Chymase-Mediated MMP Activation in Ishemia Reperfusion Injury

缺血再灌注损伤中食糜酶介导的 MMP 激活

• 批准号: 8391152
• 负责人: Louis J. Dell'Italia
• 金额: --
• 依托单位: BIRMINGHAM VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8391152
• 关键词: Acute; Acute myocardial infarction; Address; Adult; Angiotensins; Animal Model; Anterior; Area; Arrhythmia; Attenuated; Balloon Occlusion; Biopsy Specimen; Blood flow; Canis familiaris; Cardiac Myocytes; Cardiac Surgery procedures; Cardiopulmonary Bypass; Caring; Catheters; Cell Nucleus; Cell Surface Proteins; Cell Survival; Cells; Cessation of life; Chest; Chest Pain; Chronic; Chymase; Cicatrix; Cleaved cell; Clinical; Community Hospitals; Coronary Occlusions; Coupled; DNA Damage; DNA Repair Enzymes; Data; Edema; Edetic Acid; Evaluation; Event; Fibronectins; Focal Adhesion Kinase 1; Functional disorder; Gadolinium; Gel; Gelatinase A; Gelatinase B; Health; Heart Arrest; Hospitals; Hour; Immunohistochemistry; In Situ; In Vitro; Incidence; Infarction; Inflammatory; Infusion procedures; Injury; Intravenous; Intravenous infusion procedures; Ischemia; Isoelectric Focusing; Laminin; Left; Length; MAPK8 gene; Magnetic Resonance Imaging; Mass Spectrum Analysis; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Mediating; Mission; Modeling; Molecular; Molecular Weight; Morbidity - disease rate; Muscle Cells; Myocardial; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion; Myofibrils; Myosin ATPase; Nuclear; Nuclear Matrix-Associated Proteins; Nuclear Protein; Oral; Organ; Organ Transplantation; Patients; Perfusion; Pharmaceutical Preparations; Phase; Play; Poly(ADP-ribose) Polymerases; Protein Dephosphorylation; Proteins; Proteomics; Regulation; Reperfusion Injury; Reperfusion Therapy; Reporting; Risk; Role; Sepharose; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Spottings; Stress; Surface; Survivors; Testing; Thrombolytic Therapy; Time; Time Study; Tissues; Veterans; Weight; abstracting; acute coronary syndrome; base; clinically relevant; coronary angioplasty; electric impedance; enzyme activity; gadolinium oxide; improved; in vivo; inhibitor/antagonist; interstitial; mast cell; mitogen-activated protein kinase p38; mortality; novel; outcome forecast; patient population; percutaneous coronary intervention; prevent; public health relevance; response; restoration

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.

描述（由申请人提供）： 摘要（与之前提交的所有变化均在页边用线标记）缺血再灌注（I/R）损伤是由缺血后再灌注期间氧化/炎症应激急剧增加引起的，并引发一系列病理生理事件。损伤最终导致心肌细胞死亡，这些心肌细胞在心肌再灌注前立即存活，并且尽管及时再灌注，但仍会发生死亡，并可能增加梗死面积 (1)。目前，临床领域还没有药物可以预防或减轻急性冠状动脉综合征患者的 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降解有关。先前的研究表明 I/R 后间质基质金属蛋白酶 2 (MMP-2) 和 MMP-9 的激活显着增加 (3)。最近的研究表明，心肌细胞内激活的 MMP-2 可裂解肌原纤维 (4-6)，而核基质蛋白聚 ADP 核糖聚合酶 (PARP) 是 I/R 期间细胞存活中的一种 DNA 修复酶 (7)。然而，I/R 期间细胞内 MMP 的调节尚不清楚。在这里，在狗 I/R 模型中，我们报告间质肥大细胞糜酶在心肌细胞 MMP 激活中发挥重要作用。 常驻肥大细胞脱颗粒是 I/R 的早期事件，导致食糜酶释放到间质中 (8-10)。除了能够将血管紧张素 (Ang I) 转化为 Ang II (11,12) 之外，肥大细胞糜酶还能激活间质 MMP (13-18) 并直接降解细胞表面蛋白，如纤连蛋白 (19)，从而产生平滑肌细胞(20,21) 和心肌细胞 (22) 死亡。在本提案中，我们提供了初步数据，表明狗体内 I/R 后间质糜酶蛋白水解活性的增加导致心肌细胞核内细胞内 MMP 的强烈激活以及 DNA 损伤和肌球蛋白损失。通过口服活性食糜酶抑制剂预处理，MMP 活性显着减弱。我们还发现 I/R 增加了心肌细胞核中两种分子量范围为 100 - 150 kD 的高度丰富的未知 MMP 的活性。此外，我们发现添加到成年犬心肌细胞（铺在层粘连蛋白上）的食糜酶会导致层粘连蛋白的直接裂解、粘着斑激酶（FAK）去磷酸化、MMP 释放和肌球蛋白降解。广谱 MMP 抑制剂和 EDTA 可防止食糜酶诱导的肌球蛋白降解。 根据我们初步的体内和体外数据，我们假设 I/R 期间 ISF 食糜酶的增加会介导细胞-基质-表面连接的丧失，破坏 FAK 并最终通过核 MMP 激活导致肌原纤维变性。这一假设将在临床相关的 I/R 犬模型中进行测试，方法是在缺血后 30 分钟开始静脉输注（IV）食糜酶抑制剂，并在整个 I/R 期间持续输注，并在急性和慢性口服食糜酶抑制剂后评估 LV 功能。目标 1. 使用蛋白质组学和质谱方法确定 I/R 是否会导致新型心肌细胞核 MMP 的激活。新型核 MMP 的鉴定及其调控将为 I/R 损伤提供潜在的新分子/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) 进行分离。 IEF 后，将使用 2D 酶谱法分离琼脂糖凝胶上的蛋白质。将选择 2D 凝胶上对应于酶活性区域的蛋白质点，用于通过质谱法鉴定 MMP。目标 2. 检验再灌注期间 ISF 食糜酶活性增加导致 FAK 和激活心肌细胞核 MMP 的下游信号失活的假设。在犬缺血再灌注期间，将从缺血和非缺血区域采集连续活检样本。我们将研究 FAK 去磷酸化和下游信号通路 ERK、JNK、p38 MAP 激酶和 NF:B 激活的时间过程，这些信号通路已知可激活 MMP。为了以临床相关的方式解决这个问题，将在再灌注之前和之后采集连续活检样本，在缺血 30 分钟开始和整个再灌注过程中，有或没有 IV 输注食糜酶抑制剂。 I/R 期间将使用左心室阻抗导管评估左心室舒张和收缩功能。免疫组织化学原位酶谱分析将确定是否可以通过先前的 IV 食糜酶抑制剂输注来预防核 MMP 激活。目的 3. 检验以下假设：I/R 期间静脉输注食糜酶抑制剂，然后口服食糜酶抑制剂治疗 7 天可减少损伤并改善左心室功能。在我们的闭胸动物模型中，将使用经皮冠状动脉介入球囊闭塞一小时并再灌注两小时来闭塞近端左前部。缺血 30 分钟后开始静脉注射食糜酶抑制剂，并持续整个再灌注的两个小时。口服药物将在12小时内开始并持续7天。 I/R 损伤后 2 天和 7 天将进行具有组织标记和钆灌注的磁共振成像 (MRI)。 2天时的T2加权MRI将确定体内水肿面积，而7天时的钆加组织标记将确定载体与食糜酶抑制剂治疗的狗的体内心肌疤痕和功能。