Effects of Altering TGF Beta Signaling on Wound Healing

描述(申请人提供)：与各种疾病(如糖尿病)相关的慢性皮肤创伤和急性创伤后的异常愈合(如肥厚性疤痕)是主要的医疗负担。我们以前的研究表明，当转化生长因子21(TGF21)的过度表达水平与皮肤病的水平相当时，它会导致严重的皮肤炎症、伤口愈合延迟和过度的纤维化反应。相比之下，它的拮抗剂Smad7具有强大的抗炎作用，可以更快地重新上皮化，减少炎症，减少纤维化反应，促进皮肤伤口愈合。因此，我们假设：1)将Smad7蛋白输送到创面床可以治疗受损的皮肤创面愈合。2)Smad7通过其转录和蛋白质相互作用直接调节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调节皮肤创面愈合过程中肉芽组织形成和基质重塑的表皮分泌分子。为了验证这些假设，目标1将检查局部Smad7蛋白传递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重组蛋白来治疗两种受损伤口愈合模型中的切除皮肤伤口。首先，我们将利用K5.TGF21转基因伤口作为炎症相关的损伤愈合模型，这是验证Smad7靶向效应的理想模型。其次，我们将治疗糖尿病小鼠的伤口。目的2分析Smad7调控角质形成细胞迁移的分子机制。在AIM 1中生成的伤口样本中，将在病理条件下鉴定和验证Smad7转录靶点和调节表皮再上皮化的蛋白质伙伴。AIM 3将识别影响伤口基质的Smad7转录靶点。我们已经产生了含有生物素标记的Smad7转基因(Smad7 Biotin)的可诱导的、表皮特异性的Smad7小鼠。这些小鼠在伤口愈合的每个特定阶段切除的伤口将被用于高通量分析，以确定影响伤口基质的临时Smad7靶点。在AIM 1中产生的样本将用于检查Smad7治疗是否改变了这些靶点中的任何一个，以及体内击倒这些靶点是否会影响愈合。建议的研究有助于我们进一步了解皮肤创伤愈合受损的病理机制，测试局部注射Smad7或靶向Smad7下游蛋白的治疗方法。

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.