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（所有的变化从以前提交的标记线的利润）缺血和再灌注（I/R）损伤的结果急性增加的氧化/炎症应激在缺血后的再灌注过程中，并触发一系列的病理生理事件。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即刻存活，即使及时再灌注也会发生，并可增加梗死面积（1）。目前，在临床竞技场中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。先前的研究显示I/R后间质基质金属蛋白酶-2（MMP-2）和MMP-9活化显著增加（3）。最近的研究表明，在心肌细胞内激活的MMP-2切割肌原纤维（4-6）和核基质蛋白聚ADP-核糖聚合酶（PARP）-I/R期间细胞存活的DNA修复酶（7）。然而，在I/R期间细胞内MMPs的调节是未知的。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激活中起着重要作用。 驻留肥大细胞的脱粒是I/R的早期事件，并导致糜酶释放到回肠中（8- 10）。肥大细胞糜酶除了能够将血管紧张素（Ang I）转化为Ang II（11，12）之外，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），导致平滑肌细胞（20，21）和心肌细胞（22）死亡。在这个建议中，我们提出了初步的数据，增加间质糜蛋白酶蛋白水解活性后，在体内的狗I/R的结果在一个强大的激活细胞内基质金属蛋白酶的心肌细胞核沿着DNA损伤和肌球蛋白的损失。MMP活性显着衰减预处理与口服活性糜酶抑制剂。我们还发现，I/R增加活性的两个高度丰富的未知MMPs分子量范围为100 - 150 kD的心肌细胞核。此外，我们发现，糜蛋白酶添加到成年狗心肌细胞（板层粘连蛋白）导致直接切割层粘连蛋白，粘着斑激酶（FAK）脱磷酸化，MMP的释放和肌球蛋白降解。糜酶诱导的肌球蛋白降解被广谱MMP抑制剂和EDTA阻止。 基于我们初步的体内和体外数据，我们假设在I/R过程中增加的ISF糜酶介导细胞-基质-表面连接的丧失，破坏FAK并通过核MMP活化最终导致肌原纤维变性。将在临床相关的犬I/R模型中通过在缺血后30分钟开始静脉输注（IV）糜酶抑制剂并在整个I/R期间持续进行，对该假设进行检验，同时评价急性和慢性口服糜酶抑制剂后的LV功能。目的1.使用蛋白质组学和质谱方法确定I/R是否导致新型心肌细胞核MMPs的激活。新的核基质金属蛋白酶的鉴定及其调控将为I/R损伤提供潜在的新分子/蛋白靶点。将通过等电聚焦（IEF）分离I/R心肌细胞的核蛋白提取物。IEF后，琼脂糖凝胶上的蛋白质将使用2D酶谱法分离。选择2D凝胶上对应于酶活性区域的蛋白质点，通过质谱法鉴定MMP。目标2.验证再灌注期间ISF糜酶活性增加导致FAK失活和激活心肌细胞核MMPs的下游信号转导的假设。在犬I/R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，这些通路已知激活MMPs。为了以临床相关的方式解决这个问题，在缺血30分钟开始和整个再灌注期间，在有和没有IV输注糜酶抑制剂的情况下，在再灌注前后采集连续活检样本。在I/R期间使用LV阻抗导管评估LV舒张和收缩功能。原位酶谱与免疫组织化学将确定是否可以通过先前IV糜酶抑制剂输注来预防核MMP活化。目标3.检验以下假设：I/R期间静脉输注糜酶抑制剂，随后口服糜酶抑制剂治疗7天，可减少损伤并改善LV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近端左前动脉1小时，再灌注2小时。在缺血30分钟后开始静脉注射糜酶抑制剂，并在再灌注2小时内持续注射。口服药物将在12小时内开始，并持续7天。将在I/R损伤后2天和7天进行磁共振成像（MRI）和组织标记和钆灌注。第2天的T2加权MRI将确定体内水肿面积，而第7天的钆加组织标记将确定载体与糜酶抑制剂治疗的犬中的体内心肌瘢痕和功能。