Novel Aspects of the Cardiac Renin-Angiotensin System

描述（由申请人提供）：肾素血管紧张素系统（RAS）在心血管稳态和肾脏、血管和心脏病理的发展中起核心作用。血管紧张素II （Ang II）的作用主要通过内分泌和/或自分泌/旁分泌方式与AT1受体结合介导。我们和其他人最近发现了一种新的，在细胞内或细胞内，不需要Ang II-AT1相互作用的Ang ii介导的作用模式。我们报道了心肌细胞在高葡萄糖条件下在细胞内合成高水平的Ang II，并将其重新分配到细胞质和细胞核，而不影响细胞外水平。在高糖条件下，细胞内Ang II （iAng II）的合成依赖于酶，而不是血管紧张素转换酶（ACE）。细胞内Ang II的合成被肾素抑制剂完全阻断。我们还观察到心脏成纤维细胞中细胞内RAS的活化。值得注意的是，糖尿病大鼠的心脏中，ang II水平、心肌细胞凋亡、氧化应激和心脏纤维化明显增加，这是通过肾素抑制而不是通过ACE或AT1抑制而正常化的。这些观察结果表明，iAng II在糖尿病心肌病中的重要作用，这意味着AT1受体拮抗剂和ACE抑制剂在阻断iAng II的作用方面是无效的。在高糖条件下，导致细胞内RAS成分表达和Ang II合成的细胞机制以及Ang II的作用机制尚不清楚。我们已经报道过ang II引起心脏肥厚；然而，在病理生理条件下，Ang II与细胞外Ang II的相对意义尚不清楚。在本提案中，我们将验证高糖诱导的代谢变化导致细胞内合成Ang II的假设，Ang II通过与新的细胞内蛋白的相互作用在糖尿病心肌病的发展中起重要作用。我们将使用新生儿和成人心肌细胞和成纤维细胞，通过药理学和遗传学方法确定高血糖诱导的细胞事件，如己糖胺生物合成途径、蛋白质o -糖基化和氧化应激，对细胞内RAS的调节。我们将鉴定和表征新的iAng II相互作用蛋白，使用亲和结合和质谱方法。我们将利用AT1受体缺陷小鼠来确定iAng II在糖尿病心功能障碍中的具体作用。这些研究将确定Ang II在心脏中作用的新机制，并为糖尿病性心肌病的临床干预提供新的策略。公共卫生相关性：本研究将侧重于开发治疗糖尿病患者的新策略。与血管紧张素受体阻滞剂和血管紧张素转换酶抑制剂的标准治疗相比，阻断细胞内肾素-血管紧张素系统可能为预防/治疗糖尿病性心肌病提供实质性的益处。

DESCRIPTION (provided by applicant): The renin angiotensin system (RAS) has a central role in cardiovascular homeostasis and development of renal, vascular, and cardiac pathologies. The effects of angiotensin II (Ang II) are primarily mediated by binding to AT1 receptor in an endocrine and/or autocrine/paracrine manner. We and others have recently identified a novel, intracrine or intracellular, mode of Ang II-mediated actions that do not require Ang II-AT1 interaction. We reported that cardiac myocytes synthesize high levels of Ang II intracellularly, which redistributes to cytoplasm and nucleus, without affecting extracellular levels, in high glucose conditions. Intracellular Ang II (iAng II) synthesis in high glucose conditions is chymase, not angiotensin converting enzyme (ACE) dependent. Intracellular synthesis of Ang II is completely blocked by a renin inhibitor. We have also observed activation of the intracellular RAS in cardiac fibroblasts. Significantly, iAng II levels, cardiac myocyte apoptosis, oxidative stress, and cardiac fibrosis are markedly increased in the heart of diabetic rats, which are normalized by renin inhibition, not by ACE or AT1 inhibition. These observations suggest a significant role of iAng II in diabetic cardiomyopathy, with the implications that AT1 receptor antagonists and ACE inhibitors would be ineffective in blocking iAng II effects. The cellular mechanisms that lead to intracellular expression of RAS components and Ang II synthesis, in high glucose conditions, and the mechanism of iAng II actions are not known. We have reported that iAng II causes cardiac hypertrophy; however, the relative significance of iAng II versus extracellular Ang II in pathophysiological conditions is not known. In this proposal, we will test the hypothesis that metabolic changes, induced by high glucose, result in intracellular synthesis of Ang II, which has a significant role in development of diabetic cardiomyopathy, through interaction with novel intracellular proteins. We will use cardiac myocytes and fibroblasts, both neonatal and adult, to identify hyperglycemia-induced cellular events, such as the hexosamine biosynthesis pathway, protein O-glycosylation and oxidative stress, on regulation of the intracellular RAS, by pharmacological and genetic approaches. We will identify and characterize novel iAng II interacting proteins, using affinity binding and mass spectrometry approaches. AT1 receptor deficient mice will be utilized to determine the specific role of iAng II in diabetic cardiac dysfunction. The proposed studies will identify novel mechanisms of Ang II actions in the heart and provide for new strategies in clinical interventions for diabetic cardiomyopathy. PUBLIC HEALTH RELEVANCE: This research will focus on the development of novel strategies for the treatment of patients with diabetes. Blockade of the intracellular renin-angiotensin system, may provide substantial benefit for the prevention/treatment of diabetic cardiomyopathy, compared to standard therapy with angiotensin receptor blockers and angiotensin converting enzyme inhibitors.