Hiroshi Kusunoki

Osaka University, Suika, Ōsaka, Japan

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Publications (16)54.22 Total impact

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    ABSTRACT: AimThe 9p21 region has been pointed out by the genome-wide association studies as a hot spot for disease-associated variants. Most of the diseases linked with the locus are aging-related conditions, such us cardiovascular disease, diabetes and cancer. Centenarians are known to present a reduced risk and delayed onset for these conditions. Here, we aimed to assess if the 9p21 variants contribute to this protection by possibly altering basic aging mechanisms.Methods We genotyped 15 tag single-nucleotide polymorphisms (SNP) along the CDKN2A/B/ANRIL locus in 1505 individuals. The participants were divided in three groups: centenarians, septuagenarians and young controls. Centenarians were 593 participants (age range 100–116 years, mean 105.9 years), septuagenarians were 434 volunteers aged between 69 and 71 years (mean 70.1 ± 0.9 years) and the 478 young controls were under the age of 50 years (range 14–50 years, mean 41.8 years). We genotyped the SNP rs1333049 in an additional sample of 231 coronary artery disease patients to confirm the 9p21 association.ResultsThe leading coronary artery disease-associated SNP rs1333049 was associated with coronary artery disease; however, none of the 9p21 SNP evaluated in the present study were associated with extreme longevity.Conclusions Our findings suggest that the 9p21 disease-associated polymorphisms do not contribute to the life-long protection from cardiovascular and other age-related diseases observed in centenarians. It is likely that this protection is mediated by mechanisms different from the ones underlying the 9p21 association. Geriatr Gerontol Int 2014; ●●: ●●–●●.
    Geriatrics & Gerontology International 10/2014;
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    ABSTRACT: The paracrine loop involving inflammatory cytokines between adipocytes and macrophages establishes a vicious cycle that aggravates pro-inflammatory changes in adipose tissue. The serum level of hepatocyte growth factor (HGF) is increased in metabolic syndrome, but whether HGF is beneficial or detrimental in inflammatory conditions is unclear. We previously reported that HGF has strong anti-inflammatory effects. We therefore hypothesized that HGF may inhibit chronic inflammation in adipose tissue by inhibiting the vicious cycle between adipocytes and macrophages. We stimulated the macrophage cell line RAW264 with HGF and evaluated pro-inflammatory cytokines. Coculturing differentiated 3T3-L1 adipocytes with RAW264 results in marked upregulation of pro-inflammatory cytokines. We examined whether HGF suppresses the upregulation of pro-inflammatory cytokines in this coculture system. Cardiac-specific HGF transgenic mice (HGF-Tg) were crossed with ApoE KO (knockout) mice, to yield ApoE KO/HGF-Tg mice, which were treated with a high-fat diet (HFD) and received angiotensin (Ang) II. The phenotypes of ApoE KO and ApoE KO/HGF-Tg mice were compared. Treatment with HGF reduced the expression of pro-inflammatory cytokine genes (Tumor necrosis factor-α, monocyte chemoattractant protein-1 and interleukin-6) in RAW264 and the coculture system. The anti-inflammatory effects of HGF were also confirmed in in vivo studies. Macrophage infiltration in epididymal adipose and fatty liver was reduced in ApoE KO/HGF-Tg. Adipocyte diameter was reduced in ApoE KO/HGF-Tg, and the serum adiponectin level was upregulated. These beneficial effects in ApoE KO/HGF-Tg mice under HFD and Ang II infusion were abrogated by an anti-HGF neutralizing antibody. These results suggest that HGF inhibits the vicious cycle of adipocytes and macrophages through the inhibition of macrophage-mediated pro-inflammatory cytokines and upregulation of adiponectin in adipocytes. These favorable effects may suppress chronic inflammation in adipose tissue.Hypertension Research advance online publication, 13 March 2014; doi:10.1038/hr.2014.41.
    Hypertension Research 03/2014; · 2.79 Impact Factor
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    ABSTRACT: "Aldosterone breakthrough" observed in patients receiving long-term treatment with angiotensin blockade is strongly associated with increased risk of left ventricular hypertrophy, poor exercise capacity, refractory proteinuria, and declining glomerular filtration rate through the profibrotic actions of aldosterone. To overcome aldosterone breakthrough, we examined the additional organ-protective actions of irbesartan, because irbesartan is an angiotensin II type 1 receptor (AT1R) blocker (ARB) with peroxisome proliferator-activated receptor (PPAR)γ agonistic effects, which mediate organ-protective effects independent of AT1R blockade. In this study, we examined the organ-protective effects of irbesartan in a salt-sensitive hypertension model using AT1aR knockout mice. Aldosterone and 1% NaCl treatment resulted in a significant increase in severe cardiac and renal fibrosis. Irbesartan, but not losartan, significantly reduced renal fibrosis, glomerular injury through inhibition of macrophage infiltration, epithelial-mesenchymal transition, and oxidative stress. Similarly, cardiac fibrosis and myocyte hypertrophy were decreased by irbesartan, but not losartan, treatment, associated with a significant reduction in oxidative stress. Importantly, anti-hepatocyte growth factor (HGF) neutralizing antibody and a PPARγ antagonist (GW9662) attenuated these organ-protective effects of irbesartan. HGF protein level was increased by irbesartan, especially in the kidney and heart, while GW9662 treatment inhibited the increase in HGF level. In this study, we showed that irbesartan, which has not only AT1aR-blocking effects, but also PPARγ agonistic effects accompanied by HGF expression, inhibited organ damage by aldosterone and salt treatment. Second-generation ARBs such as irbesartan, which has the dual actions of AT1R blockade and PPARγ activation, may have clinical value for the treatment of hypertensive patients with aldosterone breakthrough.
    Journal of the American Heart Association. 01/2013; 2(2):e000103.
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    ABSTRACT: Irbesartan, a partial agonist of peroxisome proliferators activated receptor-γ (PPARγ), has been reported to improve insulin resistance and lipid profile in patients with diabetes mellitus or metabolic syndrome (MS). However, the down effectors of PPARγ have yet to be elucidated. Thus, in this study, we focused on the role of the hepatocyte growth factor (HGF) in the anti-metabolic effects of irbesartan, using apolipoprotein E (ApoE) knockout (KO) mice. ApoE KO mice placed on a high-fat diet (HFD) for 12 weeks were divided into four groups: i) the control (HFD only), ii) the HFD + irbesartan (5 mg/kg/day), iii) the HFD + irbesartan + GW9662, a PPARγ antagonist (0.5 mg/kg/day) and iv) the HFD + irbesartan + anti-HGF neutralizing antibody (200 μg/week). The liver and epididymal adipose tissues were evaluated histologically. Serum adiponectin and HGF levels were also measured by ELISA. Fatty liver (as detected by oil-red O staining) and macrophage infiltration were markedly reduced by irbesartan. Irbesartan treatment also reduced macrophage infiltration into epididymal adipose tissue and hypertrophy of adipocytes. However, these effects of irbesartan were attenuated by GW9662 as well as by anti-HGF neutralizing antibody. Serum and hepatic HGF levels were also markedly increased by irbesartan, whereas GW9662 decreased the HGF level. In conclusion, irbesartan, an angiotensin (Ang) receptor blocker (ARB) and partial agonist of PPARγ (metabosartan), demonstrated a reduction in fatty liver and chronic inflammation, such as macrophage infiltration, beyond its blood pressure-lowering effect. These favorable characteristics of irbesartan might be due to local HGF activation through its partial PPARγ agonistic action, in addition to Ang II blockade. Upregulation of local HGF by irbesartan might provide a novel advantage in a strategy for the prevention and treatment of cardiovascular diseases (CVDs).
    Biomedical reports. 01/2013; 1(1):65-70.
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    ABSTRACT: The purpose of this study was to investigate the effect of hepatocyte growth factor (HGF) on the pathogenesis of cardiac fibrosis induced by pressure overload in mice. Although cardiac fibrosis is attributed to excess pathological deposition of extracellular matrix components, the mechanism remains unclear. Recent reports revealed that α-smooth muscle actin-expressing myofibroblasts are primarily responsible for fibrosis. It is believed that myofibroblasts are differentiated from resident fibroblasts, whereas the transformation of vascular endothelial cells into myofibroblasts, known as endothelial-mesenchymal transition, has been suggested to be intimately associated with perivascular fibrosis. Thus, we hypothesized that HGF prevents cardiac fibrosis by blocking these pathways. We analyzed the pressure-overloaded HGF-transgenic mouse model made by transverse aortic constriction. Human coronary artery endothelial cells and human cardiac fibroblasts were examined in vitro after being treated with transforming growth factor-β1 or angiotensin II with or without HGF. The amount of cardiac fibrosis significantly decreased in pressure-overloaded HGF-transgenic mice compared with pressure-overloaded nontransgenic controls, particularly in the perivascular region. This was accompanied by a reduction in the expression levels of fibrosis-related genes and by significant preservation of echocardiographic measurements of cardiac function in the HGF-transgenic mice (P<0.05). The survival rate 2 months after transverse aortic constriction was higher by 45% (P<0.05). HGF inhibited the differentiation of human coronary artery endothelial cells into myofibroblasts induced by transforming growth factor-β1 and the phenotypic conversion of human cardiac fibroblasts into myofibroblasts. We conclude that HGF reduced cardiac fibrosis by inhibiting endothelial-mesenchymal transition and the transformation of fibroblasts into myofibroblasts.
    Hypertension 03/2012; 59(5):958-65. · 6.87 Impact Factor
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    ABSTRACT: Angiotensin (Ang) II type 1 receptor blockers have demonstrated beneficial effects beyond blood pressure control in the treatment of chronic kidney disease. There is clinical evidence that telmisartan is more effective than losartan in reducing proteinuria in hypertensive patients with diabetic nephropathy, because it is a partial agonist of peroxisome-proliferator activated receptor-γ (PPARγ), as well as an Ang II type 1 receptor blocker (AMADEO Study [A comparison of telMisartan versus losArtan in hypertensive type 2 DiabEtic patients with Overt nephropathy]). In this study, we examined the role of PPARγ activation in the renal protective actions of telmisartan using Ang II type 1 receptor-deficient mice. Renal injury was induced in Ang II type 1 receptor-deficient mice by producing unilateral ureteral obstruction, which exhibited severe renal interstitial fibrosis and inflammation. In these mice, telmisartan prevented hydronephrosis induced by unilateral ureteral obstruction more strongly than did losartan. Importantly, the prevention of renal atrophy and fibrosis by telmisartan was significantly attenuated by GW9662, a PPARγ antagonist. Interestingly, the downstream effector of PPARγ activation by telmisartan is hepatocyte growth factor (HGF), a well-known antifibrotic factor, because renal HGF expression was significantly increased by telmisartan, and a neutralizing antibody against HGF diminished the renal protective action of telmisartan. These beneficial changes by telmisartan were associated with a decrease in the expression of transforming growth factor-β1 and other proinflammatory and profibrotic cytokine genes through PPARγ/HGF activation. Our findings provide evidence of organ protective actions of telmisartan through the PPARγ/HGF pathway, independent of Ang II type 1 receptor blockade. Further development of the next generation of Ang II type 1 receptor blockers with added organ protective actions, such as PPARγ activation, might provide new beneficial drugs to treat renal and cardiovascular diseases.
    Hypertension 02/2012; 59(2):308-16. · 6.87 Impact Factor
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    ABSTRACT: 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. 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. 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. 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. 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.
    Atherosclerosis 11/2011; 220(2):449-55. · 3.71 Impact Factor
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    ABSTRACT: Serotonin (5-hydroxytryptamine, 5-HT) plays a crucial role in peripheral artery disease (PAD) and diabetes mellitus (DM). In these conditions, the balance between the 5-HT2A receptor in smooth muscle cells and the 5-HT1B receptor in endothelial cells (ECs) regulates vascular tonus. In the present study, we focused on the role of 5-HT in endothelial dysfunction using a selective 5-HT2A receptor blocker, sarpogrelate. In human EC, 5-HT markedly stimulated eNOS expression and the phosphorylation of eNOS, Akt and ERK1/2. In addition, a dose-dependent increase in tubule-formation on Matrigel was observed after 5-HT treatment. In contrast, high glucose significantly inhibited tubule formation and eNOS expression through inactivation of Akt, while 5-HT significantly attenuated these actions of high glucose (P<0.01). These results indicate that 5-HT stimulated angiogenesis through activation of Akt in ECs. However, in clinical situations, 5-HT seems to act as the "devil". To examine the role of 5-HT in diabetic PAD, a hindlimb ischemia model was created in diabetic mice. The blood flow ratio of the ischemic to non-ischemic limb was significantly lower in DM mice than in normal mice, while sarpogrelate significantly attenuated the decrease in the blood flow ratio compared to control (P<0.01). Consistently, the decrease in eNOS expression and Akt activity in DM mice was significantly attenuated by sarpogrelate. Overall, the present study demonstrated that selective inhibition of 5-HT2A by sarpogrelate significantly restored ischemic limb blood perfusion in a severe diabetic mouse model through stimulation of the eNOS/Akt pathway via the endothelial 5-HT1B receptor. Enhancement of vasodilation and angiogenesis by sarpogrelate might provide a unique treatment for PAD and DM patients.
    Atherosclerosis 11/2011; 220(2):337-42. · 3.71 Impact Factor
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    ABSTRACT: Both angiotensin II (Ang II) and transforming growth factor (TGF)-β1 are thought to be involved in the progression of chronic kidney disease. In contrast, hepatocyte growth factor (HGF) counteracts the actions of Ang II and TGF-β1. Therefore, in this study, we investigated the molecular mechanisms of how HGF antagonizes the Ang II-TGF-β axis in renal cells. In cultured human mesangial cells, TGF-β1 increased angiotensin type 1 receptor (AT(1)R) mRNA, mainly dependent on the Akt/phosphatidylinositol 3-kinase signaling pathway. Furthermore, TGF-β1 decreased the expression and phosphatase activity of phosphatase and tensin homolog, deleted on chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/Akt pathway. These data revealed positive feedback of the Ang II-TGF-β pathway, because Ang II increased TGF-β expression. In contrast, HGF significantly attenuated the increase in AT(1)R gene expression, and inhibited the decrease in PTEN induced by TGF-β1. Of importance, a PTEN-specific inhibitor significantly attenuated the reduction in TGF-β1-induced AT(1)R expression by HGF. These data suggest that HGF attenuated TGF-β1-induced AT(1)R expression through the PTEN/Akt pathway. To investigate this hypothesis, we performed in vivo experiments in mice with increased circulating levels of HGF produced by transgenically expressing HGF under control of a cardiac-specific transgene (HGF-Tg). In HGF-Tg mice, renal injury and fibrosis were significantly decreased, associated with reduction in AT(1)R expression and increase in PTEN after Ang II infusion, as compared with control mice. Moreover, these renal protective effects were abrogated by a neutralizing antibody against HGF. Thus, the present study demonstrated that HGF counteracts the vicious cycle of Ang II-TGF-β1-AT(1)R, mediating the inhibition of PTEN.
    Hypertension 06/2011; 58(2):190-6. · 6.87 Impact Factor
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    ABSTRACT: Hepatocyte growth factor (HGF) is a mesenchyme-derived pleiotropic factor which regulates cell growth, cell motility, and morphogenesis of various types of cells, and is thus considered a humoral mediator for morphogenic tissue interactions. Although HGF was originally identified as a potent mitogen for hepatocytes, it has also been identified as a member of angiogenic growth factors. Interestingly, the presence of its specific receptor, c-met, is observed in vascular cells and cardiac myocytes. On the other hand, recently, we demonstrated that HGF plasmid DNA transfer significantly improves the size of ulcer in patients with peripheral artery disease (PAD) at Phase III clinical trial, while vascular endothelial growth factor (VEGF) gene therapies for PAD at Phase III have not been succeeded yet. To further investigate this difference between HGF and VEGF, we showed that HGF but not VEGF improves the senescence EPC against oxidative stress through the inhibition of rac1. Moreover, we reported that HGF promotes SHIP-2 translocation from epithelial growth factor receptor (EGFR) to c-Met, and it would protect oxidative stress through EGFR degradation. By this anti-oxidative and anti-senescence effects of HGF would maintain the vessel so long in patients with PAD who receive much oxidative stress in real world. In this report, we discuss a potential therapeutic strategy using HGF in cardiovascular diseases.
    Current Signal Transduction Therapy 04/2011; 6(2):221-227. · 0.45 Impact Factor
  • Journal of The American College of Cardiology - J AMER COLL CARDIOL. 01/2011; 57(14).
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    ABSTRACT: The progression of chronic kidney disease (CKD) is characterized by the persistent accumulation of extracellular matrix. Especially, α-SMA-positive myofibroblasts producing large amounts of TGF-β1 are considered to play a key role in interstitial fibrosis. Although hepatocyte growth factor (HGF) improved renal fibrosis in various models, the molecular mechanisms involved are not yet fully understood. In this study, the molecular mechanisms of the inhibition of fibrosis by HGF was examined using HGF transgenic mice (HGF-Tg) with angiotensin II (Ang II) infusion in 4 weeks models. HGF-Tg mice showed significantly decreased Ang II-induced renal fibrosis and lesser numbers of interstitial myofibroblasts, whereas the antifibrotic effect of HGF was abrogated using HGF-neutralizing antibody. The antifibrotic action in HGF-Tg mice was concordant with a decrease in TGF- β1, collagen type I and IV mRNA expression and an increase in MMP-2 and MMP-9 expression. Furthermore, HGF-Tg mice treated with Ang II showed apoptosis of myofibroblasts. To further investigate the antifibrotic effect of HGF, cultured human mesangial cells were used. HGF induced apoptosis of myofibroblast. Inhibition of the FAK-ERK-MMP signaling cascade by specific inhibitor or siRNA significantly decreased HGF-induced myofibroblast apoptosis. The present study demonstrates that the increase in metalloproteinases through FAK-ERK signaling by HGF promotes myofibroblast apoptosis. Activation of metalloproteinases by HGF in the fibrotic kidney might be considered to attenuate the progression of CKD.
    Journal of Hypertension 12/2010; 28(12):2454-61. · 4.22 Impact Factor
  • Nippon rinsho. Japanese journal of clinical medicine 08/2010; 68 Suppl 8:641-5.
  • Journal of Cardiac Failure - J CARD FAIL. 01/2010; 16(9).
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    ABSTRACT: Neointimal hyperplasia contributes to atherosclerosis and restenosis after percutaneous coronary intervention. Vascular injury in each of these conditions results in the release of mitogenic growth factors and hormones that contribute to pathological vascular smooth muscle cell growth and inflammation. Hepatocyte growth factor (HGF) is known as an antiinflammatory growth factor, although it is downregulated in injured tissue. However, the precise mechanism how HGF reduces inflammation is unclear. To elucidate the mechanism how HGF and its receptor c-Met reduces angiotensin II (Ang II)-induced inflammation. HGF reduced Ang II-induced vascular smooth muscle cell growth and inflammation by controlling translocation of SHIP2 (Src homology domain 2-containing inositol 5'-phosphatase 2), which led to Ang II-dependent degradation of epithelial growth factor receptor. Moreover, the present study also revealed a preventive effect of HGF on atherosclerotic change in an Ang II infusion and cuff HGF transgenic mouse model. These data suggest that the HGF/c-Met system might regulate extrinsic factor signaling that maintains the homeostasis of organs.
    Circulation Research 09/2009; 105(7):667-75, 13 p following 675. · 11.86 Impact Factor
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    ABSTRACT: Although both hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) are potent angiogenic growth factors in animal models of ischemia, their characteristics are not the same in animal experiments and clinical trials. To elucidate the discrepancy between HGF and VEGF, we compared the effects of HGF and VEGF on endothelial progenitor cells under angiotensin II stimulation, which is a well-known risk factor for atherosclerosis. Here, we demonstrated that HGF, but not VEGF, attenuated angiotensin II-induced senescence of endothelial progenitor cells through a reduction of oxidative stress by inhibition of the phosphatidylinositol-3,4,5-triphosphate/rac1 pathway. Potent induction of neovascularization of endothelial progenitor cells by HGF, but not VEGF, under angiotensin II was also confirmed by in vivo experiments using several models, including HGF transgenic mice.
    Hypertension 01/2009; 53(1):77-82. · 6.87 Impact Factor