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Estrogen Withdrawal Prolonged Vascular Inflammation and Aggravated Diastolic Function in Pressure-overloaded Female Rats
The 10th Annual Scientific Meeting
1001 Activation of the Prostanoid Receptor (EP4) Suppresses Cardiac Remodeling after Myocardial Ischemia/Reperfusion Injury Through Inhibition of MCP-1 and MMP Activity KEIICHI HISHIKARI1, JUN-ICHI SUZUKI1, MASAHITO OGAWA1, KAZUYA ISOBE1, MITSUAKI ISOBE1, TEISUKE TAKAHASHI2, MICHIHITO ONISHI2, KIYOSHI TAKAYAMA2 1 Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Tokyo, Japan, 2Molecular Biology Laboratory, Taisho Pharmaceutical Co, Saitama, Japan Prostaglandin E2 (PGE2) and its receptor, EP4, are implicated in myocardial ischemia/ reperfusion (I/R) injury. To analyze the role of EP4, we administered EP4 agonist (EP4RAG, n56) or vehicle (PBS, n58) to rats with myocardial I/R injury. After 7 days of reperfusion, I/R rats exerted LV dilatation and contractile dysfunction with myocyte hypertrophy and interstitial fibrosis. EP4RAG significantly reduced infarction area/ischemic myocardium (72.460.7 versus 23.360.6 %; p!0.05) and improved the LV contraction and dilatation compared to that of vehicle (fractional shortening, 23.861.4 versus 48.562.4 %; p!0.05). EP4RAG also attenuated recruitment of inflammatory cells, especially macrophages, and interstitial fibrosis in hearts. Monocyte chemoattractant protein (MCP)-1 and gene expression of TNF-alpha, IL-6, and IL1beta was increased in both nonischemic (ANAR) and ischemic (AAR) myocardium, however Western blot analysis and RNase protection assay showed EP4RAG suppressed these changes. Gelatin zymography revealed EP4RAG significantly reduced MMP-2 and -9 activities in both ANAR and AAR. Chemoattractant assay demonstrated EP4RAG suppressed the migration of macrophages stimulated by IL-1beta (number of cells in 400 fields, 109.669.2 versus 24.662.2 counts; p!0.05) and decreased the production of MCP-1 in the supernatant (587.3655.3 versus 171.5647.5 pg/mL; p!0.05). The data suggest the EP4 agonist is effective for attenuation of I/R injury by suppressing MCP-1 and the infiltration of inflammatory cells, especially macrophages. EP4 agonist is a promising treatment for myocardial I/R injury.
1002 Crucumin, a Natural p300-specific Histone Acetyltransferase Inhibitor, is a Therapeutic Agent that Prevents the Development of Heart Failure in vivo MORIMOTO TATSUYA1, KAWAMURA TERUHISA2, TAKAYA TOMOHIDE2, WADA HIROMICHI2, SUNAGAWA YOICHI2, IKEMOTO MASAKI3, FUJITA MASATOSHI3, KITA TORU1, HASEGAWA KOJI2 1 Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan, 2Division of Translational Reserch, Kyoto Medical Center, National Hospital Organization, Kyoto Japan, 3School of Health Sciences, Faculty of Medicine, Kyoto University, Kyoto, Japan Purpose: One of intrinsic histone acetyltransferases, p300, serves as a coactivator of hypertrophy-responsive transcriptional factors such as a cardiac zinc finger protein GATA-4 and is involved in its hypertrophic stimulus-induced acetylation and DNA binding. Our purpose is to investigate whether a p300- HAT specific inhibitor, curcumin, represses the development of heart failure. Methods and Results: First, we examined effects of curcumin on primary neonatal rat cardiac myocytes in culture. Curcumin (5-10maicroM) repressed phenylephrine-induced hypertrophic responses such as increase in cell size and transactivation of the ANF and beta-MHC promoters. Curcumin also blocked phenylephrine-induced increase in acetylation and DNA binding activity of GATA-4 as well as acetylation of histones. Second, to determine whether curcumin can prevent the development of heart failure in vivo, we have utilized a salt-sensitive Dahl (DS) rat model of hypertension. In this model, left ventricular concentric hypertrophy at the age of 11 weeks is followed by decompensated heart failure at the age of 17 weeks. We randomized 11-week-old DS rats (n556) to oral chronic treatment with curcumin (50 mg/Kg/day) or vehicle. At the age of 17 weeks, curcumin significantly ameliorated the survival rate (curcumin: 76%, vehicle: 44%, p!0.001). Conclusion: A natural compound, curcumin represses hypertrophic responses in cardiac myocytes by perturbing a p300-dependent transcriptional pathway, and prevents the development of hypertension-induced decompensated heart failure in vivo.
1003 Estrogen Withdrawal Prolonged Vascular Inflammation and Aggravated Diastolic Function in Pressure-overloaded Female Rats TAKAHIRO MORI, HISASHI KAI, HIDEO YASUKAWA, HIROSHI KUDO, KIYOKO TAKEMIYA, YUSUKE SUGI, NARIMASA TAKAYAMA, DAISUKE FUKUI, TSUTOMU IMAIZUMI Department of Internal Medicine Division of Cardio-Vascular Medicine, Kurume University, Kurume, Japan Background: The prevalence of diastolic dysfunction is high in hypertensive women, particularly in the post-menopausal state. However, the underlying
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mechanism remains unknown. Recently, we have shown that in male Wistar rats with a supra-renal aortic constriction(AC), pressure overload induces a transient perivascular inflammation, which leads to reactive myocardial fibrosis and the resultant diastolic dysfunction. The aim of this study was to determine whether the estrogen withdrawal would aggravate the AC-induced myocardial fibrosis and the resultant diastolic dysfunction. Methods: Female Wistar rats were randomized to ovariectomy (OVX)þAC group and ShamþAC group receiving bilateral OVX and the sham operation at 6 weekold, respectively, followed by AC at 10 week-old(day0). Results: OVX had no effects on blood pressure elevation at day3, 7, 28 . There were no significant differences in LV weight/body weight ratio and myocyte diameter between the two groups .OVX enhanced the AC-induced myocardial fibrosis and diastolic dysfunction. In ShamþAC rats, AC induced transient myocardial MCP-1 induction and perivascular macrophage infiltration, peaking at day3 and returning to the baseline levels at day7. In OVXþAC rats, the AC-induced MCP-1 induction and macrophage infiltration at day3 were enhanced and remained high levels at day28. Conclusions: The estrogen withdrawal aggravated the pressure overload-induced myocardial fibrosis and diastolic dysfunction by enhancing inflammatory process.
1004 Nuclear Factor-kB Inhibitor Attenuates Hypoxia-Induced Left Ventricular Remodeling in Diabetic Rats AKIRA UKIMURA1, TETSUYA HAYASHI1, CHIKA YAMASHITA2, TATSUHIKO MORI1, YASUSHI KITAURA1 1 The Third Division, Department of Internal Medicine, Osaka Medical College, Takatsuki, Japan, 2Osaka University of Pharmaceutical, Takatsuki, Japan Background: Intermittent hypoxia relevant to sleep apnea might increase cardiac events in diabetic patients. The aim of this study was to examine the role of nuclear factor-kB (NF-kB) in the development of left ventricular (LV) remodeling in diabetic rats exposed to intermittent hypoxia. Methods: Male OLETF rats (n515) at 9 weeks of age were kept under intermittent hypoxia (oxygen, 10.060.5% for 8 hours per day) or normoxia. Rats exposed to hypoxia were treated with vehicle (V) or NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC) 80 mg/kg/day for 3 weeks. Fine structure and percent fibrosis (%Fibrosis) were examined, and oxidative stress was assessed by immunohistochemical expression of 4-hydroxy-2-nonenal protein (4-HNE). The activities of NF-E´»B and MMP-9 was evaluated by gel shift assay and zymography, respectively. Results: Intermittent hypoxia increased right ventricular systolic pressures but did not increase left LV systolic pressures. Intermittent hypoxia significantly increased hypertrophy of cardiomyocytes and %Fibrosis. Moreover, NF-kB and MMP-9 activities, 4-HNE expressions were increased by intermittent hypoxia. Treatment with PDTC preserved fine structure of LV myocardium by suppressing NF-kB activity and 4-HNE expressions. Conclusion: PDTC inhibits NF-kB activity and exhibited cardioprotection in diabetic rats exposed to intermittent hypoxia. This study suggests that NF-kB activation might contribute to the development of LV remodeling in diabetic patients with the sleep apnea syndrome.
1005 Pravastatin Improves Angiotensin II-Induced Cardiac Remodeling ZHUJIE XU, HIROSHI OKAMOTO, MASATOSHI AKINO, HIROYUKI TSUTSUI Department of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Japan In experimental settings, statins have been shown to improve cardiac and endothelial function through the anti-oxidative and anti-inflammatory actions. Some of these effects occur independently of cholesterol lowering and can be explained by the inhibition of isoprenylation of the Rho family. The purpose of this study was to examine the effect of pravastatin on angiotensin II-induced left ventricular remodeling. 8-week-old male wild type mice were subcutaneously infused with saline or angiotensin II using an osmotic mini-pump, and then treated them with either saline or pravastatin. Blood pressure and plasma cholesterol levels showed no marked differences between the Sal/AII and Pra/AII groups. After 28 days, angiotensin II-induced increase in myocyte cross-sectional area, cardiac fibrosis was significantly attenuated by pravastatin treatment. Angiotensin II-induced diastolic dysfunction was preserved by pravastatin administration. Cardiac remodeling-related gene expression of matrix metalloproteinases (MMP)-2, 3 and osteopontin (OPN) was significantly increased by angiotensin II infusion, which was significantly attenuated by pravastatin treatment. Compared with Sal/AII-treated mice, the expression of eNOS significantly increased in pravastatin-treated mice. Pravastatin attenuated cardiac remodeling and improved diastolic dysfunction in a murine model of AII-induced LV hypertrophy. The down-regulation of MMP-2, 3, OPN and the up-regulation of eNOS, may at least in part, contribute to the clinical benefits of statins in the treatment of heart failure.
1001 Activation of the Prostanoid Receptor (EP4) Suppresses Cardiac Remodeling after Myocardial Ischemia/Reperfusion Injury Through Inhibition of MCP-1 and MMP Activity KEIICHI HISHIKARI1, JUN-ICHI SUZUKI1, MASAHITO OGAWA1, KAZUYA ISOBE1, MITSUAKI ISOBE1, TEISUKE TAKAHASHI2, MICHIHITO ONISHI2, KIYOSHI TAKAYAMA2 1 Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Tokyo, Japan, 2Molecular Biology Laboratory, Taisho Pharmaceutical Co, Saitama, Japan Prostaglandin E2 (PGE2) and its receptor, EP4, are implicated in myocardial ischemia/ reperfusion (I/R) injury. To analyze the role of EP4, we administered EP4 agonist (EP4RAG, n56) or vehicle (PBS, n58) to rats with myocardial I/R injury. After 7 days of reperfusion, I/R rats exerted LV dilatation and contractile dysfunction with myocyte hypertrophy and interstitial fibrosis. EP4RAG significantly reduced infarction area/ischemic myocardium (72.460.7 versus 23.360.6 %; p!0.05) and improved the LV contraction and dilatation compared to that of vehicle (fractional shortening, 23.861.4 versus 48.562.4 %; p!0.05). EP4RAG also attenuated recruitment of inflammatory cells, especially macrophages, and interstitial fibrosis in hearts. Monocyte chemoattractant protein (MCP)-1 and gene expression of TNF-alpha, IL-6, and IL1beta was increased in both nonischemic (ANAR) and ischemic (AAR) myocardium, however Western blot analysis and RNase protection assay showed EP4RAG suppressed these changes. Gelatin zymography revealed EP4RAG significantly reduced MMP-2 and -9 activities in both ANAR and AAR. Chemoattractant assay demonstrated EP4RAG suppressed the migration of macrophages stimulated by IL-1beta (number of cells in 400 fields, 109.669.2 versus 24.662.2 counts; p!0.05) and decreased the production of MCP-1 in the supernatant (587.3655.3 versus 171.5647.5 pg/mL; p!0.05). The data suggest the EP4 agonist is effective for attenuation of I/R injury by suppressing MCP-1 and the infiltration of inflammatory cells, especially macrophages. EP4 agonist is a promising treatment for myocardial I/R injury.
1002 Crucumin, a Natural p300-specific Histone Acetyltransferase Inhibitor, is a Therapeutic Agent that Prevents the Development of Heart Failure in vivo MORIMOTO TATSUYA1, KAWAMURA TERUHISA2, TAKAYA TOMOHIDE2, WADA HIROMICHI2, SUNAGAWA YOICHI2, IKEMOTO MASAKI3, FUJITA MASATOSHI3, KITA TORU1, HASEGAWA KOJI2 1 Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan, 2Division of Translational Reserch, Kyoto Medical Center, National Hospital Organization, Kyoto Japan, 3School of Health Sciences, Faculty of Medicine, Kyoto University, Kyoto, Japan Purpose: One of intrinsic histone acetyltransferases, p300, serves as a coactivator of hypertrophy-responsive transcriptional factors such as a cardiac zinc finger protein GATA-4 and is involved in its hypertrophic stimulus-induced acetylation and DNA binding. Our purpose is to investigate whether a p300- HAT specific inhibitor, curcumin, represses the development of heart failure. Methods and Results: First, we examined effects of curcumin on primary neonatal rat cardiac myocytes in culture. Curcumin (5-10maicroM) repressed phenylephrine-induced hypertrophic responses such as increase in cell size and transactivation of the ANF and beta-MHC promoters. Curcumin also blocked phenylephrine-induced increase in acetylation and DNA binding activity of GATA-4 as well as acetylation of histones. Second, to determine whether curcumin can prevent the development of heart failure in vivo, we have utilized a salt-sensitive Dahl (DS) rat model of hypertension. In this model, left ventricular concentric hypertrophy at the age of 11 weeks is followed by decompensated heart failure at the age of 17 weeks. We randomized 11-week-old DS rats (n556) to oral chronic treatment with curcumin (50 mg/Kg/day) or vehicle. At the age of 17 weeks, curcumin significantly ameliorated the survival rate (curcumin: 76%, vehicle: 44%, p!0.001). Conclusion: A natural compound, curcumin represses hypertrophic responses in cardiac myocytes by perturbing a p300-dependent transcriptional pathway, and prevents the development of hypertension-induced decompensated heart failure in vivo.
1003 Estrogen Withdrawal Prolonged Vascular Inflammation and Aggravated Diastolic Function in Pressure-overloaded Female Rats TAKAHIRO MORI, HISASHI KAI, HIDEO YASUKAWA, HIROSHI KUDO, KIYOKO TAKEMIYA, YUSUKE SUGI, NARIMASA TAKAYAMA, DAISUKE FUKUI, TSUTOMU IMAIZUMI Department of Internal Medicine Division of Cardio-Vascular Medicine, Kurume University, Kurume, Japan Background: The prevalence of diastolic dysfunction is high in hypertensive women, particularly in the post-menopausal state. However, the underlying
JHFS
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mechanism remains unknown. Recently, we have shown that in male Wistar rats with a supra-renal aortic constriction(AC), pressure overload induces a transient perivascular inflammation, which leads to reactive myocardial fibrosis and the resultant diastolic dysfunction. The aim of this study was to determine whether the estrogen withdrawal would aggravate the AC-induced myocardial fibrosis and the resultant diastolic dysfunction. Methods: Female Wistar rats were randomized to ovariectomy (OVX)þAC group and ShamþAC group receiving bilateral OVX and the sham operation at 6 weekold, respectively, followed by AC at 10 week-old(day0). Results: OVX had no effects on blood pressure elevation at day3, 7, 28 . There were no significant differences in LV weight/body weight ratio and myocyte diameter between the two groups .OVX enhanced the AC-induced myocardial fibrosis and diastolic dysfunction. In ShamþAC rats, AC induced transient myocardial MCP-1 induction and perivascular macrophage infiltration, peaking at day3 and returning to the baseline levels at day7. In OVXþAC rats, the AC-induced MCP-1 induction and macrophage infiltration at day3 were enhanced and remained high levels at day28. Conclusions: The estrogen withdrawal aggravated the pressure overload-induced myocardial fibrosis and diastolic dysfunction by enhancing inflammatory process.
1004 Nuclear Factor-kB Inhibitor Attenuates Hypoxia-Induced Left Ventricular Remodeling in Diabetic Rats AKIRA UKIMURA1, TETSUYA HAYASHI1, CHIKA YAMASHITA2, TATSUHIKO MORI1, YASUSHI KITAURA1 1 The Third Division, Department of Internal Medicine, Osaka Medical College, Takatsuki, Japan, 2Osaka University of Pharmaceutical, Takatsuki, Japan Background: Intermittent hypoxia relevant to sleep apnea might increase cardiac events in diabetic patients. The aim of this study was to examine the role of nuclear factor-kB (NF-kB) in the development of left ventricular (LV) remodeling in diabetic rats exposed to intermittent hypoxia. Methods: Male OLETF rats (n515) at 9 weeks of age were kept under intermittent hypoxia (oxygen, 10.060.5% for 8 hours per day) or normoxia. Rats exposed to hypoxia were treated with vehicle (V) or NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC) 80 mg/kg/day for 3 weeks. Fine structure and percent fibrosis (%Fibrosis) were examined, and oxidative stress was assessed by immunohistochemical expression of 4-hydroxy-2-nonenal protein (4-HNE). The activities of NF-E´»B and MMP-9 was evaluated by gel shift assay and zymography, respectively. Results: Intermittent hypoxia increased right ventricular systolic pressures but did not increase left LV systolic pressures. Intermittent hypoxia significantly increased hypertrophy of cardiomyocytes and %Fibrosis. Moreover, NF-kB and MMP-9 activities, 4-HNE expressions were increased by intermittent hypoxia. Treatment with PDTC preserved fine structure of LV myocardium by suppressing NF-kB activity and 4-HNE expressions. Conclusion: PDTC inhibits NF-kB activity and exhibited cardioprotection in diabetic rats exposed to intermittent hypoxia. This study suggests that NF-kB activation might contribute to the development of LV remodeling in diabetic patients with the sleep apnea syndrome.
1005 Pravastatin Improves Angiotensin II-Induced Cardiac Remodeling ZHUJIE XU, HIROSHI OKAMOTO, MASATOSHI AKINO, HIROYUKI TSUTSUI Department of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Japan In experimental settings, statins have been shown to improve cardiac and endothelial function through the anti-oxidative and anti-inflammatory actions. Some of these effects occur independently of cholesterol lowering and can be explained by the inhibition of isoprenylation of the Rho family. The purpose of this study was to examine the effect of pravastatin on angiotensin II-induced left ventricular remodeling. 8-week-old male wild type mice were subcutaneously infused with saline or angiotensin II using an osmotic mini-pump, and then treated them with either saline or pravastatin. Blood pressure and plasma cholesterol levels showed no marked differences between the Sal/AII and Pra/AII groups. After 28 days, angiotensin II-induced increase in myocyte cross-sectional area, cardiac fibrosis was significantly attenuated by pravastatin treatment. Angiotensin II-induced diastolic dysfunction was preserved by pravastatin administration. Cardiac remodeling-related gene expression of matrix metalloproteinases (MMP)-2, 3 and osteopontin (OPN) was significantly increased by angiotensin II infusion, which was significantly attenuated by pravastatin treatment. Compared with Sal/AII-treated mice, the expression of eNOS significantly increased in pravastatin-treated mice. Pravastatin attenuated cardiac remodeling and improved diastolic dysfunction in a murine model of AII-induced LV hypertrophy. The down-regulation of MMP-2, 3, OPN and the up-regulation of eNOS, may at least in part, contribute to the clinical benefits of statins in the treatment of heart failure.