Converting wound scar into healing with regeneration

总结 这项资助的目的是了解SHH信号在调节再生愈合中的作用， 哺乳动物的皮肤伤口包括人类在内的大多数哺乳动物都缺乏再生皮肤愈合。后 在创伤的情况下，伤口愈合通常导致“修复”（也定义为纤维化/瘢痕形成）。这一进程未能充分 将丢失的组织恢复到原来的形式，从而形成“细胞创可贴”。哺乳动物无法再生 正常皮肤最显著地表现为在伤口疤痕中缺少包括毛囊的皮肤附属物。一 皮肤创伤中毛囊再生的主要障碍是不能重建毛囊诱导生态位 称为真皮乳头（DP）。 在这里，我们表明，SHH激活内'修复'（疤痕）伤口真皮诱导从头DP 形成HFN。这些数据清楚地表明，瘢痕伤口内的肌成纤维细胞不是 本质上无法形成再生生态位。我们假设疤痕细胞可能拥有 再生潜力，但缺少关键的发育线索，SHH。为了解决这个 假设，首先，我们将询问在纤维化伤口中活跃的Wnt信号传导是否是SHH驱动的DP所需的 形成并导致伤口中的HFN。为此，我们将对b-连环蛋白进行功能丧失研究， 在具有异位SHH激活的遗传修饰伤口中，经典Wnt信号传导的重要效应物 （目标1）。我们还将检查成熟Wnt活性成纤维细胞在Hh活化的成纤维细胞中转化为DP的潜力。 愈合后的伤口是完整的。这些实验将解决完全分化的细胞的潜在可塑性， 细胞以及Hh通路的诱导如何改变其表型（Aim 1）。二、初步单细胞 比较Hh活化的和WT伤口的RNAseq分析已经鉴定了几个基因的下游表达。 Hh活化后的BMP。我们将研究BMP信号传导在介导Hh信号传导功能中的作用， 通过对BMP受体信号传导的功能丧失和获得的研究。我们还将研究 通过Chip-Seq分析，直接Hh调节剂Gli 1和2直接结合BMP基因调节位点（Aim 2）。第三，人类伤口在许多特征上与“修复”的鼠类伤口非常相似。我们将测试 人类伤口可以通过引入病毒活性SmoM 2或可溶性SHH来响应SHH信号以产生DP 激动剂用于异种移植物和创伤模型。重要的是，我们将描绘人类真皮细胞的反应， SHH通过单细胞RNAseq分析，并将这些结果与来自小鼠伤口的结果进行比较 （目标3）。这些综合研究将促进我们对诱导毛发所需机制的理解 毛囊再生，否则无毛伤口环境，并将提供重要的新信息， 人类伤口与鼠类伤口的比较以及如何处理伤口以改善愈合。

Summary The goal of this grant is to understand the role of SHH signaling in regulation of regenerative healing in mammalian skin wounds. Most mammals including humans are deficient in regenerative skin healing. After injury, wound healing typically results in 'repair' (also defined as fibrosis/scarring). This process fails to fully restore the lost tissue to its original form, resulting in “a cellular band-aid”. The inability of mammals to regenerate normal skin is most notably illustrated by the lack of skin appendages including hair follicles in the wound scar. A major hurdle in hair follicle regeneration in skin wounds is the failure to reestablish the hair follicle inductive niche known as the dermal papilla (DP). Here, we show that SHH activation within 'repaired' (scarring) wound dermis induces de novo DP formation leading to HFN. These data clearly demonstrate that myofibroblasts within scarring wounds are not intrinsically incapable of forming the regenerative niche. We hypothesize that scarring cells may possess the regenerative potential but are missing the crucial developmental cue, SHH. To address this hypothesis, first, we will ask if Wnt signaling, which is active in fibrotic wounds, is required for SHH-driven DP formation and resultant HFN in wounds. For this we will perform loss-of-function studies on b-catenin, an essential effector of canonical Wnt signaling, in genetically modified wounds with ectopic SHH activation (Aim1). We will also examine the potential of mature Wnt-active fibroblasts to convert to DP in Hh-activated wounds after healing is complete. These experiments will address the potential plasticity of fully differentiated cells and how induction by the Hh pathway may alter their phenotype (Aim 1). Second, preliminary single cell RNAseq analyses comparing Hh-activated and WT wounds have identified downstream expression of several BMPs following Hh activation. We will examine the role of BMP signaling in mediating the function of Hh signaling through loss- and gain-of-function studies on BMP receptor signaling. We will also examine the ability of immediate Hh regulators Gli1 and 2, to directly bind BMP gene regulatory sites by Chip-Seq analysis (Aim 2). Third, human wounds strongly resemble 'repaired' murine wounds in many traits. We will test whether human wounds can respond to SHH signals to produce DP by introducing viral active-SmoM2 or soluble SHH agonists into xenograft-and-wounding models. Importantly, we will delineate the response of human dermal cells to SHH through single cell RNAseq analyses, and also compare these results with those from murine wounds (Aim3). These combined studies will advance our understanding of the mechanisms required to induce hair follicle regeneration in an otherwise hairless wound environment and will provide essential new information about how human wounds may compare with murine wounds and be manipulated for improved healing.