Development of Extracellular Heat Shock Protein-90 as a Novel Topical Wound Heali

描述（由申请人提供）：人类皮肤伤口的护理，包括瘀疮和压疮、糖尿病溃疡和烧伤伤口，每年花费美国约110亿美元。例如，在美国，由于无法愈合的糖尿病溃疡而截肢的人数接近10万，其中单次手术和住院就可能花费6.5万美元。目前可用的治疗方法显示中等或很少的疗效，但非常昂贵，如RegranexTM （PDGF-BB）。在24个月的时间里，治疗一个糖尿病足部伤口的费用高达28,000美元。因此，迫切需要开发新的更具成本效益的伤口愈合剂。直到最近，热休克蛋白90α (Hsp90a)被认为是一种atp依赖性的细胞内伴侣蛋白，在细胞内有超过100个客户靶标。然而，我们实验室过去五年的研究揭示了皮肤细胞在病理或应激条件下（如在伤口环境中发现的缺氧和新出现的细胞因子）分泌hsp90a的惊人需求。我们发现人角质形成细胞和真皮成纤维细胞都能快速分泌Hsp90a。分泌的Hsp90a通过与低密度脂蛋白受体相关蛋白-1 （LRP1）受体结合促进细胞运动。更有趣的是，促运动活性位于hsp90a的中间结构域和连接区域之间，独立于其n端atp酶活性。与传统的生长因子不同，hsp90a诱导的细胞迁移不能被TGF2所抑制，TGF2是存在于伤口中的丰富的细胞迁移和增殖抑制剂。这些新发现（已经产生了诸如Bandyopahdhay等人的出版物）。JCB, 2006，李等。EMBO, 2007， Cheng等。MCB， 2008和Woodley等人。JCS， 2009和一项美国专利，US 2010/0035815A1，重组hsp90a外用于皮肤创面愈合的申请)首次解释了究竟是哪种因素真正驱使真皮细胞（真皮成纤维细胞和内皮细胞）迁移到富含tgf2的创面床，因为这些细胞必须移动到创面沉积新的结缔组织材料和建立新的血管（重塑）。临床前，局部应用重组hsp90a可显著提高小鼠创面愈合率约45%。与此同时，fda批准的RegranexTM（用于治疗糖尿病溃疡）的效果要小得多（约17%）。这些新发现支持了我们的假设，即分泌的Hsp90a，而不是传统的生长因子，是表皮和真皮细胞迁移对抗TGF2抑制以愈合伤口的驱动力。在本研究中，我们将1)首先在体外缩小人Hsp90a与LRP-1受体结合并促进皮肤细胞运动的最低肽/氨基酸需求，作为全长Hsp90a， 2)利用猪伤口愈合模型建立候选肽，用于未来的临床研究。我们的目标是开发一种重组Hsp90a肽，使其成为一种新的、更有效的伤口愈合剂。

DESCRIPTION (provided by applicant): The care for human skin wounds, including stasis and pressure ulcers, diabetic ulcers and burn wounds, costs the United States ~$11 billion/year. For example, the number of lower extremity amputations is approaching 100,000 in the US due to non-healing diabetic ulcers, in which a single surgical procedure and hospitalization alone can cost $65,000. The currently available treatments show moderate or little efficacy and yet are very expensive, such as RegranexTM (PDGF-BB). Treating a single diabetic foot wound costs up to $28,000 within a 24-month period. Thus, there has been a pressing need to develop new and more cost-effective wound healing agents. Until recently, the heat shock protein-90alpha (Hsp90a) has been known as an ATP-dependent intracellular chaperone protein with more than 100 client targets inside the cell. However, studies from the past five years in our laboratory have unveiled a surprising need for skin cells to secrete Hsp90 a under pathological or stressful conditions like those found in the wound milieu, such as hypoxia and newly appearing cytokines. We found that both human keratinocytes and dermal fibroblasts rapidly secrete Hsp90a. The secreted Hsp90a promotes cell motility through binding to the LDL-Receptor Related Protein-1 (LRP1) receptor. More intriguingly, the pro-motility activity resides between the middle domain and the linker region of Hsp90 a, independent of its N-terminal ATPase activity. Unlike conventional growth factors, the Hsp90 a -induced cell migration cannot be inhibited by TGF2, the abundant inhibitor of cell migration and proliferation present in the wound. These new findings (which have generated publications such as Bandyopahdhay et al. JCB, 2006, Li et al. EMBO, 2007, Cheng et al. MCB, 2008 and Woodley et al. JCS, 2009 and a US patent, US 2010/0035815A1, application for the topical use of recombinant Hsp90 a for skin wound healing) provide an explanation for the first time of which factor really drives dermal cells (dermal fibroblasts and endothelial cells) to migrate into the TGF2-rich wound bed, because these cells must move into the wound to deposit new connective tissue materials and build new blood vessels (remodeling). Pre- clinically, topical application of recombinant Hsp90 a significantly accelerated the wound-healing rate by ~45% in mice. In parallel, FDA-approved RegranexTM (used to treat diabetic ulcers) showed much less (~17%) effect. These new findings led to our hypothesis that secreted Hsp90a, but not conventional growth factors, is the driving force of both epidermal and dermal cell migration against TGF2 inhibition to heal wounds. In this proposed study, we will 1) first narrow down the minimum peptide/amino acid requirements in human Hsp90a for binding to the LRP-1 receptor and promoting skin cell motility as the full-length Hsp90 a in vitro and 2) establish the candidate peptide using a pig wound healing model for future clinical studies. Our goal is to develop a recombinant Hsp90a peptide into a novel and more effective wound-healing agent.