Regulation of caspase-1 by Sod2 in the heart

描述（由申请人提供）：半胱天冬酶是一个半胱氨酸蛋白酶家族，包括至少14个成员，在哺乳动物细胞凋亡或细胞因子的蛋白水解激活中起关键作用。caspase -1是最具特征的炎性caspase，最初是根据其在成熟细胞因子中切割无活性的白介素-1b （IL-12）前体的蛋白水解活性而确定的。锰超氧化物歧化酶（MnSOD）是一种位于线粒体中的抗氧化酶，对活性氧（ROS）诱导的组织损伤提供关键保护。我们最近观察到，通过RNA干扰下调MnSOD（也称为so2）的基础水平可上调微血管内皮细胞（EC）中caspase-1的表达。此外，MnSOD下调会增加LPS或TNFa刺激caspase-1表达的增加。相反，通过RNA干扰下调CuZnSOD（也称为Sod1）并不会增加内皮细胞中单独或响应LPS刺激的caspase-1表达水平。这表明MnSOD对caspase-1的表达具有特异性的负调控作用。我们的中心假设是，caspase-1在Sod2-/-小鼠中的表达增加会导致严重的炎症和凋亡，而Sod1-/-小鼠则不会表现出caspase-1的表达增加和炎症或凋亡。在第一个特定目的中，我们将评估Sod2 -/-小鼠是否会显示caspase-1表达增加和细胞因子表达增加。此外，我们还将评估Sod2-/-小鼠心脏组织和血清中IL-12、IL-18的水平。我们还将评估caspase-1、IL-12和IL-18在Sod1 -/-小鼠中的表达。此外，我们将给Sod2-/-小鼠注射caspase-1抑制剂YVAD-CHO，并评估我们是否可以改善这些小鼠的炎症标志物。在这个目的中，我们将评估Sod2-/-小鼠是否会显示心肌细胞和内皮细胞凋亡增加。我们还将使用caspase-3、caspase-7、PARP切割和Tunel核检测作为标记来评估细胞凋亡。此外，我们将给Sod2 -/-小鼠注射广谱caspase抑制剂YVAD-CHO，并评估凋亡标志物的表达。此外，我们将确定MnSOD是否会在病理生理环境（如心脏缺血再灌注）中调节caspase-1的表达。这些研究将证明组成型MnSOD表达是否需要在体内将caspase-1维持在低水平。本研究的结果也首次显示了MnSOD在体内对caspase-1表达的阴性控制中的新作用。公共卫生相关性：心肌细胞凋亡在人类心力衰竭中增加，随后一些研究表明心肌细胞凋亡是心力衰竭的中心机制。本应用中提出的探索性研究将证明一种众所周知的抗氧化酶在控制炎症性caspase-1表达方面的新功能。这些研究的结果可能有助于在心力衰竭、内皮功能障碍或其他心血管疾病中使用MnSOD或诱导MnSOD的物质来保护心肌损伤。

DESCRIPTION (provided by applicant): Caspases are a family of cysteine proteases that include at least 14 members, and play a critical role in mammalian apoptosis or proteolytic activation of cytokines. Caspase-1, the best characterized inflammatory caspase was originally identified on the basis of its proteolytic activity for cleaving the inactive interleukin-1b (IL-12) precursor in the mature cytokine. Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme located in the mitochondria that provides critical protection against reactive oxygen species (ROS) -induced tissue injury. We recently made the novel observation that downregulation of basal level of MnSOD (also known as Sod2) by RNA interference upregulated caspase-1 expression in microvascular endothelial cells (EC). Additionally, MnSOD downregulation increased LPS or TNFa stimulated increase in caspase-1 expression. In contrast, downregulation of CuZnSOD (also known as Sod1) by RNA interference did not increase the level of caspase-1 expression alone or in response to LPS stimulation in endothelial cells. This observation suggests that MnSOD plays a specific negative regulatory role in the expression of caspase-1. Our central hypothesis is that increased caspase-1 expression in Sod2-/- mice would result in severe inflammation and apoptosis, whereas Sod1-/- mice would not show increased expression of caspase-1 and inflammation or apoptosis. In the first specific aim we will evaluate whether Sod2 -/- mice would show increased caspase-1 expression and increased cytokine expression. In addition, we will also evaluate the level of IL-12, IL-18 in the heart tissue and sera of Sod2-/- mice. We will also evaluate the expression of caspase-1, IL-12 and IL-18 in Sod1 -/- mice. In addition, we will inject Sod2-/- mice with caspase-1 inhibitor YVAD-CHO, and will evaluate whether we could ameliorate inflammation markers in these mice. In this aim, we will evaluate whether Sod2-/- mice would show increased apoptosis of cardiac cells and endothelial cells. We will also evaluate apoptosis using caspase-3, caspase-7, PARP cleavage, and detection of Tunel nuclei as markers. In addition, we will inject Sod2 -/- mice with broad spectrum caspase inhibitor YVAD-CHO, and evaluate the expression of apoptosis markers. In addition, we will determine whether MnSOD would regulate caspase-1 expression in a pathophysiological setting such as ischemia-reperfusion of heart. These studies would demonstrate whether constitutive MnSOD expression is necessary to maintain caspase-1 at a low level in vivo. The results obtained in this investigation would also show for the first time a novel role of MnSOD in the negative control of caspase-1 expression in vivo. PUBLIC HEALTH RELEVANCE: Apoptosis of cardiomyocytes is increased in human heart failure, and several studies have subsequently suggested that cardiomyocyte apoptosis is a central mechanism in heart failure. The exploratory studies proposed in this application will demonstrate a novel function of a well known antioxidant enzyme in the control of inflammatory caspase-1 expression. The outcome of these studies may help in the therapeutic use of MnSOD or substances that induce MnSOD in the protection of myocardial injury in heart failure, endothelial dysfunction or other cardiovascular diseases.