Therapeutic Modulation of Endothelial Cell Tetrahydrobiopterin

内皮细胞四氢生物蝶呤的治疗调节

• 批准号: 8379505
• 负责人: David G Harrison
• 金额: $ 33.61万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8379505
• 关键词: Affect; Alanine; Anabolism; Animal Model; Animals; Antibodies; Aorta; Area; Arginine; Arterial Fatty Streak; Aspartate; Atherosclerosis; Binding; Biological Assay; Blood Circulation; Blood Vessels; Blood flow; Cardiac; Cells; Coronary artery; Cultured Cells; Data; Detection; Development; Disease; Dissociation; Distal; Electrons; Endothelial Cells; Endothelium; Enhancing Lesion; Enzymes; Feedback; GTP Cyclohydrolase; Genetic; Guanosine Triphosphate; High Pressure Liquid Chromatography; Human; Inflammatory Response; Injury; Instruction; Knock-in Mouse; Laboratories; Lesion; Lipids; Measures; Methods; Modification; Molecular; Monitor; Mus; Nitric Oxide Synthase; Oxygen; Pharmaceutical Preparations; Pharmacology; Phenotype; Phosphorylation; Positioning Attribute; Prevention; Production; Proteins; Research; Risk Factors; Serine; Site; Superoxides; Testing; Therapeutic; Tissues; Toxicology; Vasodilation; atherogenesis; casein kinase; cofactor; embryonic stem cell; gene discovery; genetic regulatory protein; high throughput screening; in vivo; mouse model; novel; novel strategies; prevent; programs; response; shear stress; tetrahydrobiopterin; treatment strategy; tripolyphosphate

PROJECT SUMMARY (See instructions): Regions of the circulation, such as the carotid bulb, the proximal coronary arteries and the distal aorta are exposed to disturbed, often oscillatory flow and are predisposed to development of atherosclerosis. There is currently no therapy to prevent lesion development at these sites. Tetrahydrobiopterin (BH4) is a critical cofactor for the nitric oxide synthase (NOS) enzymes and in its absence, the NOS enzymes become uncoupled, so that they produce superoxide (O2-) rather than NO. Our laboratory has discovered a new mechanism by which endothelial cells modulate BH4 levels in response to shear. We found that laminar shear stimulates BH4 levels by 30-fold, and increases the activity of GTP cyclohydrolase-1 (GTPCH-1), the rate-limiting enzyme for BH4 production, by a similar extent. Shear stress dissociates GTPCH-1 from its feedback regulatory protein (GFRP), and this allows phosphorylation of GTPCH-1 on serine 81 (serine 72 in the mouse) by casein kinase alpha prime. In contrast, oscillatory shear stress does not dissociate GFRP and GTPCH-1, and does not cause GTPCH-1 phosphorylation, causing BH4 levels to be insufficient. This reduces NO production and increases O2" levels, and markedly enhances atherosclerotic lesion development in a mouse model with disturbed carotid flows. We therefore propose that GTPCH-1 phosphorylation represents a critical switch that alters endothelial cell phenotype, promotes oxidative injury, reduces NO production and predisposes to atherosclerosis. To test this hypothesis in vivo, we have successfully targeted knock-in of both an aspartate (to mimic phosphorylation) and an alanine (to prevent phosphorylation) in murine embryonic stem cells. In aims 1, we will to study mice with the aspartate knock-in (KI[GCH/S72D] mice) to determine if maintaining GTPCH-1 activation prevents atherosclerosis and preserves NO function. In aim 2, we will study mice in which GTPCH-1 cannot be activated by shear ( K I [GCH/S72A] mice), hypothesize that these animals will have enhanced atherosclerosis lesion. In the final aim, we plan to continue studies to discover new molecules that cause dissociation of GFRP and GTPCH-1. To accomplish this, we have developed a high-throughput screening assay and have already used this to screen 34,000 molecules. We have promising preliminary data to show that this approach will allow discovery of genes that can increase endothelial cell BH4 levels. We propose that agents discovered using this approach will provide of novel approach for prevention of atherosclerosis at sites of disturbed flow in vivo.

项目摘要（参见说明）： 循环区域，例如颈动脉球、近端冠状动脉和远端主动脉，暴露于受干扰的、通常是振荡的血流中，并且容易发生动脉粥样硬化。目前没有治疗方法可以预防这些部位的病变发展。四氢生物蝶呤 (BH4) 是一氧化氮合酶 (NOS) 的关键辅助因子，如果缺少它，NOS 酶就会解偶联，从而产生超氧化物 (O2-) 而不是 NO。我们的实验室发现了内皮细胞响应剪切力调节 BH4 水平的新机制。我们发现层流剪切可将 BH4 水平刺激 30 倍，并以类似程度提高 BH4 生产限速酶 GTP 环水解酶-1 (GTPCH-1) 的活性。剪切应力使 GTPCH-1 与其分离 反馈调节蛋白 (GFRP)，这使得酪蛋白激酶 α Prime 能够磷酸化 GTPCH-1 的丝氨酸 81（小鼠中的丝氨酸 72）。相反，振荡剪切应力不会解离GFRP和GTPCH-1，也不会引起GTPCH-1磷酸化，从而导致BH4水平不足。这减少了 NO 的产生并增加了 O2" 水平，并显着增强了颈动脉血流紊乱的小鼠模型中动脉粥样硬化病变的发展。因此，我们认为 GTPCH-1 磷酸化是改变内皮细胞表型、促进氧化损伤、减少 NO 产生的关键开关。为了在体内验证这一假设，我们成功地敲入了天冬氨酸（以模拟磷酸化）和天门冬氨酸。小鼠胚胎干细胞中的丙氨酸（防止磷酸化） 在目标 1 中，我们将研究天冬氨酸敲入小鼠（KI[GCH/S72D] 小鼠），以确定维持 GTPCH-1 激活是否可以预防动脉粥样硬化并保留 NO 功能。在目标 2 中，我们将研究 GTPCH-1 不能被剪切激活的小鼠（K I [GCH/S72A] 小鼠），假设这些动物将具有增强。最终目标是，我们计划继续研究发现导致 GFRP 和 GTPCH-1 解离的新分子。为了实现这一目标，我们开发了一种高通量筛选方法，并已使用该方法筛选了 34,000 个分子。我们有希望的初步数据表明，这种方法将允许发现可以增加内皮细胞 BH4 水平的基因。我们建议使用这种方法发现的药物将为体内血流紊乱部位预防动脉粥样硬化提供新方法。