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（请参阅说明）： 循环的区域，例如颈动脉，冠状动脉近端动脉和远端主动脉，暴露于受干扰的，通常振荡的流动，并且易于发展动脉粥样硬化。目前尚无预防这些部位病变发展的疗法。四氢无生物蛋白酶（BH4）是一氧化氮合酶（NOS）酶的关键辅助因子，在没有它的情况下，NOS酶变得不偶联，因此它们产生了超氧化物（O2-），而不是没有。我们的实验室发现了一种新机制，该机制通过该机制调节BH4水平响应剪切。我们发现层流剪切可刺激BH4水平30倍，并增加GTP环氢酶-1（GTPCH-1）的活性，这是BH4产生的速率限制酶，其程度相似。剪切应力将GTPCH-1从其 反馈调节蛋白（GFRP），这允许通过酪蛋白激酶alpha Prime在丝氨酸81（小鼠中的丝氨酸72）上磷酸化。相反，振荡剪切应力不会解离GFRP和GTPCH-1，并且不会引起GTPCH-1磷酸化，从而导致BH4水平不足。这可以降低没有生产并增加O2的水平，并显着增强了具有干扰的颈动脉流动的小鼠模型中的动脉粥样硬化病变的发育。因此，我们建议GTPCH-1磷酸化代表了一个关键开关，从而改变了内皮细胞表型的改变，可以促进氧化性损伤，从而促进了这种障碍，从而促进了这一成功的范围，从而使您的近距离化viv viv viv nodecor nodecor inthersisclersis。促进了该障碍。天冬氨酸（模仿磷酸化）和丙氨酸（预防鼠类胚胎干细胞）中的丙氨酸（AIM 1）。通过剪切（GCH/S72A]小鼠）激活，假设这些动物会在最终目标中增强动脉粥样硬化病变。为此，我们开发了高通量筛选测定法，并且已经将其用于筛选34,000个分子。我们已经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特工将提供新的方法，以预防体内流动障碍部位的动脉粥样硬化。