基質金屬蛋白脢(MMPs)與金屬蛋白脢組織抑制因子(TIMPs)的表現調節與活化之交互關聯性及其在左心室重塑病程中的病理機制

Abstract

心肌梗塞（myocardial infarction; MI）後發生的左心室重塑（left ventricular remodeling; LV remodeling）病程將導致患者心臟衰竭（heart failure; HF），甚至死亡。近二十年來， 研究者已知細胞間基質（extracellular matrix; ECM）的失序是LV remodeling 病理機轉中 的一個重要因素。基質金屬蛋白.（Matrix metalloproteinases; MMPs）是一群可降解組 織中ECM 的蛋白質分解酵素，而不正常ECM 的降解與許多疾病的病理生理狀況有關。 此外， 組織中存在一類內源性金屬蛋白.組織抑制因子（ tissue inhibitors of metalloproteinases; TIMPs），它們精密調控著MMPs 的活性。因此，一旦體內發生MMP 與TIMP 基因表現或酵素活化水平上的調控失衡，將導致組織中MMP 活性異常提昇， 就會誘發MI 傷害、HF 及擴張性心肌病（dilated cardiomyopathy; DCM）等等。儘管影 響ECM 的正常代謝與疾病發生關係密切，但是MMPs、TIMPs 及其調節因子間的交互 作用與在MI 或HF 病程的發生機制至今仍存在許多疑團，等待探索。 在本計畫中，我們針對解決上述疑團，擬訂三個研究策略。研究策略一，我們將構 築MMP/TIMP .promoter activity 的載體（ vector ）， 包括MMP-2/TIMP-2 與 MMP-9/TIMP-4 兩個載體，其分別將在MMP 與TIMP 基因起動子（promoter，約1.5 ~ 2 kb 長度）後端聯接報導基因（reportor）綠色螢光蛋白（green fluorescent protein; GFP） 與紅色螢光蛋白（red fluorescent protein; dsRed）。所建構的載體將利用DNA-微脂粒 （liposome）方法傳送入培養的心臟成纖維母細胞（myofibroblast）株H9c2 或由大鼠初 代培養的心臟成纖維母細胞中，用於探討在TGF-β、TNF-α、angiotensin peptides 或 hypoxia 處理下，細胞中MMP 與TIMP 基因被調節表現的交互相關性，而這些都是已知 會影響細胞啟動與ECM 代謝有關的訊號傳遞途徑（signaling pathways）的因子。研究 策略二，建構一ECM、MMPs、TIMPs、與其相關基因之寡核.酸（oligonucleotide，65 ~ 70 mer）的基因晶片CardioMP Gene Chip，此晶片將用於經TGF-β、TNF-α、angiotensin peptides 或hypoxia 處理之細胞（研究策略一），及MI 或衰竭心臟組織中ECM、MMPs、 TIMPs 及其相關基因表現的全貌與態樣（研究策略三）。CardioMP Gene Chip 將採用Cy3 與Cy5 .螢光競爭性雜交（competitive hybridization）的玻璃晶片系統行之。研究策略 三，我們將建立冠狀動脈結紮（coronary artery ligation; CAL）與注射adriamycin 方法誘 發大鼠MI 或HF 的動物模式，此將提供我們探討在LV remodeling 病程中心臟ECM、 MMPs、TIMPs 及其相關因子的交互影響網絡。大鼠在接受CAL 手術或密集注射 adriamycin 後的不同時間點，我們將利用zymography 方法測定大鼠血漿中與心臟組織中 各種MMPs 與TIMPs 活性，也將利用CardioMP Gene Chip 分析其心臟組織中各基因表現的態樣。 ECM 的代謝是一個發生在細胞與細胞間的精密且動態地調節過程，MMP/TIMP 間 的平衡狀況一旦受到破壞，ECM 代謝調節就會失序，這是許多心血管主要的病理機轉 與病變特癥。據此，我們的研究將有助於研究者更瞭解此複雜心血管疾病（特別是HF） 的致病機轉，除對心血管疾病的發病機制有進一步認識外，進而能研發出新穎且有效性 的MMPs 抑製藥物或方法。

Left ventricular (LV) remodeling after myocardial infarction (MI) contributes significantly to heart failure (HF) and death. Over last 2 decades, the researchers support that dysregulating extracellular matrix (ECM) plays a major role in the progress of LV remodeling. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes responsible for ECM degradation in tissue during various pathophysiological conditions. In addition, MMP activity is tightly controlled by endogenous inhibitors, named tissue inhibitors of metalloproteinases (TIMPs). Alteration in the balanced between MMP and TIMP in the levels of transcriptional induction and enzymatic activation, resulting in enhanced MMP activity, has been performed to occur in pathological process, such as MI, HF, and dilated cardiomyopathy (DCM). However, interplay and dynamic regulation of MMPs, TIMPs, and their effectors in cardiac ECM remodeling during MI or HF progresses are largely unclear and remain to be explored. In the present project, we propose three research strategies addressing on the above subjects. In Strategy 1, the dual MMP/TIMP promoter activity vectors, MMP-2/TIMP-2 and MMP-9/TIMP-4, will be constructed. In both vectors, green fluorescent protein (GFP) and red fluorescent protein (dsRed) will be constructed downstream of the gene promoter (approximate 1.5 - 2 kb) of MMP and TIMP as reporters, respectively. Two vectors will be transferred into cultured myofibroblast H9c2 cells or rat primary myofibroblasts by DNA-liposome method to explore the interplay of transcriptional regulation of MMP and TIMP under TGF-β, TNF-α, angiotensin peptides, or hypoxia treatments, all of the factors reported as a trigger in signaling pathways of ECM metabolism in myocardium. In Strategy 2, a CardioMP gene chip will be fabricated for studying the global and specific portfolio of ECM, MMPs, TIMPs, and their relative genes that are regulated within MI or failing myocardium (in the Strategy 3), and TGF-β, TNF-α, angiotensin peptides as well as hypoxia treated mrofibroblasts (in the Strategy 1). In the CardioMP Gene Chip, about 100 oligonucleotide probes with 65-70 mer will be designed according to the gene sequences encoding ECM proteins, MMPs, TIMPs and their regulated proteins, synthesized, and spotted onto a glass chip. A competitive hybridization with Cy3 and Cy5 labeled sequences as targets will be performed in this chip system. In Strategy 3, rat MI and HF models by coronary artery ligation (CAL) and adriamycin induction will be established and applied to study the dynamic role and network among ECM, MMPs, TIMPs, and their relative factors during LV remodeling. The profiles of plasma and cardiac MMPs and TIMPs activities (by zymography), and the transcriptional patterns of LV tissues (by CardioMP Gene Chip) sampled from rats will be assayed over a time-course following the CAL procedure and adriamycin injection. Metabolism of ECM is a tightly and dynamically regulated process occurring in and among cells. Dysregulation of ECM metabolism by the MMP/TIMP imbalance plays an important role in cardiovascular pathogenesis. Our studies will be valuable in understanding this modulation and will provide new insight into the pathogenesis of cardiovascular diseases, particular HF, and further illustrate new potential targets or methods for MMPs inhibition.