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（与之前提交的所有更改均在页边空白处用线标记） 缺血再灌注（I/R）损伤是由氧化/炎症应激急剧增加引起的 缺血后再灌注并引发一系列病理生理事件。伤害达到顶峰 在心肌再灌注之前立即存活的心肌细胞的死亡，尽管及时进行 再灌注并可能增加梗塞面积 (1)。目前尚无药物应用于临床 可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相关 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 后 MMP-9 激活 (3)。最近的研究表明心肌细胞内的 MMP-2 被激活 裂解肌原纤维 (4-6) 和核基质蛋白聚 ADP-核糖聚合酶 (PARP) - DNA 修复 I/R 期间细胞存活中的酶 (7)。然而，I/R 期间细胞内 MMP 的调节尚不清楚。 在此，在狗 I/R 模型中，我们报告间质肥大细胞糜酶在心肌细胞中发挥重要作用 MMP 激活。 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，肥大细胞食糜酶还可以激活 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 狗体内 I/R 后间质食糜酶蛋白水解活性增加，导致 心肌细胞核内的细胞内 MMP 以及 DNA 损伤和肌球蛋白丢失。基质金属蛋白酶 用口服活性食糜酶抑制剂预处理后，其活性显着减弱。我们还发现 I/R 两种高度丰富的未知 MMP（分子量范围为 100 - 150 kD）的活性增加 心肌细胞核。此外，我们发现成年狗心肌细胞中添加了食糜酶（铺在层粘连蛋白上） 导致层粘连蛋白直接裂解、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 ISF 食糜酶增加 I/R 期间介导细胞-基质-表面连接的丧失，破坏 FAK 并最终导致 通过核 MMP 激活导致肌原纤维变性。该假设将在临床上得到检验 通过在缺血后 30 分钟开始静脉输注（IV）食糜酶抑制剂来建立相关的 I/R 狗模型，并且 在整个 I/R 期间持续评估左室功能，并在慢性口服食糜酶抑制剂后进行。 目标 1. 使用以下方法确定 I/R 是否会导致新型心肌细胞核 MMP 的激活 蛋白质组学和质谱方法。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 中提取核蛋白 心肌细胞将通过等电聚焦（IEF）分离。 IEF 后，琼脂糖凝胶上的蛋白质将 使用 2D 酶谱法进行分离。 2D 凝胶上对应于酶活性区域的蛋白质点将 选择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的假设 FAK 和激活心肌细胞核 MMP 的下游信号失活。连续活检 在犬缺血再灌注期间从缺血和非缺血区域采集样本。我们将研究时间 FAK去磷酸化和下游信号通路ERK、JNK、p38 MAP激活的过程 激酶和 NF¿B，已知它们可以激活 MMP。为了以临床相关的方式解决这个问题， 在再灌注之前和之后采集连续活检样本，无论是否静脉输注食糜酶 抑制剂在缺血 30 分钟和整个再灌注过程中开始。左心室舒张和收缩功能将 在 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分析将 确定是否可以通过事先静脉注射食糜酶抑制剂来预防核 MMP 激活。 目标 3. 检验以下假设：I/R 期间静脉注射食糜酶抑制剂，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，采用经皮冠状动脉介入术封堵左前近端 球囊闭塞一小时，再灌注两小时。将开始静脉食糜酶抑制剂 缺血 30 分钟后持续整个再灌注两个小时。将开始口服药物 12小时内持续7天。具有组织标记和功能的磁共振成像 (MRI) I/R损伤后2天和7天进行钆灌注。 2 天后 T2 加权 MRI 确定体内水肿面积，而钆加上 7 天时的组织标记将确定体内 媒介物与食糜酶抑制剂治疗的狗的心肌疤痕和功能。