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

外源性 VII 型胶原蛋白对皮肤伤口愈合的作用

• 批准号: 10200645
• 负责人: DAVID T. WOODLEY
• 金额: --
• 依托单位: VA GREATER LOS ANGELES HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200645
• 关键词: Acceleration; Adherence; Animals; Applications Grants; Autoimmune; Back; Binding; Biological; Biology; Bulla; Burn injury; Cicatrix; Clinic; Clinical; Collagen; Collagen Type I; Collagen Type IV; Collagen Type VII; Connective Tissue; Deposition; Dermal; Dermis; Diabetes Mellitus; Diabetic mouse; Disease; Domestic Pig; Dose; Down-Regulation; Edema; Elderly; Elements; Epidermis; Epidermolysis Bullosa Acquisita; Epidermolysis Bullosa Dystrophica; Exhibits; FDA approved; Family suidae; Fibroblasts; Fibronectins; Genetic Diseases; Grant; Histology; Human; Immunohistochemistry; Impaired healing; Inbred HRS Mice; Intravenous; Laboratories; Laboratory Research; Laminin; Leg; Leg Ulcer; Lower Extremity; Military Personnel; Mus; Myofibroblast; Nude Mice; Obesity; Patients; Pharmaceutical Preparations; Procedures; Protein Isoforms; Recombinants; Research; Research Design; Role; Schedule; Site; Skin; Skin wound healing; Smooth Muscle Actin Staining Method; Specificity; Spinal cord injury; Standardization; Stasis Ulcer; Stasis dermatitis; Structure; Thick; Time; Topical application; Transforming Growth Factor beta; Transplantation; Ulcer; Veterans; Work; Wound models; bench to bedside; carcinogenicity; chronic wound; connective tissue growth factor; cost; db/db mouse; decubitus ulcer; diabetic; diabetic ulcer; experimental study; healing; intravenous injection; macromolecule; military veteran; milligram; novel; periostin; regenerative; reparative healing; skin disorder; skin wound; wound; wound bed; wound closure; wound healing; wound response; wound treatment

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. ! !

我们已经取得了令人惊讶的发现，VII型（锚定纤维）胶原蛋白（C7）外源性施用 局部和静脉内施用到鼠皮肤伤口，诱导伤口闭合的显著加速， 伤口愈合后留下的疤痕较少。这是一个意外的发现，因为在正常情况下， 在未受伤的皮肤中，C7被构造成被称为锚定原纤维的大结构，其被限制在 真皮和表皮之间的交界处，真皮-表皮交界处（DEJ）。多年来，我们 研究了C7的结构和功能，并惊讶地发现，在愈合皮肤的伤口床上， 在创伤中，C7不局限于DEJ，而是广泛分布在整个创伤床。我们也 开发了生产毫克量重组人VII型胶原蛋白（rhC 7）的程序。我们 将rhC 7添加到无毛小鼠背部的1 × 1 cm标准全层伤口中，并评估伤口 结束我们发现，与载体对照或未处理相比，rhC 7处理的伤口表现出 伤口愈合的急剧加速。同样的结果也发生在我们治疗皮肤伤口时， 在皮肤伤口愈合延迟的糖尿病小鼠中。我们还将人类皮肤移植到 无胸腺小鼠，损伤移植的人皮肤并施用载体或rhC 7。再次，rhC 7诱导 与载体相比，加速了人皮肤中的伤口闭合。rhC 7具有特异性活性， 其他大分子（I型胶原、IV型胶原、纤连蛋白和层粘连蛋白332）没有加速 这些动物的伤口愈合。还注意到用rhC 7处理的鼠皮肤伤口愈合， 减少临床疤痕。然后，我们通过组织学和免疫组织化学评价了载体和rhC 7处理的小鼠伤口。 免疫组织化学标记的疤痕。与溶剂处理的伤口相比， 用rhC 7处理的小鼠表现出瘢痕形成标志物包括胶原沉积的显著下调， 平滑肌肌动蛋白阳性成纤维细胞（肌成纤维细胞），结缔组织生长因子，骨膜蛋白和原- 转化生长因子β（TGF β）的纤维化同种型。鉴于这些意想不到的戏剧性结果， 这项资助的主题是C7在皮肤伤口愈合中的作用。最近，我们还建立了 第一个也是唯一一个皮肤伤口愈合延迟的糖尿病猪。在本提案中，我们希望确定 局部施用rhC 7对正常和糖尿病家猪中皮肤伤口的影响。 用猪做实验是至关重要的，因为猪皮与人类皮肤更相关。猪是 皮肤紧的动物，皮肤与人类皮肤最相似。猪是伤口的首选物种 治疗研究。我们还将确定局部rhC 7是否抑制瘢痕形成并产生 “再生”愈合，而不仅仅是“修复”愈合。我们还将确定rhC 7在 猪皮伤口作为治疗人类伤口的替代品。这对确定剂量很重要 以及rhC 7作为人类慢性皮肤创伤的新型创伤愈合剂的给药方案。一旦 建立局部rhC 7的最佳剂量和给药方案，我们将确定rhC 7是否加速伤口愈合 闭合并抑制糖尿病猪的瘢痕形成。伤口愈合是一个巨大的临床问题，特别是对于 军人和退伍军人。退伍军人经常遭受慢性皮肤伤口的诱发条件， 如糖尿病性溃疡、郁积性皮炎性腿部溃疡和由于脊髓损伤而不能移动的褥疮性溃疡。 由于肥胖和糖尿病的增加，这个问题正在升级。我们会对第一次受伤的人进行评估 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 这些关于皮肤伤口对rhC 7反应的令人兴奋的观察，但我们不知道 背后的病理生理机制。计划中的实验将告诉我们 支持这些意见的机制。他们还将推进从研究中引入rhC 7 从实验室到临床，即所谓的“从实验室到临床”的研究。 ! !