Inhibitor ameliorates oxidative and proteolytic stress

描述（由申请人提供）：内皮细胞内皮（EE）功能障碍和内皮细胞与肌肉之间氧化基质的蓄积是充血性心力衰竭（CHF）的标志。本项目的总体目标是了解CHF中氧化基质蓄积和EE功能障碍的机制。先前的研究表明，CHF中氧化应激诱导、内皮细胞密度降低、基质金属蛋白酶（MMP）活化、胶原溶解和心脏组织金属蛋白酶抑制剂（CIMP）抑制之间存在相关性。该提议的新奇在于，在所有已知的金属蛋白酶组织抑制剂（TIMP）中，TIMP-4（即CIMP）在心脏中高度表达。本提案的目的是检验一个中心假设，即氧化基质蓄积和EE功能障碍是由于MMP活性水平升高和胶原溶解所致。这些水平与EE一氧化氮和CIMP水平降低有关，CIMP是在容量超负荷的缺血/再灌注延长周期期间对活性氧（ROS）水平升高的反应。CIMP水平的增加保护EE免受氧化和蛋白水解应激。我们将通过以下三个具体目标来检验中心假设：1。确定CIMP是否通过增加EE一氧化氮浓度来降低氧化应激。将在用和不用CIMP处理的慢性容量超负荷动静脉瘘小鼠中测量一氧化氮、ROS和硝基酪氨酸的血浆和左心室水平。2.确定CIMP蛋白转移是否抑制胶原溶解。将通过原位酶谱法测量MMP活性。将通过反向酶谱法测量CIMP活性水平。使用抗胶原抗体通过蛋白质印迹分析测量总胶原及其降解片段。3.确定CIMP是否能改善内皮细胞功能障碍。将在组织肌槽中测量心环中对乙酰胆碱、缓激肽和硝普钠的收缩反应。这些研究将使我们能够确定CIMP是否改善心脏对一氧化氮供体的反应。识别参与控制氧化和蛋白水解应激的主要参与者将有助于制定预防CHF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.