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

描述（由申请人提供）：美国每年花费约110亿美元护理人类皮肤伤口，包括淤滞性溃疡和压力性溃疡、糖尿病性溃疡和烧伤伤口。例如，在美国，由于不愈合的糖尿病溃疡，下肢截肢的数量接近100，000，其中单次手术和住院治疗就可能花费65，000美元。目前可用的治疗显示出中等或很小的疗效，但非常昂贵，如RegranexTM（PDGF-BB）。在24个月内治疗一个糖尿病足伤口的费用高达28,000美元。因此，迫切需要开发新的和更具成本效益的伤口愈合剂。直到最近，热休克蛋白-90 α（Hsp90 α）已被称为ATP依赖性细胞内伴侣蛋白，在细胞内具有100多个客户端靶标。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分泌Hsp90 a的惊人需求，如在伤口环境中发现的那些，如缺氧和新出现的细胞因子。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Hsp90 α。分泌的Hsp90 a通过与LDL-受体相关蛋白-1（LRP 1）受体结合来促进细胞运动。更有趣的是，促运动活性存在于Hsp90 a的中间结构域和接头区之间，独立于其N-末端ATP酶活性。与常规生长因子不同，Hsp90 α诱导的细胞迁移不能被TGF 2抑制，TGF 2是伤口中存在的大量细胞迁移和增殖抑制剂。这些新发现（其已经产生了出版物，例如Bandyopahdhay等人，JCB，2006，Li等人，EMBO，2007，Cheng等人，MCB，2008和Woodley等人，JCS，2009和美国专利US 2010/0035815A1，局部使用重组Hsp90 a用于皮肤伤口愈合的申请）首次提供了哪种因子真正驱动真皮细胞的解释这些细胞（真皮成纤维细胞和内皮细胞）迁移到富含TGF 2的伤口床中，因为这些细胞必须移动到伤口中以存款新的结缔组织材料并构建新的血管（重塑）。在临床前，局部应用重组Hsp90 a显著加速小鼠的伤口愈合率约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%）。这些新的发现导致我们的假设，分泌的Hsp90 α，而不是传统的生长因子，是表皮和真皮细胞迁移的驱动力，对TGF 2抑制愈合伤口。在本研究中，我们将1）首先缩小人Hsp90 a与LRP-1受体结合并在体外促进皮肤细胞运动的最低肽/氨基酸需求，作为全长Hsp90 a; 2）使用猪伤口愈合模型建立候选肽，用于未来的临床研究。我们的目标是将重组Hsp90 a肽开发成一种新的，更有效的伤口愈合剂。 公共卫生相关性：由于缺乏有效的治疗，皮肤溃疡和不愈合的伤口是一个主要的医疗保健问题。最近，我们意外地发现，缺氧诱导人角质形成细胞（HK）分泌细胞内的chaparone蛋白称为热休克蛋白α（Hsp90 a）。分泌的Hsp90 α通过自分泌机制刺激HK和附近的真皮细胞，通过旁分泌机制，通过结合LDL受体相关蛋白-1（LRP 1）受体迁移，即使在TGF 2存在下。该提议将检验缺氧机制> Hsp90 α分泌> LRP 1刺激>细胞运动性是伤口愈合的必要驱动力的假设。我们将开发基于局部Hsp90a肽的治疗和基于Hsp90a分泌细胞的治疗用于愈合皮肤伤口。

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. PUBLIC HEALTH RELEVANCE: Skin ulcers and non-healing wounds are a major healthcare problem due to the lack of effective therapy. Recently, we unexpectedly found that hypoxia induces human keratinocytes (HKs) to secrete an intracellular chaparone protein called heat shock protein alpha (Hsp90 a). The secreted Hsp90 a stimulates, via an autocrine mechanism, HK and the nearby dermal cell, through a paracrine mechanism, migration by binding to the LDL Receptor-Related Protein-1 (LRP1) receptor, even in the presence of TGF2. This proposal will test the hypothesis that the mechanism of hypoxia> Hsp90 a secretion>LRP1 stimulation>cell motility is an essential driving force for wound healing. We will develop topical Hsp90a peptide-based therapy and Hsp90a -secreting cell-based therapy for healing skin wounds.