第二型血管收縮素轉換酶和基質金屬蛋白酶的活性與肺部疾病之相關性

Abstract

在腎素-血管收縮素系統中（Renin-angiotensin system; RAS），已知血管收縮素轉化酶（angiotensin-converting enzyme; ACE）/血管收縮素II（angiotensin II; Ang II）途徑（ACE/Ang II axis）與許多肺部疾病有關，但在RAS系統中的另一個ACE類似酵素，第二型血管收縮素轉化酶（angiotensin-converting enzyme II; ACE2）的生理功能卻較少被探討，ACE2可將Ang II水解成血管收縮素1-7（angiotensin 1-7; Ang 1-7），而Ang 1-7被認為有拮抗Ang II的功能。在我們過去研究結果中顯示，ACE2/Ang 1-7 axis的異常與心臟組織纖維化病程有關，而此與基質金屬蛋白酶（matrix metalloproteinases; MMPs）和基質金屬蛋白酶組織抑制因子（tissue inhibitors of MMPs; TIMPs）的調控失衡有關。 肺部疾病中常見的肋膜積液（pleural effusion）是指肋膜腔中積存過量的液體，為一種臨床上常見的病徵，其形成機制主要為心臟衰竭、炎症、惡性腫瘤、肺結核等病症所造成不同程度的病程。過去的研究中顯示肋膜積液病程中所誘發免疫反應的發炎機制，會進而導致肋膜腔纖維化。另外肺纖維化，臨床上稱之為原發性肺纖維化（idiopathic pulmonary fibrosis），是一種起因不明，長期漸進發展且不可逆轉的致命性肺部疾病。 在本研究中，我們欲探討肺部ACE2和MMPs/TIMPs平衡調控與肺部疾病和肺部組織纖維化的關係性。本研究的第一部分為探討ACE/ACE2和MMPs的變化，是否與特定之肋膜積液病變有關，進而發展可用於臨床診斷的生物性指標；研究的第二部分為利用博萊酶素（bleomycin）的胸腔注射誘發小鼠肺纖維化，用以探討肺臟組織纖維化病程中，ACE/ACE2與MMPs/TIMPs活性表現的差異性。另外本研究也利用ACE2基因剔除小鼠（ACE2 knockout (KO) mice）探討ACE2在肺纖維化病變病程中所扮演的角色。 在第一部分的臨床肋膜積液研究中，相較於濾出型積液（transudates effusion），在滲出型積液（exudates effusion）中，可測得顯著較高ACE/ACE2之活性比值與MMPs活性。再則，在滲出型積液中，肺結核患者肋膜積液中的ACE/ACE2比值、腺核苷去氨酶（adenosine deaminase; ADA）活性及MMP-9活性均顯著高於肺炎與腺癌病患肋膜積液中所測得之值。本項研究之重要結論為RAS和MMP活性與肋膜積液病變有關，而肋膜積液中ACE/ACE2比值、ADA活性及MMP-9活性可做為肺結核患者之診斷生物標誌。 在第二部分的肺纖維化研究中，我們成功建立胸腔注射1 U/kg bleomycin方式誘發實驗小鼠產生肺部纖維化病症之模式，而在小鼠誘發肺部疾病病程中（bleomycin處理後第3、7及28天），小鼠肺臟組織中所測得的ACE/ACE2與MMP-9/TIMP-1活性有顯著改變，此即實驗小鼠在注射bleomycin後第7天時，其肺部組織中的ACE、MMP-9和TIMP-1皆有顯著上升，且組織切片觀察有顯著白血球的浸潤和膠原蛋白累積的病變；再則於heterozygous、homozygous及hemizygous ACE2 KO小鼠實驗中，結果顯示在缺乏ACE2的小鼠中，於注射bleomycin的早期（第3天）就會使肺組織中的MMP-9活性有顯著表現，以及TIMP-1含量顯著下降。本項研究之重要結論為ACE2在bleomycin誘發肺臟纖維化病程中，扮演的功能與MMP-9/ TIMP-1的表現調節有關。

Angiotensin converting enzyme (ACE)/angiotensin II (Ang II) axis in renin-angiotensin system (RAS) is associated with the development of several pulmonary diseases; however, much less is known about the functions of angiotensin converting enzyme II (ACE2), an ACE homologue that hydrolyses Ang II to angiotensin 1-7 (Ang 1-7), a peptide that exerts the actions opposite to those of Ang II. In our previous studies, we showed that ACE2 dysregulation and unbalanced matrix metalloproteinases (MMPs)/tissue inhibitors of MMPs (TIMPs) are highly associated with the fibrotic damage in cardiovascular diseases. In this project, we aimed to study the molecular mechanism of ACE2 regulation on pulmonary fibrosis by experimental mouse models. We also attempted to elucidate the roles of ACE2/Ang 1-7 axis in pulmonary function and the hypothesis whether part of the antifibrotic effects of ACE2/Ang 1-7 axis is via the balancing regulation of MMPs/TIMPs? Pleural effusion is a common medical problem in the chest and involves accumulation of an abnormal amount of pleural fluid in the pleural space. Several diseases, such as congestive heart failure, liver cirrhosis, tuberculosis, adenocarcinoma and pneumonia, are common diseases that cause pleural effusions, but there is less rapid and accurate diagnostic method for pleural effusion. Recent studies showed that the inflammatory response induced by these chest diseases would cause pleural fibrosis. The major enzymes, ACE and ACE2 in RAS, may be involved in the mechanism of pleural fibrosis and pleural effusion. Another lung disease such as idiopathic pulmonary fibrosis has been known as a chronic, progressive, irreversible, and usually lethal lung disease of unknown cause. In this study, we proposed the goals to explore the relationships between ACE2 and MMPs/TIMPs balance in pulmonary disease and pulmonary fibrosis. The first part of the present studies was to detect ACE/ACE2 and MMPs activities in pleural effusions. It can identify diagnosis based on clinical variables to differentiate from pleural effusions. For the second part of the present studies, we took mice to induce pulmonary fibrosis with bleomycin in lung cavity to investigate the pathogenesis of pulmonary fibrosis, and the balancing activity ACE/ACE2 as well as MMPs/TIMPs. In addition, we used ACE2 knockout (KO) mice to investigate the role of ACE2 in the pathogenesis of pulmonary fibrosis. In the first part, the study of clinical pleural effusions, the ACE/ACE2 ratios and MMPs activity in exudate effusions were significantly higher than in transudate effusions. Furthermore, in exudative effusion, ACE/ACE2 ratio, adenosine deaminase (ADA) and MMP-9 activities in tuberculosis effusions were significantly higher than those of pneumonia effusions and adenocarcinoma effusions. The major results of the part I are that the RAS and MMPs activity are important in abnormal pleural fluid. ACE/ACE2 ratio, ADA activity and MMP-9 activity can be used as biomarkers in the diagnosis of tuberculosis patients. In the second part, we have successfully established the mouse model of pulmonary inflammation and fibrosis induced by 1 U/kg bleomycin injection. The profiling of lung ACE/ACE2 and MMP-9/TIMP-1 activity in the mice challenged with bleomycin at 3-day, 7-day and 28-day after the treatments have been performed. The data showed that ACE, MMP-9 activities and TIMP-1 concentration in the lung tissue were significantly increased in wild-type (WT) mice injected bleomycin at 7-day, and the histological sections showed significant leukocyte infiltration and collagen accumulation. Furthermore, in heterozygous, homozygous and hemizygous ACE2 KO mice, the results showed that the lung tissue MMP-9 activity is significantly higher than Control in early stage (3-day), and TIMP-1 concentration is significantly decreased. The major results of the part II indicate that the important role of ACE2 on bleomycin-induced pulmonary fibrosis may be via the regulation of MMP-9/TIMP-1 expression.