血管收縮素II與血管收縮素1-7於人類心臟纖維母細胞中對心臟血管收縮素轉化酶II的表現調節

Interplay of Angiotensin II and Angiotensin 1-7 in the Modulation of Cardiac Angiotensin-Converting Enzyme II of Human Cardiofibroblasts

溫証皓(Cheng-Hao Wen)

指導教授 ： 林志生

國立交通大學/生物科技學院/生物科技系所/碩士(2009年)

https://doi.org/10.6842/NCTU.2009.00363

摘要

腎素-血管收縮素系統 (renin-angiotensin system, RAS) 中的組成要素已經被廣泛使用在作為治療多種不同疾病的藥物標靶。這些治療方式常藉由抑制特定的受器 (receptor) 或其合成酵素來減低血管收縮素II (angiotensin II, Ang II) 的胜肽含量並達到抑制高血壓的效應。在西元兩千年，一個嶄新的酵素被發現，並被命名為血管收縮素轉化酶II (angiotensin-converting enzyme II, ACE2)。ACE2與其耳熟能詳的類似物血管收縮素轉化酶I (ACE) 同樣成為眾所矚目的焦點。與ACE相同的是，ACE2同樣是一種第一型跨膜金屬肽酶 (type I transmembrane metallopeptidase)，並可作為一羧基胜肽水解酶 (carboxypeptidase)，切除特定受質 (substrate) C端的殘基 (residue)，但兩者所切除的residue數目不同。ACE2之所以可以作為一個調節心血管疾病的潛力標靶，是由於其可扮演將Ang II代謝成具有保護血管效用的另一胜肽血管收縮素1-7 (angiotensin 1-7, Ang 1-7)。Ang II和Ang 1-7同為RAS中的重要調控胜肽。
Ang II已經被證實在心臟重塑 (remodeling) 過程中扮演要角，在許多疾病例如心肌梗塞 (myocardial infarction, MI)、心臟衰竭 (heart failure, HF) 或是心房顫動 (atrial fibrillation, AF) 的病理狀態下，都可以同時測得Ang II以及ACE2的高量表現。有研究顯示，心臟內高量表現的ACE2可能參與防止Ang II異常表現所引起的高血壓或心臟纖維化，這些現象指出ACE2直接參與心臟保護的角色，並提供在醫療上可能的新契機。更進一步的證據指出，在心臟衰竭病患的心臟組織中可同時測得高量表現的ACE2以及Ang 1-7，這也顯示了ACE2在心血管疾病中可能是藉由調節Ang II的含量來維持體內的自我平衡。因此，我們提出一個假設，ACE2表現量的提升可能是心臟為抵抗異常高量表現的Ang II所產生的自我保護機制。
在目前的研究中，我們使用人類心臟纖維母細胞 (human cardiofibroblast, HCF) 作為探討Ang II以及Ang 1-7對於ACE2在轉錄及轉譯調節上的重要模型。而當前的實驗結果也證實Ang II可以提高ACE2在人類心臟纖維母細胞的表現量，而此活化機制是經由血管收縮素II第一型受器 (angiotensin II type 1 receptor, AT1R) 進行調控。Ang II所引起的ACE2高量表現更可以被AT1R及其下游諸如菸醯胺腺嘌呤二核酸磷酸氧化酵素 (Nicotinamide adenine dinucleotide phosphate oxidase, NADPH oxidase)、Extracellular signal-regulated kinase - Mitogen-activated protein kinase, ERK讣MAPK的拮抗劑所阻斷，這樣的結果更確立Ang II對於ACE2調控可能經由的訊號傳遞路徑。此外，promoter assay的結果顯示在Ang II的刺激下，ACE2 promoter活性顯著提升，而此提升的效應也可在加入AT1R的阻斷劑纈沙坦 (Valsartan) 後被阻斷，顯示出Ang II參與調控ACE2啟動子 (promoter) 的活性。
除此之外，我們的研究結果更發現Ang 1-7也可以提升ACE2在心臟纖維母細胞的表現量，而ACE2的向上調控則可在加入Mas receptor的抑制劑A779後被阻斷。我們的結果推論，Ang 1-7對於ACE2的調控是經由Mas receptor並可以透過其下游的NADPH oxidase以及ERK-MAPK的訊息路徑調控ACE2的表現。共軛焦螢光顯微鏡的影像結果也更進一步提供Ang II和Ang 1-7對ACE2調控的證據，並呈現出ACE2及AT1R在心臟纖維母細胞的實際分布，而此影像的結果也與先前的發現一致。
簡而言之，據我們的實驗結果證實了Ang II所誘導產生的ACE2可以增加Ang II代謝成Ang 1-7的量，而增量的Ang 1-7又更進一步增強ACE2的表現。根據這樣的結果，我們提出ACE2在心臟調控中具備一正回饋機制 (positive feedback loop)，以維持人體內RAS的穩定平衡。我們的結果也提供產學界一個可發展治療因RAS失常所引起心血管疾病的潛力新標靶。

關鍵字

血管收縮素II ； 血管收縮素1-7 ； 血管收縮素轉化酶II ； 人類心臟纖維母細胞 ； 腎素-血管收縮素系統

並列摘要

Components of renin–angiotensin system (RAS) are well established targets for pharmacological intervention in a variety of disorders. Many such therapies abrogate the effects of the hypertensive and mitogenic peptide, angiotensin II (Ang II), by antagonising its interaction with its receptor, or by inhibiting its formative enzyme, angiotensin-converting enzyme (ACE). At the turn of the millennium, a novel homologous enzyme, termed ACE2, was identified which increasingly shares the limelight with its better-known homologue. In common with ACE, ACE2 is a type I transmembrane metallopeptidase; however, unlike ACE, ACE2 functions as a carboxypeptidase, cleaving a single C-terminal residue from a distinct range of substrates. ACE2 is a potential therapeutic target for the control of cardiovascular disease owing to its key role in the formation of vasoprotective peptides angiotensin 1-7 (Ang 1-7) from Ang II [cleavage from angiotensin I by angiotensin-converting enzyme (ACE)]. Ang II and Ang 1-7 are both critical regulatory peptides in RAS.
Ang II has been documented to play important role in the progression of cardiac remodeling. Elevated Ang II paralleled to cardiac ACE2 upregulation was reported in some pathophysiological conditions, such as myocardial infarction, heart failure and atrial fibrillation. Intracardiac overexpression of ACE2 prevents Ang II induced hypertension and cardiac fibrosis, implicating a direct in vivo cardioprotective role for ACE2, in addition to suggesting possible therapeutic utility. Further evidence for a role of ACE2 in maintaining cardiovascular homeostasis is via Ang II regulation which detected increased ACE2 and Ang 1-7 forming activity in failing human hearts. Hence, we tested the hypothesis that upregulation of ACE2 may provide cardio-protection effects to counteract the elevated Ang II.
In the present study, human cardiofibroblast (HCF) cells were used to test the regulatory effects of Ang II and Ang 1-7 on the ACE2 expression at transcriptional and translational level. The results show that Ang II could upregulate ACE2 expression and this action may modulate through the activation of Ang II type I receptor (AT1R). Ang II-mediated ACE2 upregulation could be blocked by the antagonists of downstream targets of AT1R, NADPH oxidase and ERK讣MAPK cascades. To test the Ang II mediated ACE2 promoter activity, our result showed that human cardiac ACE2 promoter activity was significantly upregulation with Ang II stimulation. Additionally, Ang II-induced ACE2 promoter activity could be abolished when the HCF cells pretreated with Valsartan.
Furthermore, Ang 1-7 also could up-regulate ACE2 expression in the HCF cells and this upregulation could be inhibited by Mas receptor blocker, A779. Our result shows that the Ang 1-7讣depedent ACE2 upregulation is via Mas receptor signaling pathway and even go through the NADPH oxidase and ERK-MAPK cascades. The confocal fluorescence imaging results provide further validation for Ang II讣 and Ang 1-7讣mediated ACE2 expression and an actual presentation of AT1R and ACE2 localization in HCF cells. Additionally, the image data also show the consistent results with our previous data.
In conclusion, our observation implicate that Ang II-induced ACE2 may increase Ang 1-7 formation from Ang II and then the ACE2 expression is further enhanced by the Ang 1-7. According to the results, we proposed a positive feedback-like loop on the cardiac ACE2 regulation for heart to maintain a steady state of RAS. Our results may point out new targets and possibilities for developing novel therapeutic strategies in cardiovascular diseases induced by the dysfunction of RAS.

並列關鍵字

angiotensin II ； angiotensin 1-7 ； angiotensin-converting enzyme II ； human cardiofibroblasts ； renin-angiotensin system

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