Atherosclerosis Mechanisms: Angiotensin II production and action

动脉粥样硬化机制：血管紧张素 II 的产生和作用

• 批准号: 9903447
• 负责人: Alan Daugherty
• 金额: $ 50.12万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9903447
• 关键词: Address; Amino Acid Sequence; Androgens; Angiotensin II; Angiotensin Type 1a Receptor; Angiotensinogen; Arterial Fatty Streak; Atherosclerosis; Binding; Breeding; Cell Culture System; Cleaved cell; Color; Conserved Sequence; Data; Development; Elements; Endocrine system; Endothelial Cells; Enzymes; Fibroblasts; Genes; Hepatocyte; Human; In Vitro; Infusion procedures; Kidney; LDL-Receptor Related Protein 2; Lesion; Leukocytes; Literature; Location; Low Density Lipoprotein Receptor; LoxP-flanked allele; Mediating; Methods; Mus; Mutagenesis; Nephrons; Pathway interactions; Peptidyl-Dipeptidase A; Pharmacology; Play; Production; Proteins; Renin; Renin-Angiotensin System; Reproducibility; Role; Site; Smooth Muscle Myocytes; Source; Surface Plasmon Resonance; System; Techniques; Testing; Therapeutic; Transgenes; Transgenic Mice; Viral; base; cell type; enzyme deficiency; experimental study; hypercholesterolemia; insight; member; mouse model; mutant; novel therapeutic intervention; prevent; promoter; protein structure; receptor; urinary

Abstract The renin angiotensin system (RAS) plays a critical role in the development of atherosclerosis. Mechanisms by which the RAS contributes to atherosclerosis have been focusing on effects of angiotensin II (AngII) production and its action through AT1a receptors within atherosclerotic lesions. On the basis of our preliminary data, we challenge this concept and propose a new hypothesis: Renal AngII production through a megalin-mediated pathway in proximal convoluted tubules (PCTs) promotes atherosclerosis via its local stimulation of AT1a receptors. Angiotensinogen (AGT), the substrate of the RAS, derived from hepatocytes is filtered through glomeruli and retained by megalin, a member of LDL receptor superfamily, in PCTs. Our protein sequence and structure analyses identified two conserved sequences that may associate with its binding to megalin. Aim 1 will define how hepatocyte-derived AGT regulates renal AngII production and contributes to atherosclerosis. We will use site-mutagenesis, surface plasmon resonance, and cell culture system to determine how AGT and megalin interact in vitro. Subsequently, we will use an adeno-associated viral (AAV) system to manipulate conserved sequences of AGT in hepatocyte-specific AGT deficient mice to determine whether conserved sequences of AGT influence renal AngII production and atherosclerosis. Effects of megalin on renal AngII production and atherosclerosis will be determined using PCT-specific megalin deficient mice. Since AGT is cleaved by two critical enzymes, renin and angiotensin-converting enzyme (ACE), to produce AngII, Aim 2 will first determine whether renin or ACE derived from PCTs contributes to renal AngII production and atherosclerosis using PCT-specific renin and ACE deficient mice, respectively. Studies proposed in Aim 1 for AGT and megalin interaction and Aim 2 for the two enzymes (renin and ACE) do not provide direct evidence whether AngII produced in PCTs contributes to atherosclerosis. To answer this question, we will use a transgenic mouse model that has restricted production of AngII in PCTs. AngII promotes atherosclerosis through AT1a receptor- mediated mechanism. Therefore, subsequent experiments will determine whether PCTs are the location for AT1a receptors to promote atherosclerosis. Completion of proposed studies will provide evidence whether renal PCTs are the major source for each classic RAS component to promote atherosclerosis. Demonstration of this new concept may change our understanding of the AngII/AT1a receptor-mediated mechanisms of atherosclerosis, which may also provide insights into developing new therapeutic strategies.

摘要 肾素血管紧张素系统（RAS）在血管紧张素转换酶的发生发展中起着关键作用。 动脉粥样硬化RAS促进动脉粥样硬化的机制已经被 关注血管紧张素II（AngII）的产生及其通过AT 1a受体的作用 动脉粥样硬化病变中。根据我们的初步数据，我们对这一概念提出质疑 并提出了一个新的假说：肾血管紧张素II的产生是通过巨蛋白介导的， 近曲小管（PCTs）中的通路通过其局部 刺激AT 1a受体。血管紧张素原（AGT）是RAS的底物， 通过肾小球过滤，并被巨蛋白（LDL的一个成员）保留 受体超家族。我们的蛋白质序列和结构分析确定了两个 可能与其与巨蛋白结合相关的保守序列。目标1将定义如何 肝细胞衍生的AGT调节肾AngII的产生并促进动脉粥样硬化。 我们将使用定点突变、表面等离子体共振和细胞培养系统来确定 AGT和megalin在体外如何相互作用。随后，我们将使用腺相关病毒 (AAV)在肝细胞特异性AGT缺陷中操纵AGT保守序列的系统 小鼠以确定AGT的保守序列是否影响肾AngII的产生， 动脉粥样硬化巨蛋白对肾血管紧张素II生成和动脉粥样硬化的影响将是 使用PCT特异性巨蛋白缺陷小鼠进行测定。由于AGT被两个关键的 酶，肾素和血管紧张素转换酶（ACE），以产生AngII，Aim 2将首先 确定来源于PCT的肾素或ACE是否有助于肾AngII的产生， 分别使用PCT特异性肾素和ACE缺陷小鼠的动脉粥样硬化。研究 在目标1中提出了AGT和巨蛋白相互作用，目标2中提出了两种酶（肾素和 ACE）没有提供直接证据证明PCT中产生的AngII是否有助于 动脉粥样硬化为了回答这个问题，我们将使用一个转基因小鼠模型， 限制PCT中AngII的生产。血管紧张素II通过AT 1a受体促进动脉粥样硬化- 中介机制。因此，随后的实验将确定PCTs是否是 AT 1a受体促进动脉粥样硬化的位置。完成拟议的研究将 提供证据证明肾脏PCT是否是每种经典RAS组分的主要来源， 促进动脉粥样硬化。这一新概念的演示可能会改变我们对 AngII/AT 1a受体介导的动脉粥样硬化机制，这也可能提供 对开发新的治疗策略的见解。