Tetrahydrobiopterin & eNOS uncoupling regulation of EPC function and wound healin

四氢生物蝶呤

• 批准号: 8838109
• 负责人: Alex F Chen
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-10-01 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8838109
• 关键词: Affect; Aging; Amputation; Angiogenic Proteins; Autologous; Blood flow; Bone Marrow; Cell Therapy; Cell physiology; Cells; Clinical; Complication; Data; Defect; Diabetes Mellitus; Diabetic mouse; Diabetic wound; Enzymes; Functional disorder; GTP Cyclohydrolase I; General Population; Generations; Goals; Healed; Health; Home environment; Hyperglycemia; Impaired wound healing; Insulin-Dependent Diabetes Mellitus; Knockout Mice; Knowledge; Laboratories; Lead; Leg Ulcer; Mediating; Microvascular Dysfunction; Mus; Nitric Oxide; Oxidative Stress; Pathway interactions; Play; Population; Publishing; Reactive Oxygen Species; Refractory; Regulation; Reporting; Research; Role; Site; Stem cells; Streptozocin; Superoxides; Testing; Therapeutic; Time; Transgenic Mice; Trauma; Treatment outcome; Veterans; Work; Wound Healing; angiogenesis; base; cofactor; combat; design; diabetic; diabetic patient; diabetic wound healing; healing; human NOS3 protein; improved; in vivo; novel; overexpression; postnatal; prevent; restoration; tetrahydrobiopterin; therapeutic angiogenesis; vasculogenesis; wound

DESCRIPTION (provided by applicant): Refractory wounds in diabetes are a particularly challenging clinical problem that often leads to amputations. It is a major health problem in diabetic population including many veterans. Angiogenesis is a rate-limiting step in normal wound repair but is severely impaired as a diabetic microvascular complication, resulting in diminished blood flow in the non-healing wounds. The circulating endothelial progenitor cells (EPCs), derived from the bone marrow, home to the wounding site to form new vessels, are dysfunctional in diabetes. The cellular mechanisms underlying diabetic EPC dysfunction, however, are poorly understood. Recent studies show that EPCs from endothelial nitric oxide synthase (eNOS) knockout mice displays markedly impaired angiogenic function, suggesting that one mechanism of EPC dysfunction is decreased nitric oxide (NO). In contrast, a cardinal feature - in diabetes is hyperglycemia-mediated superoxide anion (O2 ) overproduction, which has been reported to impair EPC function. Accumulating evidence indicates that eNOS is a bi-functional enzyme that - becomes "uncoupled" and will produce O2 instead of NO when its essential cofactor tetrahydrobiopterin (BH4) is oxidized by reactive oxygen species (ROS). Recent studies indicate that eNOS is uncoupled in streptozotocin (STZ)-induced type 1 diabetes. In this setting, uncoupled eNOS exaggerates oxidative stress. Therefore, it is extremely important to understand the dynamic regulation of BH4 synthesis on EPC function in vivo. BH4 synthesis is controlled by its rate-limiting enzyme GTP cyclohydrolase I (GTPCH I). Recent studies demonstrate that circulating EPCs in diabetic patients have reduced BH4 level, resulting in excessive ROS and decreased NO. Yet remarkably little is known about how GTPCH/BH4 pathway regulates EPC function in diabetes. Lack of such knowledge is a significant problem, because without it, acquiring the ability to rescue EPC dysfunction to augment therapeutic angiogenesis is highly unlikely. Our long-term goal is to understand how impaired wound healing in diabetes can be therapeutically ameliorated. The objective of this proposal, which is a step toward attaining that goal, is to determine how GTPCH/BH4 pathway regulates EPC angiogenesis in diabetic wound healing. Our central hypothesis is that eNOS uncoupling contributes to EPC dysfunction in STZ-induced diabetic mice via inducing anti-angiogenic protein thrombospodin-1 (TSP-1), which may be retarded by GTPCH overexpression in vivo, resulting in eNOS recoupling and improved efficacy of EPC cell therapy on refractory diabetic wounds. Thus, the rationale for the proposed research is that discovery of the defects in diabetic EPCs and the means to correct them could lead to autologous cell therapies for diabetic wounds. Our hypothesis was formulated after a careful analysis of published work in the field and the generation of some key preliminary data in our own laboratory. We plan to test our central hypothesis and accomplish our objective by pursuing two Specific Aims, using some novel planned approaches including the endothelial-specific GTPCH I transgenic mice (Tg-GCH) and GTPCH/BH4 deficient hph-1 mice. In Aim 1, we will elucidate how eNOS uncoupling impairs EPC angiogenesis. In Aim 2, we will determine if preventing eNOS uncoupling by increasing BH4 in EPCs improves the efficacy of EPC cell therapy on diabetic wounds. The major significance of the proposed research is that it will, for the first time, determine how eNOS uncoupling and GTPCH/BH4 pathway regulate EPC function and wound repair in an integrated fashion, which may provide a mechanistic basis for the restoration of EPC function and therapeutic angiogenesis to combat refractory diabetic wounds, a devastating complication that affects thousands of aging veterans as well as the general population.

描述（由申请人提供）： 糖尿病难治性伤口是一个特别具有挑战性的临床问题，常常导致截肢。这是糖尿病人群（包括许多退伍军人）的一个主要健康问题。血管生成是正常伤口修复的限速步骤，但作为糖尿病微血管并发症，血管生成受到严重损害，导致不愈合伤口中的血流减少。循环内皮祖细胞（EPC）源自骨髓，在受伤部位形成新血管，在糖尿病中功能失调。然而，糖尿病 EPC 功能障碍的细胞机制尚不清楚。最近的研究表明，内皮一氧化氮合酶 (eNOS) 敲除小鼠的 EPC 表现出明显受损的血管生成功能，这表明 EPC 功能障碍的机制之一是一氧化氮 (NO) 减少。相比之下，糖尿病的一个主要特征是高血糖介导的超氧阴离子 (O2) 过量产生，据报道这会损害 EPC 功能。越来越多的证据表明，eNOS 是一种双功能酶，当其必需的辅助因子四氢生物蝶呤 (BH4) 被活性氧 (ROS) 氧化时，它会“解偶联”并产生 O2 而不是 NO。最近的研究表明，eNOS 在链脲佐菌素 (STZ) 诱导的 1 型糖尿病中呈解偶联状态。在这种情况下，未偶联的 eNOS 会加剧氧化应激。因此，了解BH4合成对体内EPC功能的动态调节极为重要。 BH4 的合成由其限速酶 GTP 环水解酶 I (GTPCH I) 控制。最近的研究表明，糖尿病患者的循环 EPC 降低了 BH4 水平，导致 ROS 过多和 NO 减少。然而，人们对 GTPCH/BH4 通路如何调节糖尿病中 EPC 功能知之甚少。缺乏此类知识是一个重大问题，因为如果没有这些知识，就不太可能获得挽救 EPC 功能障碍以增强治疗性血管生成的能力。 我们的长期目标是了解如何通过治疗方法改善糖尿病患者的伤口愈合受损。该提案的目的是确定 GTPCH/BH4 通路如何调节糖尿病伤口愈合中的 EPC 血管生成，这是实现该目标的一步。我们的中心假设是，eNOS 解偶联通过诱导抗血管生成蛋白血小板球蛋白-1 (TSP-1) 导致 STZ 诱导的糖尿病小鼠的 EPC 功能障碍，TSP-1 可能会因体内 GTPCH 过度表达而延迟，从而导致 eNOS 重新偶联并提高 EPC 细胞治疗难治性糖尿病伤口的疗效。因此，这项研究的基本原理是，发现糖尿病 EPC 的缺陷以及纠正这些缺陷的方法可能会导致糖尿病伤口的自体细胞疗法。我们的假设是在仔细分析该领域已发表的工作并在我们自己的实验室生成一些关键的初步数据后制定的。我们计划通过追求两个特定目标来测试我们的中心假设并实现我们的目标，使用一些新的计划方法，包括内皮特异性 GTPCH I 转基因小鼠 (Tg-GCH) 和 GTPCH/BH4 缺陷 hph-1 小鼠。在目标 1 中，我们将阐明 eNOS 解偶联如何损害 EPC 血管生成。在目标 2 中，我们将确定通过增加 EPC 中的 BH4 来预防 eNOS 解偶联是否可以提高 EPC 细胞疗法对糖尿病伤口的疗效。该研究的主要意义在于，它将首次确定eNOS解偶联和GTPCH/BH4通路如何以整合的方式调节EPC功能和伤口修复，这可能为恢复EPC功能和治疗性血管生成以对抗难治性糖尿病伤口提供机制基础，难治性糖尿病伤口是一种影响数千名老年退伍军人和普通大众的毁灭性并发症。