Inhibitor ameliorates oxidative and proteolytic stress

描述(由申请人提供)：心内膜内皮功能障碍和内皮与肌肉之间的氧化基质堆积是充血性心力衰竭(CHF)的特征。本项目的总体目标是了解CHF中氧化基质堆积和EE功能障碍的机制。以往的研究表明，CHF时氧化应激的诱导、内皮细胞密度的降低、基质金属蛋白酶(MMPs)的激活、胶原酶的降解和心肌组织金属蛋白酶抑制物(CIMP)的抑制之间存在关联。这一提议的新颖之处在于，在所有已知的金属蛋白酶组织抑制物(TIMP)中，TIMP-4(即CIMP)在心脏中高表达。这项建议的目的是验证一个中心假设，即氧化基质堆积和EE功能障碍是由于基质金属蛋白酶活性增加和胶原酶分解水平增加所致。这些水平与EE、NO和CIMP水平的降低有关，以应对在容量超负荷的长时间缺血/再灌注周期中活性氧物种(ROS)水平的升高。CIMP水平的增加可以保护EE免受氧化和蛋白质降解压力。我们将通过以下三个具体目标来验证这一中心假说：1.确定CIMP是否通过增加EE一氧化氮浓度来降低氧化应激。使用和不使用CIMP治疗慢性容量超负荷性动静脉瘘小鼠，将测量血浆和左心室一氧化氮、ROS和硝基酪氨酸水平。2.确定CIMP蛋白转移是否抑制胶原酶的降解。用原位酶谱法检测基质金属蛋白酶活性。CIMP活性的水平将通过反向酶谱来测量。用抗胶原抗体进行蛋白印迹分析，测定总胶原蛋白及其降解片段。3.确定CIMP是否能改善心内膜内皮细胞功能障碍。心脏环对乙酰胆碱、缓激肽和硝普钠的收缩反应将在组织肌层中进行测量。这些研究将使我们能够确定CIMP是否改善了对一氧化氮供体的心脏反应。确定参与控制氧化和蛋白分解应激的主要因素将有助于制定预防心力衰竭的策略。

DESCRIPTION (provided by applicant): Endocardial endothelial (EE) dysfunction and accumulation of oxidized-matrix between endothelial and muscle are the hallmarks of congestive heart failure (CHF). The overall objective of this project is to understand the mechanism of oxidized-matrix accumulation and EE dysfunction in CHF. Previous studies have suggested an association between induction of oxidative stress, decrease in endothelial cell density, activation of matrix metalloproteinase (MMP), collagenolysis, and repression of cardiac tissue inhibitor of metalloproteinase (CIMP) in CHF. The novelty of this proposal is that among all known tissue inhibitors of metalloproteinase (TIMP), the TIMP-4 (i.e. CIMP) is highly expressed in the heart. The purpose of this proposal is to test a central hypothesis that oxidized-matrix accumulation and EE dysfunction are due to increased levels of MMP activity, and collagenolysis. These levels are associated with decreased levels of EE nitric oxide, and CIMP in response to increased levels of reactive oxygen species (ROS) during protracted cycles of ischemia/reperfusion in volume overload. The increased levels of CIMP protects EE against oxidative and proteolytic stresses. We will test the central hypothesis by the following three specific aims: 1. To determine whether CIMP decreases oxidative stress by increasing EE nitric oxide concentration. Plasma and left ventricle levels of nitric oxide, ROS, and nitrotyrosine will be measured in chronic volume overload arteriovenous fistula mice treated with and without CIMP. 2. To determine whether CIMP protein transfer inhibits collagenolysis. MMP activity will be measured by in situ zymography. The levels of CIMP activity will be measured by reverse zymography. Total collagen and its degradation fragments will be measured by Western-blot analysis using anti-collagen antibody. 3. To determine whether CIMP ameliorates endocardial endothelial dysfunction. Contractile responses to acetylcholine, bradykinin, and nitroprusside in cardiac rings will be measured in a tissue myobath. These studies will enable us to determine whether CIMP improves the hearts response to nitric oxide donors. Identification of major players involved in the control of oxidative and proteolytic stresses will help to develop strategies to prevent CHF.