Exosomes in wound healing

摘要 慢性伤口是主要的医疗负担。一个常见的并发症的非- 慢性糖尿病伤口的愈合是停滞的炎症。这种脱轨的机制 因此，免疫应答特别令人感兴趣。这项建议是以我们的小说为基础的 观察伤口边缘组织的角质形成细胞（κ）与来访的巨噬细胞的相互作用 通过κ-起源的外泌体，携带遗传信息到伤口部位的巨噬细胞， 有组织和有指导的方式。基因中的EXOmotif选择它们进行外泌体包装。我们 提出miR-21是一种这样的非编码基因，其前体pre-miR-21是 包装在κ源性外泌体中并递送至伤口巨噬细胞以溶解 及时消炎。这种串扰过程在糖尿病中受到损害。我们 观察到特定κ-起源的外泌体子集的表面蛋白化学，即存在 甘露糖有利于伤口巨噬细胞的摄取。糖尿病改变这种表面蛋白 化学引起甘露糖修饰，从而损害这些外来体的摄取， 伤口巨噬细胞因此，提出了以下三个具体目标：目标1。确定 外泌体miR-21从角质形成细胞向巨噬细胞的转移是否在糖尿病患者中受损 1.1加载的角质形成细胞来源的miR-21（前和成熟）的摄取 创伤部位巨噬细胞（WMφ）的外泌体在糖尿病条件下受损。 糖尿病伤口中受损的角质形成细胞-巨噬细胞串扰损害 炎症目标2.测试是否角质形成细胞衍生的外来体的表面糖蛋白来自 伤口部位在糖尿病损害巨噬细胞靶向的条件下被糖基化。 图2.1伤口部位κ-GFPEXO表面上的甘露糖在以下条件下被糖基化： 糖尿病，使得巨噬细胞的摄取受损。2.2伤口边缘κ-GFPEXO 在糖尿病条件下发生甘露糖异构化。目标3.分离角质形成细胞- 来源于来自患有以下疾病的患者的人慢性伤口液（cWF）的外来体（HuK 14 EXO） 糖尿病，以确定这些外来体是否含有较低的miR-21（前和成熟） 与非糖尿病患者相比。目的3检验以下发现的意义： 目标1在患者环境中。这份由早期研究者重新提交的提案旨在 为专门研究外泌体细胞特异性起源的实验室奠定了基础 体液中的亚群、它们在其他细胞/组织中的靶向/归巢以及功能意义。 这项工作的前提是，这些过程的功能障碍与糖尿病患者的发病有关。 并发症，并且只有在基本过程正确的情况下才能制定救援策略 明白

ABSTRACT Chronic wounds represent a major healthcare burden. One of the common complications of non- healing chronic diabetic wounds is stalled inflammation. Mechanisms underlying such derailed immune responses are therefore of extraordinary interest. This proposal rests on our novel observation that keratinocytes (κ) of the wound-edge tissue cross-talk with visiting macrophages via κ-originating exosomes which carry genetic information to wound-site macrophages in a highly organized and directed manner. EXOmotif in genes selects them for exosomal packaging. We propose that miR-21 is one such non-coding gene the precursor of which, pre-miR-21, is packaged in κ-originating exosomes and delivered to wound macrophages to resolve inflammation in a timely manner. This process of cross-talk is compromised in diabetes. We observed that surface protein chemistry of a specific κ-originating exosomal subset, i.e. presence of mannose favors uptake by wound macrophages. Diabetes modifies this surface protein chemistry causing mannose modifications thus compromising the uptake of these exosomes by wound macrophages. The following three specific aims are thus proposed: Aim 1. Determine whether exosomal miR-21 transfer from keratinocyte to macrophage is compromised in diabetic wound-edge tissue 1.1 Uptake of keratinocyte-originating miR-21 (pre and mature) loaded exosomes by wound-site macrophages (wmφ) is compromised under conditions of diabetes. 1.2 Compromised keratinocyte-macrophage cross-talk in diabetic wound impairs resolution of inflammation. Aim 2. Test whether surface glycoproteins of keratinocyte-derived exosomes from the wound site are glycosylated under conditions of diabetes impairing macrophage targeting. 2.1 Mannose, on the surface of κ-GFPEXO at the wound site, is glycosylated under conditions of diabetes such that the uptake by macrophages is impaired 2.2 Wound-edge κ-GFPEXO undergoes mannose isomerization under conditions of diabetes. Aim 3. Isolate keratinocyte- originating exosomes (HuK14EXO) from human chronic wound fluid (cWF) from patients with diabetes to determine whether these exosomes contain lower miR-21 (pre and mature) compared to those from patients without diabetes. Aim 3 tests the significance of the findings of Aim 1 in a patient setting. This resubmitted proposal from an early-stage investigator is aimed at laying the foundation of a laboratory dedicated to studying the cell-specific origin of exosomal subsets in bodily fluids, their targeting/homing in other cells/tissues, and functional significance. The premise of such work is that malfunction of such processes is implicated in diabetic complications and that rescue strategies can only be developed if underlying processes are understood.