Chymase-Mediated MMP Activation in Ishemia Reperfusion Injury

摘要(与上次提交的文件相比，所有更改均在页边空白处用行标记) 缺血再灌注(I/R)损伤是由氧化/炎症应激急剧增加所致 缺血后再灌流并触发一连串的病理生理事件。伤病的最终结果是 在心肌再灌注前立即存活的心肌细胞死亡，尽管及时 并可增加梗塞面积(1)。目前，还没有在临床领域使用的药物。 这可以预防或减轻急性冠脉综合征患者的I/R损伤(1，2)。 I/R导致心肌细胞死亡，并与核变性和肌原纤维有关 退化。以往的研究表明，间质基质金属蛋白酶-2(MMP2)显著增加 I/R后基质金属蛋白酶-9活化(3)。最近的研究表明，心肌细胞内有激活的基质金属蛋白酶-2 切割肌原纤维(4-6)和核基质蛋白多聚ADP-核糖聚合酶(PARP)-DNA修复 I/R期间细胞存活中的酶(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中的重要作用 基质金属蛋白酶的激活。 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。在这项提案中，我们提供了初步数据， 犬在体I/R后间质糜酶蛋白水解酶活性增强 心肌细胞核内的MMPs以及DNA损伤和肌球蛋白的丢失。明尼苏达 口服活性的乳糜酶抑制剂可显著降低活性。我们还发现，I/R 两种相对分子质量在100-150kD范围内的高度丰富的未知MMPs的活性增强 心肌细胞核。此外，我们还发现在成年犬心肌细胞中加入了凝乳酶(层粘连蛋白)。 导致层粘连蛋白、粘着斑激酶(FAK)脱磷、基质金属蛋白酶的释放和肌球蛋白的直接裂解 退化。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ISF糜酶增加 在I/R期间，介导细胞-基质-表面连接的丢失，扰乱FAK并最终导致 肌原纤维通过核基质金属蛋白酶的激活而变性。这一假设将在临床上得到验证 犬缺血30min后静脉注射糜酶抑制剂建立犬I/R模型 持续至整个I/R期间，急性和慢性口服糜乳酶抑制剂后评价左心功能。 目的1.确定I/R是否导致新的心肌细胞核MMPs的激活 蛋白质组学和质谱学方法。新型核基质金属蛋白酶的鉴定及其调控 将为I/R损伤提供潜在的新的分子/蛋白质靶点。缺血再灌流中核蛋白的提取 用等电聚焦(IEF)分离心肌细胞。经过离子交换后，琼脂糖凝胶上的蛋白质将 用二维酶谱分离。2D凝胶上对应于酶活性区域的蛋白质斑点将 被选作基质金属蛋白酶的质谱学鉴定。 目的2.验证再灌流期间ISF糜酶活性增加会导致 激活心肌细胞核MMPs的FAK及其下游信号失活。连续活检术 在犬I/R期间，分别从缺血区和非缺血区采集标本。我们会研究时间 FAK去磷酸化及其下游信号通路ERK、JNK、p38MAP激活的过程 KK和NFB，它们被认为可以激活MMPs。为了以临床相关的方式解决这个问题， 在使用和不使用静脉注射凝乳酶的情况下，分别在再灌注前后采集连续的活检样本。 抑制剂在缺血30min和整个再灌流过程中开始起效。左室舒张和收缩功能将 I/R期间使用LV阻抗导管进行评估免疫组织化学原位酶谱检查 明确是否可以通过预先静脉输注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的假设 口服糜酶抑制剂治疗7天，可减轻损伤，改善左心功能。在我们的 闭胸动物模型，采用经皮冠状动脉介入技术阻断左前壁近端 球囊闭塞1小时，再灌注2小时。静脉注射乳糜酶抑制剂将启动 缺血30分钟后，持续至再灌流2小时。口服药将启动 在12小时内，并持续7天。磁共振成像(MRI)与组织标记和 分别于I/R损伤后2天和7天进行Gd灌流。2天的T2加权MRI将 在活体内测定水肿面积，而7天的Gd+组织标记将在活体内确定 赋形剂与乳糜酶抑制剂治疗犬的心肌瘢痕和功能。

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.