Effects of ACE2 gene therapy on Diabetes

描述（由申请人提供）：糖尿病是一个日益严重的问题，在世界各地。I型和II型糖尿病的临床试验和动物模型已经表明，血管紧张素-II（Ang-II）信号通路的过度活跃有助于糖尿病和糖尿病并发症的发展。具有临床意义的是，阻断肾素-血管紧张素系统（RAS）可预防新发糖尿病并降低糖尿病并发症的风险。血管紧张素转换酶（ACE）2是新近发现的一种单羧肽酶，是ACE的第一个同源物.认为其主要通过切割Ang-II产生Ang-（1-7）来抑制Ang-II信号级联，其作用对抗Ang-II并且由Mas受体介导。这种酶存在于各种组织和器官中，包括肾脏、肝脏、脂肪组织和胰腺。其表达在糖尿病的内分泌胰腺和糖尿病肾病的早期阶段升高。胰岛表达RAS组分和NADPH氧化酶（Nox）组分，它们是介导氧化应激的关键元件。在胰岛中，Ang-II和氧化应激都能够减少胰岛素基因的表达和分泌。假设ACE 2对抗ACE/Ang-II/AT 1受体轴，并可通过抑制Nox活性和Ang-II介导的胰岛素基因表达和分泌减少来保护胰腺β细胞功能。为了操纵ACE 2的表达，我们产生了一种新的腺病毒编码ACE 2，并报告增加ACE 2的mRNA，蛋白质和活性在细胞和组织。我们假设胰腺中ACE 2的过度表达将减少氧化应激并改善β细胞功能，从而改善糖尿病小鼠的葡萄糖稳态。为了验证这一假设，我们将致力于以下具体目标：1）确定ACE 2表达和/或活性与糖尿病之间的关系的存在; 2）评估ACE 2过表达在糖尿病小鼠中的后果; 3）确定ACE 2过表达是否改善糖尿病中的胰腺β细胞功能。为了实现这些目标，我们将首先使用ACE 2敲除和db/db小鼠（2型糖尿病）模型来建立ACE 2与糖尿病之间的关系。然后，我们将结合联合收割机最先进的分子，药理学和生理学工具，在糖尿病前期和年轻的糖尿病db/db小鼠中进行体外（分离的胰岛）和体内（胰腺）基因治疗。最后，我们将讨论ACE 2可能抵消糖尿病中过度活跃的RAS的有害作用的机制。该提案将显示ACE 2过表达对血糖正常化的有益作用的证据，并突出ACE 2作为糖尿病治疗的新靶点。 公共卫生相关性：大约8%的美国人受到糖尿病的影响，糖尿病是心血管疾病的一个众所周知的危险因素，并且由于目前西方世界的肥胖流行病，预计糖尿病将增长。在2型糖尿病小鼠模型中使用基因治疗，本申请将描述一种新酶ACE 2调节血糖水平的能力。如果得到证实，ACE 2可能成为治疗2型糖尿病的新靶点。

DESCRIPTION (provided by applicant): Diabetes is a growing problem in all parts of the World. Clinical trials and animal models of type I and type II diabetes have shown that hyperactivity of angiotensin-II (Ang-II) signaling pathways contribute to the development of diabetes and diabetic complications. Of clinical relevance, blockade of the renin-angiotensin system (RAS) prevents new-onset diabetes and reduces the risk of diabetic complications. Angiotensin converting enzyme (ACE) 2 is a recently discovered mono- carboxypeptidase and the first homolog of ACE. It is thought to inhibit Ang-II signaling cascades mostly by cleaving Ang-II to generate Ang-(1-7), which effects oppose Ang-II and are mediated by the Mas receptor. The enzyme is present in various tissues and organs, including the kidney, liver, adipose tissue and pancreas. Its expression is elevated in the endocrine pancreas in diabetes and in the early phase during diabetic nephropathy. Pancreatic islets express both RAS components and NADPH oxidase (Nox) components, which are key elements in mediating oxidative stress. In the islet Ang-II and oxidative stress are both capable of decreasing insulin gene expression and secretion. ACE2 is hypothesized to oppose the ACE/Ang-II/AT1 receptor axis and may protect pancreatic beta- cell function by inhibiting both Nox activity and the Ang-II-mediated reduction of insulin gene expression and secretion. To manipulate ACE2 expression, we generated a novel adenovirus coding for ACE2 and reported increased ACE2 mRNA, protein and activity in cells and tissues. We hypothesize that ACE2 over-expression in the pancreas will reduce oxidative stress and ameliorate beta-cell function, thus leading to improved glucose homeostasis in diabetic mice. To test this hypothesis, we will address the following specific aims: 1) Determine the existence of a relationship between ACE2 expression and/or activity and diabetes; 2) Evaluate the consequences of ACE2 over-expression in diabetic mice; 3) Establish whether ACE2 over-expression improves pancreatic beta-cell function in diabetes. To achieve these goals, we will first use ACE2 knockout and db/db mouse (type 2 diabetes) models to establish the relationship between ACE2 and diabetes. Then we will combine state of the art molecular, pharmacological and physiological tools for in vitro (isolated islets) and in vivo (pancreas) gene therapy in pre-diabetic and young diabetic db/db mice. Finally, we will address the mechanisms by which ACE2 could potentially counterbalance the deleterious effects of the hyperactive RAS in diabetes. This proposal will show evidence of the beneficial effects of ACE2 over-expression on the normalization of blood glucose and highlight ACE2 as a new target for the treatment of diabetes. PUBLIC HEALTH RELEVANCE: Approximately 8% of Americans are affected by diabetes, a well known risk factor for cardiovascular diseases, and it is expected to grow due to the current obesity epidemic in the Western World. Using gene therapy in a mouse model of type 2 diabetes, this application will describe the ability of a new enzyme, ACE2, to regulate blood glucose levels. If confirmed, ACE2 could become a new target for the treatment of type 2 diabetes.