The Role of Exogenous Type VII Collagen on the Healing of Skin Wounds

我们有一个令人惊讶的发现，III型(锚定纤维)胶原(C7)是由外源性的 对小鼠皮肤创伤，无论是局部还是静脉注射，都能显著加速伤口闭合和 伤口愈合时结疤较少。这是一个意想不到的发现，因为在正常情况下 没有受伤的皮肤，C7被配置成称为锚定纤维的大结构，这些结构局限于 真皮和表皮之间的连接处，真皮-表皮连接处(DEJ)。多年来，我们一直在 研究了C7的结构和功能，惊讶地发现在愈合皮肤的伤口床上 对于伤口，C7并不局限于DEJ，而是广泛分布于整个伤口床。我们也 开发了制造毫克量的重组人VII型胶原(RhC7)的程序。我们 在无毛小鼠背部1x1厘米标准全层创面上添加重组人C7，并评估创面 结案了。我们发现，与车辆对照或不处理相比，重组人C7处理的伤口表现出 伤口愈合速度急剧加快。当我们处理皮肤伤口时，同样的结果也发生了 表现出皮肤伤口愈合延迟的糖尿病小鼠。我们还将人的皮肤移植到 无菌小鼠，损伤移植的人皮肤，并应用赋形剂或重组人C7。再一次，重组人C7诱导 与车辆相比，人体皮肤的伤口愈合速度更快。重组人C7具有特定的活性， 其他大分子(I型胶原、IV型胶原、纤维连接蛋白和层粘连蛋白332)没有加速 这些动物的伤口闭合。还注意到，用重组人C7处理的小鼠皮肤伤口在 减少了临床上的疤痕。然后，我们通过组织学和通过 瘢痕形成标志物的免疫组织化学检测。与赋形剂处理的伤口相比，小鼠皮肤创伤 用重组人C7治疗后，瘢痕形成的标志物包括胶原沉积显著下调， 平滑肌肌动蛋白阳性的成纤维细胞(肌成纤维细胞)、结缔组织生长因子、Periostin和Pre. 转化生长因子β(TGFb)的纤维化亚型。鉴于这些意想不到的戏剧性结果， 这笔赠款的主题是C7在皮肤伤口愈合中的作用。最近，我们还建立了 第一只也是唯一一只皮肤伤口延迟愈合的糖尿病猪。在这项提案中，我们希望确定 局部应用重组人C7对正常和糖尿病家猪皮肤创面的影响。 在猪身上进行实验是至关重要的，因为猪皮与人的皮肤更相关。猪是 动物皮肤紧致，皮肤与人类皮肤最相似。猪是伤口的首选物种 治疗研究。我们还将确定外用重组人C7是否能抑制瘢痕形成并产生 “再生”治疗，而不仅仅是“修复”治疗。我们还将建立重组人C7在 猪皮伤口作为治疗人类伤口的替代品。这对于确定剂量是很重要的 作为一种新的创面愈合剂，用于人类慢性皮肤创面的给药方案。一旦 确定了外用rhC7的最佳剂量和给药方案，我们将确定rhC7是否会加速伤口 并抑制糖尿病猪的疤痕形成。伤口愈合是一个巨大的临床问题，尤其是对 军人和退伍军人。退伍军人经常遭受慢性皮肤创伤的诱因条件，如 如糖尿病溃疡、淤积性皮炎、腿部溃疡和卧位性溃疡等因脊髓损伤而导致的不能动弹。 由于肥胖症和糖尿病的增加，这个问题正在升级。我们将评估第一次受伤的情况 在与人类最相关的动物中，正常皮肤与糖尿病皮肤的愈合机制。我们有 这些令人兴奋的观察关于皮肤创伤对rh C7的反应，但我们不知道 它们背后的病理生理机制。计划中的实验将告诉我们基本的 支持这些观察的机制。他们还将提前从研究中引进重组人C7 实验室到诊所为患者服务，即所谓的“床到床”研究。 好了！ 好了！

We have made the surprising finding that type VII (anchoring fibril) collagen (C7) exogenously administered to murine skin wounds, both topically and intravenously, induces dramatic acceleration of wound closure and the wounds heal with less scarring. This was an unexpected finding because, under normal conditions in unwounded skin, C7 is configured into large structures called anchoring fibrils which are confined to the junction between the dermis and epidermis, the dermal-epidermal junction (DEJ). For many years, we have studied the structure and function of C7 and were surprised to find that in the wound bed of healing skin wounds, C7 is not confined to the DEJ, but, rather, is widely distributed throughout the wound bed. We also developed procedures for making milligram quantities of recombinant human type VII collagen (rhC7). We added rhC7 to 1 x 1 cm standard full-thickness wounds on the backs of hairless mice and evaluated wound closure. We found that compared with vehicle control or no treatment, rhC7-treated wounds exhibited dramatic acceleration of wound closure. The same results also occurred when we treated skin wounds made in diabetic mice who exhibit delayed skin wound healing. We also transplanted human skin onto the backs of athymic mice, wounded the transplanted human skin and applied either vehicle or rhC7. Again, rhC7 induced accelerated wound closure in the human skin compared with the vehicle. rhC7 had specific activity since other large macromolecules (type I collagen, type IV collagen, fibronectin, and laminin 332) did not accelerate wound closure in these animals. It was also noted that the murine skin wounds treated with rhC7 healed with less clinical scarring. We then evaluated vehicle- and rhC7-treated murine wounds by histology and by immunohistochemistry for markers of scarring. Compared with vehicle-treated wounds, murine skin wounds treated with rhC7 exhibited a dramatic down-regulation of markers of scarring including collagen deposition, smooth muscle actin positive fibroblasts (myofibroblasts), connective tissue growth factor, periostin and pro- fibrotic isoforms of transforming growth factor beta (TGFb). Given these unexpected dramatic results, the subject of this grant is the role of C7 in the healing of skin wounds. Recently, we have also established the first and only diabetic pig with delayed healing of skin wounds. In this proposal, we wish to determine the influence of topically administered rhC7 on skin wounds made in domestic pigs, both normal and diabetic. Performing experiments with pigs is critical because pigskin is much more relevant to human skin. Pigs are tight-skinned animals and have skin the most similar to human skin. Pigs are the species of choice for wound healing studies. We will also determine if topical rhC7 inhibits scarring and generates elements of “regenerative” healing rather than just “reparative” healing. We will also establish the stability of rhC7 in pigskin wounds as a surrogate for healing human wounds. This will be important for determining the dose and dosing schedule of rhC7 as a new wound healing agent for humans with chronic skin wounds. Once the optimal dose and dosing schedule of topical rhC7 is established, we will determine if rhC7 accelerates wound closure and inhibits scarring in diabetic pigs. Wound healing is an enormous clinical problem, especially for military personnel and Veterans. Veterans often suffer predisposing conditions for chronic skin wounds such as diabetic ulcers, stasis dermatitis leg ulcers and decubitus ulcers from immobility due to spinal cord injuries. The problem is escalating due to rises in obesity and diabetes. We will evaluate for the first-time wound healing mechanisms in normal skin versus diabetic skin in the animal most relevant to humans. We have these exciting observations regarding skin wound responses to rhC7, but we do not know the pathophysiological mechanisms behind them. The planned experiments will inform us of the basic mechanisms underpinning these observations. They will also advance bringing rhC7 from the research laboratory to the clinic for patients, so-called “bench-to-bedside” research. ! !