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1 第 16 回助成研究発表会要旨集 ( 平成 16 年 7 月 ) 発表番号 65(0343) 食塩感受性高血圧から心不全への移行 および移行後の心不全の表現系を規定する因子の遺伝子発現レベルにおける検索 助成研究者 : 山本一博 ( 大阪大学大学院医学系研究科病態情報内科 ) 共同研究者 : 増山理 ( 兵庫医科大学循環器内科 ) 真野敏昭 ( 大阪大学医学部付属病院循環器内科 ) 心不全には 左室収縮機能低下と左室拡大を伴う収縮不全と 左室拡大や左室収縮機能低下を伴わず左室拡張機能障害を主たる病態とする拡張不全という異なる表現系がある 我々はダール食塩感受性ラットを用いて 同じ遺伝的背景にありながら後天的要因により これら異なる表現系に至る心不全モデルの作成に成功した 本研究では 食塩感受性高血圧症において 移行する心不全の表現系を規定する因子の同定と 食塩感受性高血圧から心不全への移行予防法 心不全の各表現系に対する特異的治療法の確立を目標とする ダール食塩感受性ラットに 7 週齢より高食塩食を投与する拡張不全モデル 8 週齢より高食塩食を投与する収縮不全モデルに加え 正常食で飼育したダール食塩感受性ラットを正常群として用いた 両心不全モデルにおいて細胞外マトリックスの分解をつかさどる matrix metalloproteinase-2(mmp-2) および MMP-9 の遺伝子発現が亢進していた これらの発現亢進は収縮不全モデルにおいて より顕著であった しかし zymography において活性を検討したところ MMP-2 の活性は両心不全モデルに差はなく MMP-9 の活性が収縮不全モデル> 拡張不全モデルとなった 収縮不全モデルでは 左室拡大を生じる前から MMP-2 および MMP-9 の遺伝子発現は亢進していた 降圧効果を示さない低用量のアンジオテンシン変換酵素 (ACE) 阻害薬投与は MMP-2 および MMP-9 の遺伝子発現を抑制し 左室拡大および心不全発症も阻止した 以上より 食塩感受性高血圧から左室拡大を伴う収縮不全に移行する機序は MMP-2 に加え MMP-9 の遺伝子発現ならびに活性亢進が重要な役割を担っていると考えられる また ACE 阻害薬による左室拡大阻止は MMPs の遺伝子発現阻止によりもたらされる可能性が示された 現在 ヒト心不全における ACE 阻害薬の効果も十分とは言いがたく MMPs 特に MMP-9 をターゲットとした治療は 収縮不全に対する新たな治療戦略となる可能性があると考える 一方 食塩感受性高血圧から左室拡大を伴わない拡張不全に移行する機序は MMP-9 の活性亢進前に コラーゲン産生亢進が進み 左室スティフネス亢進が起こることにあると考えられ コラーゲン産生阻止が拡張不全における重要な治療ターゲットと考えられる
2
3 0343 (ACE)
4 ACE mrna gelatin zymography mrna real-time quantitative PCR ACE 5mg/kg/day ACE 5mg/kg/day
5 ACE ACE mrna gelatin zymography mrna real-time quantitative PCR ±SEM one-factor ANOVA LSD Table 1 DHF modelshf model Control [1] mid-wall mid-wall
6 Figure 1 Gene expression of MMPs, TIMPs and collagens in each model *p<0.05 vs Control (19 weeks)#p<0.05 vs Control (26 weeks)p<0.05 vs DHF
7 Figure 1 matrix metalloproteinases (MMPs) MMP-2 MMP-9 MMPs MMPs tissue inhibitor of matrix metalloproteinase (TIMP) TIMPs TIMP-1 TIMP-2 MMPs TIMPs MMPs in vitro gelatin zymography Figure 2 MMP-9 Figure 2. A: Representative gelatinolytic bands at 92 kda (MMP-9). a, b: control rats at 19 weeks. c, d: DHF rats. e, f: control rats at 26 weeks. g, h: SHF rats. i: positive control for MMP-2. B: Summary data of activity in 92 kda gelatinase. C: Representative gelatinolytic bands at 72 kda (MMP-2). a, b: control rats at 19 weeks. c, d: DHF rats. e, f: control rats at 26 weeks. g, h: SHF rats. i: positive control for MMP-9. D: Summary data of activity in 72 kda gelatinase. *: p<0.05 versus control rats at 19 weeks, #: p<0.05 versus control rats at 26 weeks, :p<0.05 versus the DHF model rats
8 MMP-2 in vitro gelatin zymography ACE ACE ACE I III MMP-2 MMP-9 mrna Figure 3TIMP-1 TIMP-2 ACE MMP-2 MMP-9 TIMP-1 TIMP-2 mrna Figure 3. mrna level of MMP-2, MMP-9, ACE TIMP-1 and TIMP-2. *p<0.05 vs 23wN, p<0.05 vs 23w ACE(-), p<0.05 vs 26wN, p<0.05 vs 26w ACE(-) ACE MMPs in vitro gelatin zymography Figure 4 72 kda gelatinase ACE
9 Figure 4. Representative gelatinolytic bands at 72-kDa region (MMP-2) and 92-kDa region (MMP-9). B: Summary data of 72-kDa gelatinase activity. *p<0.05 vs 23wN group, p<0.05 vs 23wACEI(-) group p<0.05 vs 26wN group, p<0.05 vs 26wACEI(-) group at same time point. Values are means ± SEM. MMP-2 MMP-9 MMPs MMP-2MMP-9 MMP-2 MMP-9 MMP-2 MMP-9 MMPs [3] MMPs
10 MMPs MMPs [4-8] MMPs MMPs MMPs ACE MMPs MMPs ACE ACE MMPs MMPs MMPs 20 in vitro gelatin zymography MMP-2 MMP-9 MMP-272 kda gelatinase MMP-992 kda gelatinase MMP-9 MMP-2 MMP-9 Ducharme MMP-9 MMP-2 [9] Longo MMP-2 MMP-9 [10] MMP-2 MMP-9 MMP-9 MMP
11 MMPs MMPs TIMPs TIMP MMP 1:1 [11] MMPs TIMPs MMPs MMPs MMPs MMP-2 MMP-9 ACE MMPs ACE MMPs MMP-9 MMP-9 [1] MMPs TIMPs 1. Doi R, Masuyama T, Yamamoto K, et al. Development of different phenotypes of hypertensive heart failure: systolic versus diastolic failure in Dahl salt-sensitive rats. J. Hypertens. 2000; 18:
12 2. Sakata Y, Masuyama T, Yamamoto K, et al. Renin angiotensin system-dependent hypertrophy as a contributor to heart failure in hypertensive rats: different characteristics from renin angiotensin system-independent hypertrophy. J. Am. Coll. Cardiol. 2001; 37: Yamamoto K, Masuyama T, Sakata Y, et al. Myocardial stiffness is determined by ventricular fibrosis, but not by compensatory or excessive hypertrophy in hypertensive heart. Cardiovasc. Res. 2002; 55: Thomas CV, Coker ML, Zellner JL, et al. Increased matrix metalloproteinase activity and selective upregulation in LV myocardium from patients with end-stage dilated cardiomyopathy. Circulation 1998; 97: Spinale FG, Coker ML, Heung LJ, et al. A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure. Circulation 2000; 102: Iwanaga Y, Aoyama T, Kihara Y, et al. Excessive activation of matrix metalloproteinases coincides with left ventricular remodeling during transition from hypertrphy to heart failure in hypertensive rats. J. Am. Coll. Cardiol. 2002; 39: Reinhardt D, Sigusch HH, Henbe J, et al. Cardiac remodeling in end stage heart failure: upregulation of matrix metalloproteinase (MMP) irrespective of the underlying disease, and evidence for a direct inhibitory effect of ACE inhibitors on MMP. Heart 2002; 88: Mujumdar VS, Smiley LM, Tyagi SC. Activation of matrix metalloproteinase dilates and decreases cardiac tensile strength. Int. J. Cardiol. 2001; 79: Ducharme A, Frantz S, Aikawa M, et al. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J. Clin. Invest. 2000; 106: Longo GM, Xiong W, Greiner TC, et al. Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. J. Clin. Invest. 2002; 110: Mann DL, Spinale FG. Activation of matrix metalloproteinases in the failing human heart. Breaking the tie that binds. Circulation 1998; 98:
13 Determinants of the transition to heart failure or of phenotypes of heart failure in salt-sensitive hypertension Kazuhiro Yamamoto 1, Tohru Masuyama 2, Toshiaki Mano 3 1 Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, 2 Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine, 3 Cardiovascular Division, Osaka University Hospital Summary Systolic heart failure (SHF) is associated with systolic dysfunction and left ventricular (LV) dilatation, and diastolic heart failure (DHF) is not. We developed models of SHF and DHF in Dahl salt-sensitive rats. This study aimed to investigate determinants of the transition to heart failure or of phenotypes of heart failure in salt-sensitive hypertension, and to get insights into new therapeutic strategy for each type of heart failure. We used Dahl salt-sensitive rats fed high salt diet since 7 weeks as DHF model and since 8 weeks as SHF model. Those fed a normal chow served as control. In the both models, gene expression of collagens was enhanced. That of MMP-2 and MMP-9 was enhanced in the DHF rats, and the enhancement was greater in the SHF rats. The gelatin zymography demonstrated that there was no difference in the MMP-2 activity between the DHF and SHF rats, and that the MMP-9 activity was higher in the SHF rats than in the DHF rats. In the SHF rats, increases in mrna levels of MMP-2 and MMP-9 preceded LV dilatation. The administration of angiotensin-converting-enzyme inhibitor (ACEI) at a subdepressor dose inhibited their gene expression and prevented LV dilatation and the transition to overt heart failure. The current study suggests that MMP-9 and MMP-2 work in concert to induce SHF with LV dilatation in salt-sensitive hypertension. The ACEI-induced attenuation of LV dilatation was likely provided through the suppression of MMPs expression. Prognosis of patients with heart failure is still poor even under the treatment with ACEI. This study suggests that MMPs, particularly MMP-9, are a therapeutic target in SHF to further improve the prognosis. The promotion of collagen production may precede the enhancement of MMP-9 gene expression, leading to rapid accumulation of extracellular matrix and the transition to overt DHF. The inhibition of collagen production may be a therapeutic target of DHF
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助成番号 1641 高血圧性臓器障害における鉄取り込み受容体の関与と治療への応用 内藤由朗, 増山理 兵庫医科大学内科学循環器内科 概要 目的 鉄は生体にとって必須の微量元素であるが, 慢性的な鉄過剰状態は酸化ストレスの原因となる 我々はこれまでに食塩感受性高血圧モデル動物であるダール食塩感受性高血圧ラットや 5/6 腎臓摘出慢性腎臓病 (CKD) モデルラットを用いた検討において食塩感受性高血圧の病態形成における鉄の関与,
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