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: 8195546
• 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/8195546
• 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

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.

摘要（以前提交中的所有更改均由边距中的线路标记） 缺血和再灌注（I/R）损伤导致氧化/炎症应激急性增加 缺血后的再灌注并触发一系列病理生理事件。伤害最终 心肌再灌注前立即可行的心肌细胞死亡，并暂时发生 再灌注并可以增加梗塞大小（1）。目前，在临床领域没有使用的药物 这可以防止或减轻患有急性冠状动脉综合征的患者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变性有关 降解。先前的研究表明，间质基质金属蛋白酶2（MMP-2）显着增加 I/R（3）之后的MMP-9激活。最近的研究表明，心肌细胞中激活的MMP-2 裂解肌原纤维（4-6）和核基质蛋白聚ADP-核糖聚合酶（PARP）-A DNA修复 I/R期间细胞存活中的酶（7）。但是，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，我们报告说间隙肥大细胞嵌合酶在心肌细胞中起重要作用 MMP激活。 居民肥大细胞的脱粒是I/R的早期事件，导致嵌合酶释放到间质中（8-- 10）。除了将血管紧张素（ANG I）转换为ANG II（11,12）的能力外，肥大细胞壳酶激活 间质MMP（13-18），直接降解细胞表面蛋白，例如纤连蛋白（19），导致 平滑肌细胞（20,21）和心肌细胞（22）死亡。在此提案中，我们提供了初步数据 I/R在体内I/R之后增加的间质嵌合酶蛋白水解活性会导致强大的激活 心肌细胞核中的细胞内MMP以及DNA损伤和肌球蛋白的丧失。 MMP 用口服活性壳酶抑制剂预处理可显着减弱活性。我们还发现I/R 两个高度丰富的未知MMP的活性增加，分子量范围为100-150 kd。 心肌细胞核。此外，我们发现添加到成年狗心肌细胞（粘液层上的粘液素）中的嵌合酶 导致层粘连蛋白直接切割，局灶性激酶（FAK）dephopsphorylation，MMP释放和肌球蛋白 降解。 Chymase诱导的肌球蛋白降解是通过广谱MMP抑制剂和EDTA预防的。 根据我们的初步体内和体外数据，我们假设增加ISF嵌合酶 在I/R中介导细胞矩阵 - 表面连接的损失，破坏FAK并最终导致 通过核MMP激活的肌原纤维变性。该假设将在诊所进行检验 I/R的相关狗模型通过静脉输注（IV）的嵌合酶抑制剂（IV）在缺血后30分钟开始 贯穿整个I/R，通过评估LV功能急性和慢性口服嵌合酶抑制剂。 目标1。确定I/R是否会导致使用新型心肌细胞核MMP激活 蛋白质组学和质谱方法。识别新型核MMP及其调节 将在I/R损伤中提供潜在的新分子/蛋白质靶标。从I/R提取核蛋白 心肌细胞将通过等电聚焦（IEF）分开。在IEF之后，琼脂糖凝胶上的蛋白质将是 使用2D Zymography分离。与酶活性区域相对应的2D凝胶上的蛋白质斑点将 通过质谱法选择以识别MMP。 目标2。检验以下假设，即再灌注过程中ISF嵌合酶活性的增加导致 激活心肌细胞核MMP的FAK和下游信号的失活。连续活检 在狗I/R期间，样品将从缺血性和非缺血性区域中采集。我们将研究时间 FAK去磷酸化和下游信号通路ERK，JNK，p38 MAP的激活过程 激酶和nF¿B，已知会激活MMP。为了以临床相关的方式解决这个问题， 连续活检样品将在再灌注之前和之后，并在静脉注入嵌合酶的情况下进行。 抑制剂从缺血30分钟开始，整个再灌注开始。 LV舒张期和收缩功能将是 在I/R期间使用LV阻抗导管进行评估。具有免疫组织化学的原位Zymography将 定义是否可以通过先前的IV壳酶抑制剂输注来预防核MMP激活。 目标3。检验以下假设：I/R期间静脉注射嵌合酶抑制剂输注 口服壳酶抑制剂治疗7天导致损伤降低和LV功能改善。在我们的 封闭的胸部动物模型，近端左前将使用经皮冠状动脉介入 气球阻塞一个小时，再灌注两个小时。静脉注射酶抑制剂将开始 经过30分钟的缺血，并在整个两个小时的再灌注过程中继续进行。口服药物将开始 在12小时内持续7天。带有组织标签的磁共振成像（MRI）和 I/R受伤后2天7天，将进行灌注。 T2加权MRI在2天将 确定体内水肿面积，而Gadolinium Plus在7天后标记将确定体内 心肌疤痕和车辆与壳酶抑制剂治疗的狗的功能。