Tetrahydrobiopterin & eNOS uncoupling regulation of EPC function and wound healin

四氢生物蝶呤

• 批准号: 7870828
• 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/7870828
• 关键词: Affect; Aging; Amputation; Angiogenic Proteins; Autologous; Biological Availability; Blood Vessels; Blood flow; Bone Marrow; Cell Therapy; Cell physiology; Cells; Clinical; Complication; Data; Defect; Diabetes Mellitus; Diabetic Angiopathies; Diabetic mouse; Diabetic wound; Enzymes; Equilibrium; 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; Mus; Nitric Oxide; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Play; Population; Publishing; Reactive Oxygen Species; Refractory; Regulation; Reporting; Research; Role; Site; Stem cells; Streptozocin; Superoxides; Testing; Therapeutic; Time; Tissues; Transgenic Mice; Trauma; Treatment outcome; Vascular Diseases; 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. 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.

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.