Tetrahydrobiopterin & eNOS uncoupling regulation of EPC function and wound healin

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，通常会导致截肢。这是糖尿病人群中的一个主要健康问题，包括许多退伍军人。血管生成是正常伤口修复的限速步骤，但作为一种糖尿病微血管并发症，血管生成严重受损，导致未愈合伤口的血流量减少。循环内皮祖细胞(EPC)来源于骨髓，是形成新血管的损伤部位的家园，在糖尿病患者中功能失调。然而，糖尿病内皮祖细胞功能障碍的细胞机制却知之甚少。最近的研究表明，内皮型一氧化氮合酶(ENOS)基因敲除小鼠的EPC表现出明显的血管生成功能受损，提示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，当其必需的辅因子四氢生物蝶呤(BH4)被活性氧(ROS)氧化时，eNOS会解偶联，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型糖尿病中是解偶联的。在这种情况下，去偶联的eNOS夸大了氧化应激。因此，了解体内BH4合成对EPC功能的动态调节是非常重要的。BH4的合成受其限速酶GTP环水解酶I(GTPCH I)的控制。最近的研究表明，糖尿病患者循环内皮祖细胞降低了BH4水平，导致了过量的ROS和NO的减少。然而，值得注意的是，关于GTPCH/BH4通路如何调控糖尿病患者的EPC功能，人们知之甚少。缺乏这样的知识是一个重大的问题，因为如果没有这些知识，获得拯救EPC功能障碍以增强治疗性血管生成的能力是非常不可能的。我们的长期目标是了解如何通过治疗改善糖尿病患者受损的伤口愈合。这项建议的目的是确定GTPCH/BH4通路如何调控糖尿病伤口愈合中的EPC血管生成，这是朝着实现这一目标迈出的一步。我们的中心假设是eNOS解偶联通过诱导抗血管生成蛋白血栓蛋白-1(TSP-1)参与STZ诱导的糖尿病小鼠的EPC功能障碍，而TSP-1可能被体内过表达的GTPCH延缓，从而导致eNOS重新偶联，提高EPC细胞治疗难治性糖尿病创面的疗效。因此，提出这项研究的理由是，发现糖尿病内皮祖细胞的缺陷并纠正它们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了该领域已发表的工作并在我们自己的实验室中生成一些关键的初步数据后提出的。我们计划使用一些新的计划方法来验证我们的中心假设，并通过追求两个特定的目标来实现我们的目标，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解偶联是如何损害EPC血管生成的。在目标2中，我们将确定通过增加EPC中的BH4来防止eNOS解偶联是否提高了EPC细胞治疗糖尿病伤口的疗效。这项研究的主要意义在于，它将首次确定eNOS解偶联和GTPCH/BH4通路如何以综合的方式调节EPC功能和伤口修复，这可能为EPC功能的恢复和治疗性血管生成提供机制基础，以对抗糖尿病难治性伤口，这是一种毁灭性的并发症，影响着成千上万的老年退伍军人和普通人群。 公共卫生相关性： 与退伍军人健康的相关性许多退伍军人正在遭受衰老和糖尿病的折磨，这两种疾病经常导致血管生成受损和创伤愈合延迟，从而导致腿部溃疡和截肢。内皮祖细胞是一种新的内源性细胞群，是出生后血管生成的关键细胞效应因子，在创面愈合中起核心作用。我们目前的建议旨在探索eNOS解偶联和四氢生物蝶呤如何调控糖尿病EPC介导的血管生成。一旦获得了这些知识，就有了理论和机制基础来揭示这样一种可能性，即纠正糖尿病相关EPC缺陷的治疗策略组合可能会协同作用，并导致退伍军人糖尿病创伤的更成功的治疗结果。

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. PUBLIC HEALTH RELEVANCE: Relevance to Veterans' Health Many Veterans are suffering aging and diabetes, both of which frequently lead to impaired angiogenesis and delayed trauma healing that result in lower extremity ulcers and amputations. Endothelial progenitors cells (EPCs) are a new endogenous cell population which are the key cellular effectors of postnatal vasculogenesis and play a central role in wound healing. Our present proposal is designed to explore how eNOS uncoupling and tetrahydrobiopterin regulate EPC-mediated angiogenesis in diabetes. Once such knowledge is gained, there will be theoretic and mechanism basis to reveal the possibility that combination of therapeutic strategies to correct diabetes-related EPC deficits will likely synergize and lead to a more-successful treatment outcome for diabetic trauma in Veterans.