Regulation of caspase-1 by Sod2 in the heart

描述(申请人提供)：半胱氨酸蛋白酶是一个至少包括14个成员的半胱氨酸蛋白酶家族，在哺乳动物细胞凋亡或细胞因子的蛋白水解性激活中发挥关键作用。Caspase-1是最具特征性的炎症性caspase，最初是基于其裂解成熟细胞因子中不活跃的白介素1b(IL-12)前体的蛋白分解活性而被鉴定的。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)诱导的组织损伤具有重要的保护作用。我们最近进行了一项新的观察，通过RNA干扰下调MnSOD(也称为Sod2)的基础水平，上调了微血管内皮细胞(EC)中caspase-1的表达。此外，MnSOD下调内毒素或TNFa刺激的caspase-1表达增加。相反，通过RNA干扰下调CuZnSOD(也称为SOD1)并不能单独增加caspase-1的表达水平，也不能增加对内皮细胞对内毒素刺激的反应。这一观察结果表明，MnSOD在caspase-1的表达中起着特异性的负调控作用。我们的中心假设是，Sod2-/-小鼠caspase-1的表达增加会导致严重的炎症和凋亡，而SOD1-/-小鼠不会出现caspase-1的表达增加和炎症或凋亡。在第一个特定目标中，我们将评估Sod2-/-小鼠是否会表现出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，并将评估我们是否可以改善这些小鼠的炎症标志物。在这一目标中，我们将评估Sod2-/-小鼠是否会出现心肌细胞和内皮细胞凋亡增加。我们还将以caspase-3、caspase-7、PARP裂解和TUNEL检测为标记物来评估细胞凋亡。此外，我们还将向Sod2-/-小鼠注射广谱caspase抑制剂YVAD-CHO，并检测凋亡标志物的表达。此外，我们还将确定在心脏缺血-再灌注等病理生理环境下，MnSOD是否会调节caspase-1的表达。这些研究将证明结构性的MnSOD表达是否是维持体内低水平caspase-1所必需的。这项研究的结果也将首次显示MnSOD在体内负调控caspase-1表达中的新作用。公共卫生相关性：在人类心力衰竭中，心肌细胞的凋亡增加，随后的几项研究表明，心肌细胞的凋亡是心力衰竭的中心机制。在这项申请中提出的探索性研究将展示一种众所周知的抗氧化酶在控制炎症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.