Tissue reprogramming in diabetic wound healing

摘要 据估计，2%的美国人口受到慢性伤口的影响。大约15-20%的糖尿病患者在其一生中会出现皮肤伤口，大多数人会发展为慢性。据估计，只有56%的糖尿病溃疡性伤口在其最初表现后存活超过5年。该提案旨在利用非病毒体内组织重编程的新再生原理实现糖尿病伤口的成功血管化。组织纳米转染技术（TNT），最近报告的PI，实现组织重编程，在体内免疫监视下，使用局部电泳传递系统，使一种新的纳米制造的硅芯片硬件。所提出的研究源自申请人先前关于TNT递送特异性转录因子Etv 2、Foxc 2和Fli 1（EFF）以实现体内血管生成重编程的作用的工作。因此，EFF被发现为能够使缺血组织血管化的血管生成质粒鸡尾酒。所提出的工作是基于第一个证据，而不是使用质粒鸡尾酒已被许多团体用来实现细胞重编程，一个单一的反义寡核苷酸（阿索）是非常有效的生产诱导血管生成细胞（iV）从皮肤成纤维细胞。初步数据显示，在体内条件下，阿索可以改善糖尿病伤口组织的灌注。由于已知糖尿病会导致内皮功能障碍和血管病变，因此将测试阿索的TNT递送以改善伤口结局。阿索的血管生成作用是通过打开一个主要的血管开关Fli 1来实现的。这项工作将阐明糖尿病皮肤伤口灌注的ASO诱导灌注的分子机制（目的1）。为了能够进行侵入性机制研究，因此在目的2中提出了人源化小鼠模型。本研究的主要目的有两个：目的1：阐明阿索促进糖尿病小鼠伤口愈合的意义及其分子机制。1.1在糖尿病的鼠模型中，通过TNT的阿索递送挽救了皮肤伤口的愈合;阿索递送通过从miR-200 b依赖性基因沉默中挽救Flil来改善糖尿病伤口血管化; 1.3ASO治疗后，伤口部位成纤维细胞的亚群通过获得血管生成内皮样特征来促进预先存在的脉管系统。目的2：在人源化糖尿病小鼠模型中测试阿索重编程的显著性。2.1在人源化NSG糖尿病小鼠模型中，通过TNT递送阿索加速伤口愈合并改善伤口血管化。 这项工作为未来尝试重新编程人类皮肤基质以改善伤口部位的灌注奠定了基础。

ABSTRACT An estimated 2% of US population is affected by chronic wounds. About 15–20% of all diabetic patients develop skin wounds across their lifespan, the majority evolving toward chronicity. It is estimated that only 56% of diabetics with ulcerative wounds survive more than 5 years after their initial manifestation. This proposal seeks to achieve successful vascularization of diabetic wounds utilizing the novel regenerative principles of non-viral in vivo tissue reprogramming. Tissue nanotransfection technology (TNT), recently reported by the PI, achieves tissue reprogramming, under immune surveillance in vivo, using a topical electrophoretic delivery system enabled by a novel nanofabricated silicon chip hardware. The proposed studies emanate from prior work by the applicant on the role of TNT delivery of specific transcription factors Etv2, Foxc2, and Fli1 (EFF) to achieve vasculogenic reprograming in vivo. EFF was thus discovered as a vasculogenic plasmid cocktail capable of vascularizing ischemic tissue. The proposed work is based on first evidence that instead of the use of plasmid cocktails that have been used by many groups to achieve cell reprogramming, a single anti-sense oligonucleotide (ASO) is highly effective in producing induced vasculogenic cells (iV) from skin fibroblasts. Preliminary data show that under in vivo conditions, ASO can improve perfusion of diabetic wound tissue. Because diabetes is known to cause endothelial dysfunction and vasculopathy, TNT delivery of ASO will be tested to improve wound outcomes. Vasculogenic effects of ASO is achieved by turning on a major vascular switch Fli1. The proposed work will delineate the molecular mechanisms of ASO-induced perfusion of diabetic cutaneous wound perfusion (Aim 1). To enable invasive mechanistic studies, a humanized mouse model has been thus proposed in Aim 2. The following two specific aims are proposed: Aim 1: Elucidate the significance and molecular mechanisms by which ASO induces accelerated wound closure in diabetic mice. 1.1 ASO delivery by TNT rescues healing of cutaneous wounds in murine models of diabetes; 1.2 ASO delivery improves diabetic wound vascularization by rescuing Fli1 from miR-200b dependent gene silencing; 1.3 A subset of wound-site fibroblasts contribute to the pre-existing vasculature by acquiring vasculogenic endothelial-like characteristics post-ASO treatment. Aim 2: Test significance of ASO reprogramming in a humanized diabetic mouse model. 2.1 ASO delivery by TNT accelerates wound healing and improves wound vascularization in a humanized NSG diabetic mouse model. The proposed work lays the foundation stone to future trials attempting to reprogram human skin stroma towards improved perfusion of the wound-site.