Effects of altering TGF beta signaling on wound healing

描述（由申请人提供）：与各种疾病（如糖尿病）相关的慢性皮肤伤口和急性伤口（如增生性疤痕）的异常愈合是一个主要的卫生保健负担。我们之前的研究表明，当转化生长因子21 （TGF21）过表达到与皮肤疾病相当的水平时，它会引起严重的皮肤炎症，延迟伤口愈合和过度的纤维化反应。相反，其拮抗剂Smad7具有强效的抗炎作用，能加速皮肤伤口愈合，加速再上皮化，减少炎症和纤维化反应。因此，我们假设：1)Smad7蛋白递送至创面可治疗受损皮肤创面愈合。2) Smad7通过转录和蛋白相互作用直接调控创面愈合过程中角质形成细胞的迁移和表皮再上皮化。3) Smad7调节参与肉芽组织形成和皮肤创面愈合基质重塑的表皮分泌分子。为了验证这些假设，Aim 1将检查局部Smad7蛋白递送是否促进皮肤伤口的愈合和重塑。我们将使用带有Tat细胞可渗透标签的人Smad7重组蛋白治疗两种受损伤口愈合模型的切除性皮肤伤口。首先，我们将利用K5。TGF21转基因创面作为炎症相关的损伤愈合模型，是验证Smad7靶向作用的理想模型。其次，我们将治疗糖尿病小鼠的伤口。目的2将分析Smad7调节角质形成细胞迁移的分子机制。Smad7转录靶点和调节表皮再上皮化的蛋白伙伴将在Aim 1中生成的伤口样本的病理条件下进行鉴定和验证。目的3将确定影响伤口基质的Smad7转录靶点。我们培育了可诱导的表皮特异性Smad7小鼠，其中含有生物素标记的Smad7转基因（Smad7biotin）。这些小鼠在伤口愈合的每个特定阶段的切除伤口将用于高通量分析，以确定影响伤口基质的时间Smad7靶点。Aim 1中生成的样本将用于检查Smad7治疗是否改变了这些靶点，以及体内敲除这些靶点是否影响愈合。这些研究有助于我们进一步了解受损皮肤伤口愈合的病理机制，通过局部递送Smad7或靶向Smad7下游蛋白来测试治疗方法。

DESCRIPTION (provided by applicant): Chronic skin wounds associated with various diseases (e.g., diabetes) and aberrant healing from acute wounding (e.g., hypertrophic scarring) is a major health care burden. Our previous studies show that when transforming growth factor 21 (TGF21) is overexpressed at a level comparable to that found in skin diseases, it induces severe skin inflammation, delayed wound healing and excessive fibrotic response. In contrast, its antagonist, Smad7, has a potent anti-inflammatory effect and accelerated skin wound healing with faster re- epithelialization, reduced inflammation and reduced fibrotic response. Hence, we hypothesize: 1) Smad7 protein delivery to the wound bed can treat impaired skin wound healing. 2) Smad7 directly regulates keratinocyte migration and epidermal re-epithelialization during wound healing through its transcription- and protein-interaction activities. 3) Smad7 regulates secreting molecules in the epidermis involved in granulation tissue formation and stromal remodeling of skin wound healing. To test these hypotheses, Aim 1 will examine if local Smad7 protein delivery promotes healing and remodeling of skin wounds. We will use human Smad7 recombinant protein with a Tat cell permeable tag to treat excisional skin wounds in two impaired wound healing models. First, we will utilize K5.TGF21 transgenic wounds as an inflammation-related impaired healing model, which is an ideal model for validation of the targeted effect of Smad7. Second, we will treat wounds in diabetic mice. Aim 2 will analyze molecular mechanisms by which Smad7 regulates keratinocyte migration. Smad7 transcriptional targets and protein partners regulating epidermal re-epithelialization will be identified and validated under pathological conditions in wound samples generated in Aim 1. Aim 3 will identify Smad7 transcriptional targets affecting the wound stroma. We have generated inducible, epidermal-specific Smad7 mice containing a biotin-tagged Smad7 transgene (Smad7biotin). Excisional wounds from these mice at each specific stage of wound healing will be used for high throughput analyses to identify temporal Smad7 targets affecting the wound stroma. Samples generated in Aim 1 will be used to examine if any of these targets are altered by Smad7 treatment and if in vivo knock down of these targets affects healing. The proposed studies help us further understand pathological mechanisms of impaired skin wound healing, test therapeutic approaches by either local delivery of Smad7 or targeting proteins downstream from Smad7. PUBLIC HEALTH RELEVANCE: Chronic skin wounds affect 6.5 million patients in the United States. This application performs experimental therapeutics to treat impaired skin wound healing and analyzes the molecular mechanisms involved.