Combined effects of aging and type 2 diabetes on wound healing in a humanized mouse model

摘要 衰老是2型糖尿病患者发生慢性、不愈合足部溃疡的重要危险因素 糖尿病（T2 DM）。老年T2 DM患者的糖尿病足溃疡具有破坏性后果， 截肢和全因死亡的风险增加。有一个迫切的，未满足的需求，以开发治疗 治疗该群体中的这些不愈合的伤口;然而， 开发对人类疾病具有高度可转化性的生物疗法和干细胞疗法 不存在.因此，本提案的总体目标是开发老年T2 DM人源化小鼠 模型，以研究衰老和T2 DM的联合作用使人类糖尿病发生失调的机制。 T2 DM患者在伤口愈合过程中的免疫系统，并基于这些新的见解测试治疗方法。 为实现这一目标，提出了两个具体目标。具体目标1的目的是开发一种人性化的 用来自老年T2 DM人供体的CD 34+造血干细胞（HSC）移植的小鼠模型研究 伤口愈合假设人源化小鼠移植来自老年人的CD 34 + HSC， 糖尿病忠实地概括了不愈合的伤口表型和伤口的偏斜极化， 在人类T2 DM患者中已经记录了浸润性巨噬细胞。该模型基于我们的 已发表和未发表的研究结果表明，T2 DM和衰老通过氧化应激损害伤口愈合， 依赖HSC自治机制。因此，与年龄相关的T2 DM损伤的伤口愈合将 由来自老年T2 DM患者的供体HSC赋予。具体目标2的目的是确定 ATLAS疗法对移植有老化T2 DM HSC的人源化小鼠中伤口愈合的影响。的 假设是，在移植入T2 DM供体之前，ATLAS处理来源于老年T2 DM人供体的HSC， NSG-SGM 3小鼠降低HSC氧化应激并增加一氧化氮生物利用度， 伤口中的巨噬细胞数量和极化。我们的研究结果表明，在T2 DM小鼠创伤模型中， 治疗ATLAS，L-精氨酸，四氢生物蝶呤和L-抗坏血酸的组合，降低HSC的氧化 应激和增加HSC NO生物利用度，恢复正常的伤口愈合。的发展 提出的人源化小鼠模型弥合了鼠伤口愈合模型和人伤口愈合模型之间的主要差距。 老年T2 DM患者的不愈合足部溃疡;因此，创建了一个高度可翻译的工具， 治疗老年T2 DM患者不愈合伤口的新型生物疗法满足了一个主要的未满足需求 对于这些患者。由于这个项目的结果，我们希望大大减少痛苦， 这个毁灭性的问题不成比例地影响着全世界的老年人。

Abstract Aging is a powerful risk factor for the development of chronic, non-healing foot ulcers in people with type 2 diabetes (T2DM). Diabetic foot ulcers in older adults with T2DM have devastating consequences, leading to increased risks of amputation and all-cause mortality. There is a compelling, unmet need to develop therapies to treat these non-healing wounds in this population; however, an appropriate animal model for use in the development of biotherapeutics and stem cell therapies with a high degree of translatability to human disease does not exist. Therefore, the overall goal of this proposal is to develop an aged-T2DM humanized mouse model to study the mechanisms by which the combined effects of aging and T2DM dysregulate the human immune system of T2DM patients during wound healing and to test therapeutics based on these new insights. To achieve this goal, two Specific Aims are proposed. The purpose of Specific Aim 1 is to develop a humanized mouse model engrafted with CD34+ hematopoietic stem cells (HSCs) from aged-T2DM human donors to study wound healing. The hypothesis is that humanized mice engrafted with CD34+ HSCs from older adults with diabetes faithfully recapitulate the non-healing wound phenotype and skewed polarization of wound- infiltrating macrophages that has been documented in human T2DM patients. This model is based on our published and unpublished findings that T2DM and aging impair wound healing by an oxidant stress- dependent HSC autonomous mechanism. Thus, the aging-T2DM-associated impairment in wound healing will be conferred by the donor HSCs from aged-T2DM patients. The purpose of Specific Aim 2 is to determine the effect of ATLAS therapy on wound healing in humanized mice engrafted with aged-T2DM HSCs. The hypothesis is that ATLAS treatment of HSCs derived from aged T2DM human donors prior to engraftment into NSG-SGM3 mice reduces HSC oxidant stress and increases nitric oxide bioavailability that restores normal macrophage number and polarization in wounds. Our results show that in a T2DM murine model of wound healing ATLAS, a combination of L-arginine, tetrahydrobiopterin, and L-ascorbate, decreases HSC oxidative stress and increases HSC NO bioavailability that restores normal wound healing. The development of the proposed humanized mouse model bridges a major gap between murine wound healing models and human non-healing foot ulcers in older adults with T2DM; thus, the creation of a highly translatable tool to develop novel biological therapies to treat non-healing wounds in older adults with T2DM fulfills a major unmet need for these patients. As a consequence of the results of this project, we hope to greatly reduce the suffering from this devasting problem that disproportionately affects older adults worldwide.