腎素血管收縮素系統在肋膜腔積液和肺纖維化病程機制中的作用

Abstract

在肺部疾病中，肋膜腔積液是一種常見的醫療問題，主要肇因於肋膜腔有異常液體的蓄積；肋膜腔積液的體積多寡和成份影響了臨床症狀表現，而不同的潛在病因甚至會影響疾病的預後。發生於肋膜間皮細胞表面上的漿膜炎和白血球細胞的趨化作用被認為與胸腔積液的形成機轉有關。此外，局部組織“腎素血管收縮素系統”似乎也在“損傷/修復反應”機轉中發揮了關鍵作用。目前已知“腎素血管收縮素系統”中的angiotensin converting enzyme (ACE)/angiotensin II (Ang II) /AT1R axis 和 angiotensin converting enzyme II (ACE2)/Ang-(1-7)/Mas axis與組織結構的維護、肺部纖維化疾病以及肺間質纖維化的進展關係密切。另外，基質金屬蛋白酶（matrix metalloproteinases; MMPs）和其基質金屬蛋白酶組織抑制因子（tissue inhibitors of MMPs; TIMPs）的表現經常被用來作為細胞外基質（extracellular matrix; ECM）結構重塑的指標。在此研究中，我們探索了ACE/Ang II/AT1R axis、ACE2/Ang- (1-7)/Mas axis、MMPs/TIMPs和肋膜疾病之間的相關性。本項研究還探討了MMP-2和MMP-9在肋膜腔積液患者的基因多態性。 在實驗的第一部分中，我們分析人體肋膜腔積液中ACE/ACE2和MMPs的活性，進而探討ACE/ACE2和MMPs/ TIMPs間的平衡，與肋膜腔積液和肺纖維化的對應關係。 本研究亦針對不同的病患族群，分析其−1059G/A MMP-2、 −735C/T MMP-2和−1562C/T MMP-9的基因多態性。在第二部分中，我們成功建立小鼠肺纖維化模型，用來研究肺纖維化的發病機制，同時也探討ACE/ACE2和MMPs/ TIMPs間的平衡關係。 本研究有幾個結果。首先我們發現：（1）在滲出性肋膜腔積液中，ACE活性顯著升高使得ACE/ACE2的比例明顯升高；（2）在滲出性肋膜腔積液中，MMP-9的活性顯著增加；（3）結核性積液存在較高的ADA、 MMP-9和ACE活性，以及減少ACE2的活性。其次，在漏出性肋膜腔積液、肺炎或腺癌積液中，−1059G/A MMP-2、−735C/T MMP-2和−1562C/T MMP-9的基因多態性並無明顯差別；在homozygous −735CC MMP-2的患者肋膜液中，MMP-2有較高的活性表現。在heterozygous −1562CT MMP-9的患者肋膜液中，MMP-9有較高的活性表現。再者，在動物實驗中，接受bleomycin注射7日後的野生型（WT）小鼠，肺部組織測得較高的ACE和MMP-9活性及TIMP-1濃度，且其組織切片顯示較明顯的白血球細胞浸潤和膠原蛋白堆積。相較於對照組，ACE2基因剔除小鼠於早期（3日）肺部組織中，有較高的MMP-9活性表現和較低的TIMP-1濃度。這些結果顯示，ACE2在bleomycin導致肺纖維化過程中所扮演的重要角色，可能是透過調節MMP-9/TIMP-1的表現來達成。

Pleural effusion is a common medical problem in the chest and involves accumulation of an abnormal amount of pleural fluid in the pleural space. The severity of these symptoms depends on the volume and composition of the fluid, while the final outcome usually depends on the underlying disease responsible for the effusion. Pleural serosal inflammation on the surface of mesothelial cells and recruitment of leukocyte into the pleural cavity were considered related to the pleural effusion formation. In addition, a local tissue-based RAS has also been described and appears to play a key role in injury/repair responses. The known angiotensin converting enzyme (ACE)/angiotensin II (Ang II) /AT1R axis of RAS system and angiotensin converting enzyme II (ACE2)/Ang- (1-7) / Mas receptor axis were closely related to tissue structural maintenance, fibrosis of lung disease, and pulmonary fibrosis progression. Also, the performance of matrix metalloproteinases (MMPs) and its endogenous inhibition factor (tissue inhibitors of MMPs; TIMPs) are often used as the indicators of assessing extracellular matrix (ECM) structural remodeling. In this proposal, we tried to explore the correlation between ACE/Ang II/AT1R axis, ACE2/Ang-(1-7)/Mas axis, MMPs/TIMPs and pleural diseases. This study also investigated the frequency of the polymorphisms of MMP-2 and MMP-9 genes in the patients with pleural effusions. In the first part of experiment, we had analyzed the activity of ACE/ACE2 and MMPs in human pleural effusions in order to explore the connections between ACE/ACE2 and MMPs/TIMPs balance in the pleural effusion and pulmonary fibrosis. The −1059G/A MMP-2, −735C/T MMP-2 and −1562C/T MMP-9 polymorphisms were analyzed in the patients with different disease. In the second part, we successfully established a mouse model of pulmonary fibrosis to investigate the pathogenesis of pulmonary fibrosis, and the balancing activity ACE/ACE2 as well as MMPs/TIMPs. There were several results of our study. First of all, we found (1) a significant higher ACE activity resulting a significantly higher ratio of ACE/ACE2, in the exudative effusions; (2) an increased MMP-9 activity was found in the exudates; (3) in the tuberculous effusions, a significantly higher ADA activity combined with elevated MMP-9 and ACE levels, and reduced ACE2 activity were detected. Second, None of the polymorphisms, including genotypes distribution and allele frequency in −1059G/A MMP-2, −735C/T MMP-2 and −1562C/T MMP-9 was significantly associated with either adenocarcinoma or pneumonia exudates compared with transudates. Pleural MMP-2 activity was higher in homozygous −735CC MMP-2 patients. Pleural MMP-9 activity was higher in heterozygous −1562CT MMP-9 patients. Third, in the animal study, 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. The lung tissue MMP-9 activity is significantly higher in heterozygous, homozygous and hemizygous ACE2 KO mice than Control in early stage (3-day), and TIMP-1 concentration is significantly decreased. Those findings implied that the important role of ACE2 on bleomycin-induced pulmonary fibrosis may be via the regulation of MMP-9/TIMP-1 expression.