Converting wound scar into healing with regeneration

摘要 这项资助的目标是了解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在再生皮肤愈合方面都存在缺陷。之后 损伤、伤口愈合通常导致“修复”(也被定义为纤维化/疤痕形成)。这一过程未能完全 将丢失的组织恢复到原来的形式，产生了一个“细胞创可贴”。哺乳动物不能再生 正常皮肤最明显的例证是伤口疤痕中缺少皮肤附属物，包括毛囊。一个 皮肤创面毛囊再生的主要障碍是未能重建毛囊诱导性生态位 被称为真皮乳头(DP)。 在这里，我们展示了修复的(疤痕形成)真皮中SHH的激活诱导新的DP。 导致HFN的队形。这些数据清楚地表明，疤痕伤口内的肌成纤维细胞 本质上不能形成再生的生态位。我们推测疤痕细胞可能拥有 再生潜力，但没有关键的发育线索，SHH。要解决这个问题 假设，首先，我们将询问在纤维化伤口中活跃的Wnt信号是否对于SHH驱动的DP是必需的 伤口中HFN的形成和结果。为此，我们将进行b-连环蛋白的功能丧失研究，以及 SHH异位激活的转基因创面中规范Wnt信号的基本效应因子 (目标1)。我们还将研究成熟的Wnt活性的成纤维细胞在HH激活的情况下转化为DP的可能性 伤口愈合后已完全愈合。这些实验将解决全分化的潜在可塑性。 HH途径如何诱导细胞以及如何改变它们的表型(目标1)。第二，初步的单细胞 比较HH激活和WT创伤的RNAseq分析发现了几个下游表达 HH激活后的BMPS。我们将研究BMP信号在调节HH信号功能中的作用 通过对BMP受体信号的功能丧失和功能获得的研究。我们还将检查 即时HH调节剂Gli1和Gli2，通过芯片序列分析(AIM)直接结合BMP基因调节位点 2)。第三，人类的伤口在许多特征上与“修复”的小鼠伤口非常相似。我们将测试一下 人体伤口可通过引入病毒活性SmoM2或可溶性SHH来响应SHH信号而产生DP 将激动剂转化为异种移植和创伤模型。重要的是，我们将描绘人类皮肤细胞的反应 对SHH进行单细胞RNAseq分析，并将这些结果与小鼠伤口的结果进行比较 (目标3)。这些联合研究将促进我们对诱导毛发所需机制的理解。 在没有毛发的伤口环境中的毛囊再生，将提供关于 人类的伤口如何与老鼠的伤口相比较，以及如何被操纵以改善愈合。

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.