Mechanisms Underlying Impaired Diabetic Wound Healing

摘要 持续未解决的炎症损害糖尿病伤口愈合。创伤的功能与转归 单核细胞/巨噬细胞（单核/巨噬细胞）是伤口炎症结果的关键。该提案基于 来自活的功能性伤口巨噬细胞（WM）和来自慢性炎症的伤口液的观察结果 然后使用实验模型进一步开发患者的伤口。鉴于目前的模糊性， 巨噬细胞命名法，并提出了与M1/M2命名法不匹配的wm命名法，对于这一建议，我们 基于促炎（MIBINF）或促消退/愈合（MIBHEAL）功能状态对WM进行分类。我们 已经报道了i）成功的胞浆细胞增多症（Eff）有助于消除炎症;和ii）胞浆细胞增多症 糖尿病伤口愈合严重受损（Efflo），导致未解决的炎症，和iii）Efflo的纠正 重组MFG-E8（rMFG-E8）在糖尿病伤口愈合中缓解炎症并促进伤口愈合 治愈我们的实验室是第一个报道miRNA-21在调节创伤中的关键作用的实验室。 炎症我们最近报道了一个新范式的框架，提出了wm的可塑性， 伤口部位是伤口炎症状态的主要决定因素。目前的建议旨在 重点在两个新方面表征该范例：（i）在细胞外捕获miR货物， 伤口炎症部位的囊泡（EV）决定伤口炎症的命运和炎症状态;和（ii） 在糖尿病伤口炎症部位，miR表观遗传学沉默（甲基化）， 炎症持续存在。提出了以下三个目标：目标1：检测miR-21和红细胞增多症的关键性 伤口部位单核细胞/巨噬细胞命运的决定因素。§1.1从m inf转换的唯一的wm子集 miR-21包封在伤口部位的细胞外囊泡（EV）中，被递送到伤口，引起这种损伤。 转换.§1.2 wm的另一个子集经历了m inf m分解;这个子集在分解中起着关键作用。 伤口发炎和愈合的过程目的2：确定糖尿病如何重定向导致肥胖的命运 治愈的脱轨§2.1糖尿病条件导致wm中的miR-21表观遗传沉默（miR-21 lo）。等 缺陷，与受损的红细胞增多症（Efflo）结合，使糖尿病患者的体重下降; §2.2在糖尿病患者中， miR-21和红细胞增多症促进糖尿病性微血管炎/微血管炎恢复愈合。新型巨噬细胞靶向 脂质纳米颗粒（LNPmφ）将纠正糖尿病miR-21 lo。目的3：研究活组织和伤口边缘组织活检 从患者的糖尿病伤口中分离，测试：§3.1从wm到m F（或m heal）的转变是否 在控制不佳的糖尿病患者（HbA 1c>9）中受损，其中miR-21在表观遗传学上是沉默的; §3.2校正 miR-21和红细胞增多症使糖尿病患者从微血管炎/微血管炎进展。

ABSTRACT Persistent unresolved inflammation impairs diabetic wound healing. The function and fate of wound monocyte/macrophages (mϕ) hold the key to the outcome of wound inflammation. This proposal builds on observations originating from live functional wound macrophages (wmϕ) and wound fluid derived from chronic wounds of patients which were then developed further using experimental models. Given current ambiguity in macrophage nomenclature, and proposed misfit of wmϕ with the M1/M2 nomenclature, for this proposal we classify wmϕ based on the pro-inflammatory (mϕinf) or pro-resolution/healing (mϕheal) functional states. We have reported that i) successful wmϕ efferocytosis (Eff) helps resolve inflammation; and ii) Efferocytosis severely impaired (Efflo) in wmϕ of diabetic wounds causing unresolved inflammation, and iii) correction of Efflo with recombinant MFG-E8 (rMFG-E8) in wmϕ of diabetic wounds resolves inflammation and promotes wound healing. Our laboratory was the first to report a critical role of miRNA-21 in the regulation of wound inflammation. We recently reported the framework of a new paradigm proposing that the plasticity of wmϕ at the wound-site is a major determinant of the state of wound inflammation. The current proposal seeks to characterize this paradigm with emphasis on two novel aspects: (i) that miR cargo captured in extracellular vesicles (EVs) at the site of wound inflammation determine the fate of wmϕ and state of inflammation; and (ii) that at the site of diabetic wound inflammation miR is epigenetically silenced (methylated) such that inflammation persists. The following three aims are proposed: Aim 1: Test miR-21 and efferocytosis as critical determinants of monocyte/macrophage fate at the wound-site. §1.1 A unique subset of wmϕ convert from mϕinf  mϕF; miR-21 encapsulated in wound-site extracellular vesicles (EV) are delivered to wmϕ to cause such conversion. §1.2 Another subset of wmϕ undergoes mϕinfmϕheal; this subset plays a critical role in resolution of wound inflammation and healing. Aim 2: Determine how diabetes redirects the fate of wmϕ causing derailment of healing. §2.1 Diabetic conditions cause miR-21 epigenetic silencing in wmϕ (miR-21lo). Such deficit, in combination with impaired efferocytosis (Efflo), stalls wmϕ in mϕinf; §2.2 In diabetic wmϕ, correction of miR-21 and efferocytosis advances diabetic mϕinfmϕheal/mϕF resuming healing. Novel macrophage-targeted lipid nanoparticle (LNPmφ) will correct diabetic miR-21lo. Aim 3: Study live wmϕ and wound-edge tissue biopsies isolated from diabetic wounds of patients testing whether: §3.1 transition of wmϕ to mϕF (or mϕheal) is compromised in poorly controlled diabetics (HbA1c>9) where miR-21 is epigenetically silent; §3.2 correction of miR-21 and efferocytosis advances diabetic wmϕ from mϕinfmϕheal/mϕF.