of lymphedema, which is a serious clinical problem. Adiponectin is a circulating adipokine that is down‐regulated in obese states. We investigated the effects of adiponectin on lymphatic vessel formation in a model of lymphedema and dissected its mechanisms.
Methods and Results
A mouse model of lymphedema was created via ablation of tail surface lymphatic network. Adiponectin‐knockout mice showed the greater diameter of the injured tail compared with wild‐type mice, which was associated with lower numbers of lymphatic endothelial cells (LECs). Systemic delivery of adiponectin reduced the thickness of the injured tail and enhanced LEC formation in wild‐type and adiponectin‐knockout mice. Adiponectin administration also improved the edema of injured tails in obese KKAy mice. Treatment with adiponectin protein stimulated the differentiation of human LECs into tubelike structures and increased LEC viability. Adiponectin treatment promoted the phosphorylation of AMP‐activated protein kinase (AMPK), Akt, and endothelial nitric oxide synthase n LECs. Blockade of AMPK or Akt activity abolished adiponectin‐stimulated increase in LEC differentiation and viability and endothelial nitric oxide synthase phosphorylation. Inhibition of AMPK activation also suppressed adiponectin‐induced Akt phosphorylation in LECs. In contrast, inactivation of Akt signaling had no effects on adiponectin‐mediated AMPK phosphorylation in LECs. Furthermore, adiponectin administration did not affect the thickening of the damaged tail in endothelial nitric oxide synthase–knockout mice.
Conclusions
Adiponectin can promote lymphatic vessel formation via activation of AMPK/Akt/endothelial nitric oxide synthase signaling within LECs, thereby leading to amelioration of lymphedema.
doi:10.1161/JAHA.113.000438
PMCID: PMC3835259  PMID: 24052499
adiponectin; Akt; AMPK; eNOS; lymphangiogenesis
16.  Transient Receptor Potential Canonical-3 Channel–Dependent Fibroblast Regulation in Atrial Fibrillation 
Circulation  2012;126(17):2051-2064.
Background
Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)–promoting remodeling. Here, we investigated fibroblast regulation by Ca2+-permeable transient receptor potential canonical-3 (TRPC3) channels.
Methods and Results
Freshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (INSC) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 μmol/L). Pyrazole-3 suppressed angiotensin II-induced Ca2+ influx, proliferation, and α-smooth muscle actin protein expression in fibroblasts. Ca2+ removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, α-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5′ promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression.
Conclusions
TRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca2+ influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.
doi:10.1161/CIRCULATIONAHA.112.121830
PMCID: PMC3675169  PMID: 22992321
arrhythmia; calcium; ion channels; fibrillation; remodeling
17.  Stress Augments Insulin Resistance and Prothrombotic State 
Diabetes  2012;61(6):1552-1561.
Stressors contribute to thrombosis and insulin resistance. Since obesity-related adipose inflammation is also involved in these pathological states, we assumed that stress correlates with adipose inflammation. Male mice were subjected to 2-week intermittent restraint stress. Expression of plasma lipids, monocyte/macrophage markers (CD11b, CD68, and F4/80), proinflammatory cytokines (monocyte chemoattractant protein-1 [MCP-1], tumor necrosis factor-α, and interleukin-6), adiponectin, heat shock protein 70.1 (HSP70.1), and coagulation factors (plasminogen activation inhibitor-1 [PAI-1] and tissue factor [TF]) in blood and inguinal white adipose tissue (WAT) was determined using immunohistochemistry, enzyme-linked immunosorbent assay, and RT-PCR, respectively. Glucose metabolism was assessed by glucose tolerance tests (GTTs) and insulin tolerance tests, and expression of insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) in WAT. To examine effects of MCP-1 blockade, animals were treated with control or neutralizing antibody, or transplanted with control or 7ND (dominant-negative form of MCP-1)-overexpressing adipose-derived stromal cells (ADSCs). Stress increased monocyte accumulation, free fatty acids, proinflammatory cytokine, and HSP70.1 and reduced adiponectin. Adipose stromal cells highly expressed MCP-1. The stress-induced adipose inflammation increased PAI-1 and TF but did not give rise to thrombus formation. Without any changes in GTT, stress worsened insulin sensitivity and decreased IRS-1 and GLUT4 in WAT. Neutralizing antibody and 7ND-ADSCs reversed stress-induced adipose inflammation, procoagulant state, and insulin resistance. Stress evoked adipose inflammation to increase coagulation factors and impair insulin sensitivity through adipose-derived MCP-1.
doi:10.2337/db11-0828
PMCID: PMC3357288  PMID: 22396205
18.  Impact of a Single Intracoronary Administration of Adiponectin on Myocardial Ischemia/Reperfusion Injury in a Pig Model 
Background
Adiponectin plays a protective role in the development of obesity-linked disorders. We demonstrated that adiponectin exerts beneficial actions on acute ischemic injury in mice hearts. However, the effects of adiponectin treatment in large animals and its feasibility in clinical practice have not been investigated. This study investigated the effects of intracoronary administration of adiponectin on myocardial ischemia-reperfusion (I/R) injury in pigs.
Methods and Results
The left anterior descending coronary artery was occluded in pigs for 45 minutes and then reperfused for 24 hours. Recombinant adiponectin protein was given as a bolus intracoronary injection during ischemia. Cardiac functional parameters were measured by a manometer-tipped catheter. Apoptosis was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining. Tumor necrosis factor-α and interleukin-10 transcripts were analyzed by real-time polymerase chain reaction. Serum levels of derivatives of reactive oxygen metabolites and biological antioxidant potential were measured. Adiponectin protein was determined by immunohistochemical and Western blot analyses. Intracoronary administration of adiponectin protein led to a reduction in myocardial infarct size and improvement of left ventricular function in pigs after I/R. Injected adiponectin protein accumulated in the I/R-injured heart. Adiponectin treatment resulted in decreased tumor necrosis factor-α and increased interleukin-10 mRNA levels in the myocardium after I/R. Adiponectin-treated pigs had reduced apoptotic activity in the I/R-injured heart and showed increased biological antioxidant potential levels and decreased derivatives of reactive oxygen metabolite levels in the blood stream after I/R.
Conclusions
These data suggest that adiponectin protects against I/R injury in a preclinical pig model through its ability to suppress inflammation, apoptosis, and oxidative stress. Administration of intracoronary adiponectin could be a useful adjunctive therapy for acute myocardial infarction.
doi:10.1161/CIRCINTERVENTIONS.109.872044
PMCID: PMC3668696  PMID: 20332381
adiponectin; myocardial infarction; reperfusion
19.  iPS cell sheets created by a novel magnetite tissue engineering method for reparative angiogenesis 
Scientific Reports  2013;3:1418.
Angiogenic cell therapy represents a novel strategy for ischemic diseases, but some patients show poor responses. We investigated the therapeutic potential of an induced pluripotent stem (iPS) cell sheet created by a novel magnetite tissue engineering technology (Mag-TE) for reparative angiogenesis. Mouse iPS cell-derived Flk-1+ cells were incubated with magnetic nanoparticle-containing liposomes (MCLs). MCL-labeled Flk-1+ cells were mixed with diluted extracellular matrix (ECM) precursor and a magnet was placed on the reverse side. Magnetized Flk-1+ cells formed multi-layered cell sheets according to magnetic force. Implantation of the Flk-1+ cell sheet accelerated revascularization of ischemic hindlimbs relative to the contralateral limbs in nude mice as measured by laser Doppler blood flow and capillary density analyses. The Flk-1+ cell sheet also increased the expressions of VEGF and bFGF in ischemic tissue. iPS cell-derived Flk-1+ cell sheets created by this novel Mag-TE method represent a promising new modality for therapeutic angiogenesis.
doi:10.1038/srep01418
PMCID: PMC3593218  PMID: 23475393
20.  Therapeutic Impact of Follistatin-Like 1 on Myocardial Ischemic Injury in Preclinical Models 
Circulation  2012;126(14):1728-1738.
Background
Acute coronary syndrome is a leading cause of death in developed countries. Follistatin-like 1 (FSTL1) is a myocyte-derived secreted protein that is upregulated in the heart in response to ischemic insult. Here, we investigated the therapeutic impact of FSTL1 on acute cardiac injury in small and large preclinical animal models of ischemia/reperfusion and dissected its molecular mechanism.
Methods and Results
Administration of human FSTL1 protein significantly attenuated myocardial infarct size in a mouse or pig model of ischemia/reperfusion, which was associated with a reduction of apoptosis and inflammatory responses in the ischemic heart. Administration of FSTL1 enhanced the phosphorylation of AMP-activated protein kinase in the ischemia/reperfusion–injured heart. In cultured cardiac myocytes, FSTL1 suppressed apoptosis in response to hypoxia/reoxygenation and lipopolysaccharide-stimulated expression of proinflammatory genes through its ability to activate AMP-activated protein kinase. Ischemia/reperfusion led to enhancement of bone morphogenetic protein-4 expression and Smad1/5/8 phosphorylation in the heart, and FSTL1 suppressed the increased phosphorylation of Smad1/5/8 in ischemic myocardium. Treating cardiac myocytes with FSTL1 abolished the bone morphogenetic protein-4 –stimulated increase in apoptosis, Smad1/5/8 phosphorylation, and proinflammatory gene expression. In cultured macrophages, FSTL1 diminished lipopolysaccharide-stimulated expression of proinflammatory genes via activation of AMP-activated protein kinase and abolished bone morphogenetic protein-4 – dependent induction of proinflammatory mediators.
Conclusions
Our data indicate that FSTL1 can prevent myocardial ischemia/reperfusion injury by inhibiting apoptosis and inflammatory response through modulation of AMP-activated protein kinase– and bone morphogenetic protein-4 – dependent mechanisms, suggesting that FSTL1 could represent a novel therapeutic target for post-myocardial infarction, acute coronary syndrome.
doi:10.1161/CIRCULATIONAHA.112.115089
PMCID: PMC3548325  PMID: 22929303
apoptosis; inflammation; ischemia; myocytes; cardiac; reperfusion
21.  Effects of Aged Garlic Extract on Left Ventricular Diastolic Function and Fibrosis in a Rat Hypertension Model 
Experimental Animals  2013;62(4):305-310.
Daily consumption of garlic is known to lower the risk of hypertension and ischemic heart disease. In this study, we examined whether aged garlic extract (AGE) prevents hypertension and the progression of compensated left ventricular (LV) hypertrophy in Dahl salt-sensitive (DS) rats. DS rats were randomly divided into three groups: those fed an 8% NaCl diet until 18 weeks of age (8% NaCl group), those additionally treated with AGE (8% NaCl + AGE group), and control rats maintained on a diet containing 0.3% NaCl until 18 weeks of age (0.3% NaCl group). AGE was administered orally by gastric gavage once a day until 18 weeks of age. LV mass was significantly higher in the 8% NaCl + AGE group than in the 0.3% NaCl group at 18 weeks of age, but significantly lower in the 8% NaCl + AGE group than in the 8% NaCl group. No significant differences were observed in systolic blood pressure (SBP) between the 8% NaCl and 8% NaCl + AGE groups at 12 and 18 weeks of age. LV end-diastolic pressure and pressure half-time at 12 and 18 weeks of age were significantly lower in the 8% NaCl + AGE group compared with the 8% NaCl group. AGE significantly reduced LV interstitial fibrosis at 12 and 18 weeks of age. Chronic AGE intake attenuated LV diastolic dysfunction and fibrosis without significantly decreasing SBP in hypertensive DS rats.
doi:10.1538/expanim.62.305
PMCID: PMC4160953  PMID: 24172194
aged garlic extract; blood pressure; hypertension; left ventricular function; left ventricular hypertrophy
22.  Effects of Carperitide on Contrast-Induced Acute Kidney Injury with a Minimum Volume of Contrast in Chronic Kidney Disease Patients 
Nephron Extra  2012;2(1):303-310.
Background/Aims
Although contrast-induced acute kidney injury (CIAKI) is a major complication associated with angiography, the prophylaxis is not well established. Use of a low dose of carperitide for preventing CIAKI remains controversial. We examined the protective effect of carperitide on CIAKI after coronary angiography with a small contrast volume in chronic kidney disease (CKD) patients with coronary artery disease.
Methods
We randomly assigned 112 consecutive patients to a carperitide or a control group. The contrast volume was kept under 150 ml. The primary endpoint was the incidence of CIAKI defined by a serum creatinine of ≥25% or a serum creatinine of ≥0.5 mg/dl from baseline within 48 h. The secondary endpoint was a change in renal function at 1 week after the procedure. Results: The baseline characteristics and contrast volumes (carperitide group: 67.4 ± 38.2 ml vs. control group: 64.8 ± 20.5 ml, p = 0.661) were comparable in the two groups. The incidence of CIAKI was similar in the two groups (carperitide group: 8.5% vs. control group: 5.7%, p = 0.564). A multivariate analysis revealed that a hypotension ≥20 mm Hg was a significant predictor of developing CIAKI in the carperitide group (p = 0.015). The incidence of CIAKI in the carperitide group without hypotension was rare, but not significantly different (carperitide group: 2.4% vs. control group: 5.7%, p = 0.432). Conclusions: This study indicated that the use of a small contrast volume suppressed the incidence of CIAKI and that carperitide had no prophylactic effect against CIAKI. Our results also revealed the impact of hypotension on the development of CIAKI in the carperitide group.
doi:10.1159/000345483
PMCID: PMC3551384  PMID: 23341832
Contrast-induced acute kidney injury; Carperitide; Chronic kidney disease; Contrast medium volume
23.  The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration 
Molecular Biology of the Cell  2012;23(13):2593-2604.
Polarization in motile cells requires the coordination of several key signaling molecules, including RhoA small GTPases and phosphoinositides. It is found that SHIP2 interacts with RhoA in a GTP-dependent manner and this interaction is required for proper localization of PI(3,4,5)P3 and regulation of cell polarization and migration.
Cell migration is essential for various physiological and pathological processes. Polarization in motile cells requires the coordination of several key signaling molecules, including RhoA small GTPases and phosphoinositides. Although RhoA participates in a front–rear polarization in migrating cells, little is known about the functional interaction between RhoA and lipid turnover. We find here that src-homology 2–containing inositol-5-phosphatase 2 (SHIP2) interacts with RhoA in a GTP-dependent manner. The association between SHIP2 and RhoA is observed in spreading and migrating U251 glioma cells. The depletion of SHIP2 attenuates cell polarization and migration, which is rescued by wild-type SHIP2 but not by a mutant defective in RhoA binding. In addition, the depletion of SHIP2 impairs the proper localization of phosphatidylinositol 3,4,5-trisphosphate, which is not restored by a mutant defective in RhoA binding. These results suggest that RhoA associates with SHIP2 to regulate cell polarization and migration.
doi:10.1091/mbc.E11-11-0958
PMCID: PMC3386222  PMID: 22593208
24.  Combinational risk factors of metabolic syndrome identified by fuzzy neural network analysis of health-check data 
Background
Lifestyle-related diseases represented by metabolic syndrome develop as results of complex interaction. By using health check-up data from two large studies collected during a long-term follow-up, we searched for risk factors associated with the development of metabolic syndrome.
Methods
In our original study, we selected 77 case subjects who developed metabolic syndrome during the follow-up and 152 healthy control subjects who were free of lifestyle-related risk components from among 1803 Japanese male employees. In a replication study, we selected 2196 case subjects and 2196 healthy control subjects from among 31343 other Japanese male employees. By means of a bioinformatics approach using a fuzzy neural network (FNN), we searched any significant combinations that are associated with MetS. To ensure that the risk combination selected by FNN analysis was statistically reliable, we performed logistic regression analysis including adjustment.
Results
We selected a combination of an elevated level of γ-glutamyltranspeptidase (γ-GTP) and an elevated white blood cell (WBC) count as the most significant combination of risk factors for the development of metabolic syndrome. The FNN also identified the same tendency in a replication study. The clinical characteristics of γ-GTP level and WBC count were statistically significant even after adjustment, confirming that the results obtained from the fuzzy neural network are reasonable. Correlation ratio showed that an elevated level of γ-GTP is associated with habitual drinking of alcohol and a high WBC count is associated with habitual smoking.
Conclusions
This result obtained by fuzzy neural network analysis of health check-up data from large long-term studies can be useful in providing a personalized novel diagnostic and therapeutic method involving the γ-GTP level and the WBC count.
doi:10.1186/1472-6947-12-80
PMCID: PMC3469424  PMID: 22853735
Data mining; Combinational risk factor; Fuzzy neural network; Glutamyltranspeptidase; Lifestyle disease; Personalized diagnostic method; White blood cell
25.  Therapeutic Lymphangiogenesis With Implantation of Adipose‐Derived Regenerative Cells 
Background
Lymphedema is one of the serious clinical problems that can occur after surgical resection of malignant tumors such as breast cancer or intra‐pelvic cancers. However, no effective treatment options exist at present. Here, we report that implantation of adipose‐derived regenerative cells (ADRCs) can induce lymphangiogenesis in a mouse model of reparative lymphedema.
Methods and Results
ADRCs were isolated from C57BL/6J mice. To examine the therapeutic efficacy of ADRC implantation in vivo, we established a new mouse model of tail lymphedema. Lymphedema was improved significantly by local injection of ADRCs (P<0.05). Histological analysis revealed that lymphatic capillary density was greater in the ADRC group than in the phosphate‐buffered saline control group (P<0.01). Tissue expression of vascular endothelial growth factor C mRNA and plasma levels of vascular endothelial growth factor C were greater in the ADRC group than in the control group (P<0.01 and P<0.05, respectively). ADRCs released vascular endothelial growth factor C, which directly stimulated lymphangiogenesis. Implantation of ADRCs also enhanced recruitment of bone marrow–derived M2 macrophages, which served as lymphatic endothelial progenitor cells.
Conclusions
Implantation of autologous ADRCs could be a useful treatment option for patients with severe lymphedema via mediation of lymphangiogenesis. (J Am Heart Assoc. 2012;1:e000877 doi: 10.1161/JAHA.112.000877.)
doi:10.1161/JAHA.112.000877
PMCID: PMC3487362  PMID: 23130156
adipose tissue; adipose‐derived regenerative cells; lymphedema; lymphangiogenesis; vascular endothelial growth factor C; macrophages

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