Chymase-Mediated MMP Activation in Ishemia Reperfusion Injury

描述(由申请人提供)： 摘要(与以前提交的文件相比，所有变化都在页边标出)缺血和再灌注(I/R)损伤是由于缺血后再灌注期间氧化/炎症应激的急剧增加而引起的，并触发了一连串的病理生理事件。这种损伤最终导致心肌细胞死亡，这些细胞在心肌再灌流前立即存活，即使及时再灌流也会发生，并可能增加梗塞范围[1]。目前，临床上还没有用于预防或减轻急性冠脉综合征患者I/R损伤的药物(1，2)。I/R导致心肌细胞死亡，并与核变性和肌原纤维降解有关。以往的研究表明，I/R后间质基质金属蛋白酶-2(MMP2)和MMP9的活性显著增加(3)。最近的研究表明，心肌细胞内激活的基质金属蛋白酶-2裂解肌原纤维(4-6)和核基质蛋白多聚ADP-核糖聚合酶(PARP)-一种DNA修复酶在I/R期间细胞存活(7)。然而，细胞内MMPs在I/R过程中的调节尚不清楚。在犬I/R模型中，我们报道了间质肥大细胞糜酶在心肌细胞基质金属蛋白酶激活中的重要作用。驻留肥大细胞脱颗粒是I/R的早期事件，并导致糜酶释放到间质(8-10)。除了能够将血管紧张素(Ang I)转化为Ang II(11，12)外，肥大细胞糜酶还能激活间质MMPs(13-18)，并直接降解细胞表面蛋白，如纤维连接蛋白(19)，导致平滑肌细胞(20，21)和心肌细胞(22)死亡。在这项建议中，我们提供了初步的数据，即犬在体I/R后间质糜酶蛋白水解酶活性增加导致心肌细胞核内细胞内MMPs的强烈激活以及DNA损伤和肌球蛋白的丢失。用一种口服活性的乳糜酶抑制剂预处理可显著降低基质金属蛋白酶的活性。我们还发现，I/R增加了心肌细胞核中两个高度丰富的未知MMPs的活性，它们的相对分子质量在100-150kD之间。此外，我们还发现，在成年犬心肌细胞中加入凝乳酶后，层粘连蛋白被直接切割，粘着斑激酶(FAK)去磷酸化，基质金属蛋白酶的释放和肌球蛋白的降解。一种广谱的基质金属蛋白酶抑制剂和EDTA可阻止糜酶诱导的肌球蛋白降解。根据我们在体内和体外的初步数据，我们假设I/R过程中ISF糜酶的增加介导了细胞-基质-表面连接的丢失，破坏了FAK，并通过核基质金属蛋白酶的激活最终导致肌纤维变性。这一假说将在临床相关的犬I/R模型上得到验证，静脉注射糜酶抑制剂(IV)从缺血30min开始，持续到整个I/R期间，急性和慢性口服糜乳酶抑制剂后评估左心功能。目的1.用蛋白质组学和质谱学方法确定I/R是否导致新的心肌细胞核基质金属蛋白酶的激活。新的核MMPs的发现及其调控将为I/R损伤提供潜在的新的分子/蛋白质靶点。从I/R心肌细胞中提取的核蛋白将通过等电聚焦(IEF)分离。离子交换后，琼脂糖凝胶上的蛋白质将用2D酶谱分离。2D凝胶上与酶活性区域相对应的蛋白质斑点将被选择用于通过质谱学鉴定基质金属蛋白酶。目的2.验证ISF糜酶活性升高导致FAK失活及其下游信号通路激活心肌细胞核MMPs的假说。在犬I/R期间，分别从缺血区和非缺血区采集连续活检标本。我们将研究FAK去磷酸化和下游信号通路ERK、JNK、p38MAPK和NF：B激活的时间进程，这些信号通路是已知激活MMPs的。为了以临床相关的方式解决这个问题，在缺血30min开始和整个再灌流过程中，在有和没有静脉注射糜酶抑制剂的情况下，在再灌流之前和之后采集连续的活检样本。在I/R期间，将使用LV阻抗导管来评估LV的舒缩功能。免疫组织化学原位酶谱分析将确定是否可以通过预先静脉注射糜酶抑制剂来阻止核基质金属蛋白酶的激活。目的3.验证在I/R期间静脉注射糜酶抑制剂，然后口服糜酶抑制剂治疗7天可减少损伤，改善左心功能的假说。在我们的闭胸动物模型中，用经皮冠状动脉介入性球囊闭塞1小时，再灌流2小时，闭塞左前叶近端。静脉注射糜酶抑制剂将在缺血30分钟后启动，并持续至再灌流2小时。口服药物将在12小时内开始，持续7天。分别于I/R损伤后2天和7天行磁共振成像(MRI)和组织标记及Gd灌注检查。第2天的T2加权MRI将确定体内的水肿面积，而第7天的Gd+组织标记将确定赋形剂与糜酶抑制剂治疗的犬的体内心肌疤痕和功能。

DESCRIPTION (provided by applicant): Abstract (All changes from the previous submission are marked by line in the margins) Ischemia and reperfusion (I/R) injury results from an acute increase in oxidative/inflammatory stress during reperfusion after ischemia and triggers a cascade of pathophysiological events. The injury culminates in the death of cardiomyocytes that were viable immediately before myocardial reperfusion and occurs despite timely reperfusion and can increase infarct size (1). Currently, there is no drug that is utilized in the clinical arena that prevents or attenuates I/R injury in the patient presenting with acute coronary syndrome (1,2). I/R results in cardiomyocyte death and is associated with nuclear degeneration and myofibrillar degradation. Previous studies showed a significant increase of interstitial matrix metalloproteinase-2 (MMP-2) and MMP-9 activation after I/R (3). Recent studies demonstrated activated MMP-2 within the cardiomyocyte that cleaved myofibrils (4-6) and nuclear matrix protein poly ADP-ribose polymerase (PARP)-a DNA repair enzyme in cell survival during I/R (7). However, the regulation of intracellular MMPs during I/R is not known. Here, in a dog I/R model, we report that interstitial mast cell chymase plays an important role in cardiomyocyte MMP activation. Degranulation of resident mast cells is an early event in I/R and results in chymase release into the interstitium (8- 10). In addition to its ability to convert angiotensin (Ang I) to Ang II (11,12), mast cell chymase activates interstitial MMPs (13-18) and directly degrades cell surface proteins such as fibronectin (19), resulting in smooth muscle cell (20,21) and cardiomyocyte (22) death. In this proposal, we present preliminary data that increased interstitial chymase proteolytic activity after I/R in the dog in vivo results in a robust activation of intracellular MMPs within the cardiomyocyte nucleus along with DNA damage and loss of myosin. MMP activity was significantly attenuated by pretreatment with an orally active chymase inhibitor. We also found that I/R increased activity of two highly abundant unknown MMPs with molecular weight range 100 - 150 kD in the cardiomyocyte nucleus. Further, we found that chymase added to adult dog cardiomyocytes (plated on laminin) resulted in direct cleavage of laminin, focal adhesion kinase (FAK) dephopsphorylation, MMP release and myosin degradation. Chymase-induced myosin degradation was prevented by a broad spectrum MMP inhibitor and EDTA. Based on our preliminary in vivo and in vitro data, we hypothesize that increased ISF chymase during I/R mediates loss of cell-matrix-surface connections, disrupting FAK and culminating in myofibrillar degeneration through nuclear MMP activation. This hypothesis will be tested in a clinically relevant dog model of I/R by intravenous infusion (IV) of chymase inhibitor started 30 min after ischemia and continued throughout I/R with evaluation of LV function acutely and after chronic oral chymase inhibitor. Objective 1. Determine whether I/R results in activation of novel cardiomyocyte nuclear MMPs using proteomics and mass spectrometry approaches. Identification of novel nuclear MMPs and their regulation will provide a potential new molecular/protein target in I/R injury. Nuclear protein extraction from I/R cardiomyocytes will be separated by isoelectric focusing (IEF). After IEF, proteins on agarose gels will be separated using 2D zymography. Protein spots on the 2D gel corresponding to the areas of enzyme activity will be selected for identification of the MMP by mass spectrometry. Objective 2. Test the hypothesis that increase in ISF chymase activity during reperfusion leads to inactivation of FAK and downstream signaling that activate cardiomyocyte nuclear MMPs. Serial biopsy samples will be taken from the ischemic and nonischemic areas during I/R in dogs. We will study the time course of FAK dephosphorylation and activation of downstream signaling pathways ERK, JNK, p38 MAP kinase and NF:B, which are known to activate MMPs. To address this question in a clinically relevant fashion, serial biopsy samples will be taken before and after reperfusion with and without IV infusion of chymase inhibitor started at 30 min of ischemia and throughout the reperfusion. LV diastolic and systolic function will be assessed using the LV impedance catheter during I/R. In situ zymography with immunohistochemistry will define whether nuclear MMP activation can be prevented by prior IV chymase inhibitor infusion. Objective 3. Test the hypothesis that intravenous chymase inhibitor infusion during I/R followed by oral chymase inhibitor treatment for 7 days results in reduced injury and improved LV function. In our closed chest animal model, the proximal left anterior will be occluded using percutaneous coronary intervention balloon occlusion for one hour and reperfusion for two hours. Intravenous chymase inhibitor will be started after 30 minutes of ischemia and continued throughout the two hours of reperfusion. Oral drug will be initiated within 12 hours and continued for 7 days. Magnetic resonance imaging (MRI) with tissue tagging and gadolinium perfusion will be performed at 2 days and 7 days after I/R injury. T2 weighted MRI at 2 days will determine area of edema in vivo, while gadolinium plus tissue tagging at 7 days will determine in vivo myocardial scar and function in vehicle vs. chymase inhibitor treated dogs.