Advance of this project after previous two funding cycles can no longer be fully reflected in its initial title ("Mechanisms of Human Dermal Fibroblast Migration"), which nonetheless is unalterable according to NIH roles. This continuation application has evolved into preclinical stage and aims at developing a novel wound- healing factor, the secreted form of heat shock protein-90alpha (Hsp90?), into an effective treatment of human chronic wounds. At the beginning of the previous funding cycle, we raised a fundamental question: What is the nature of the physiological factor(s) that drives inward migration of dermal and epidermal cells into the wound bed during wound healing? Specifically, what factor(s) is responsible for the lateral migration of epidermal keratinocytes over the wound bed to close the wound and inward migration of dermal fibroblasts and microvascular endothelial cells into the wound bed to remodel the damaged tissue and to build a new vascularized neodermis? For decades, the unchallenged answer and the focus for the therapeutic development are "growth factors". Despite clinical trials on more than a dozen of these proteins, only recombinant PDGF-BB received the FDA approval (RegranexTM) in 1997 for treatment of diabetic ulcers and beyond. However, its modest efficacy, high cost and fivefold higher risk of dying from cancer for patients have limited the use of Regranex in clinical practice. We now know, at least partially, why. In humans, i) PDGF-BB only acts on dermal fibroblasts (no detectable PDGF receptors on other skin cell types) and ii) PDGF-BB-induced dermal fibroblast migration is completely blocked by TGF?, present throughout wound healing. Other reported conventional growth factors face the same hurdles. These findings challenge the importance of growth factors in wound healing for the first time. For the past four years, our laboratory has searched for a factor that can overcome these defects. 18 months of protein purification allowed us to make a surprising discovery that injury-associated stress, in particular hypoxia, triggers human dermal fibroblasts (and epidermal keratinocytes) to secrete Hsp90? to outside of the cells. The secreted Hsp90? quickly builds up to 5M working concentrations, drives both the dermal and the epidermal cell migration against the presence of TGF?. Topical recombinant Hsp90? proteins accelerates wound healing by 60% faster than the placebo and three fold faster than Regranex in nude mice (Li et a. 2007, Cheng et al. 2008, Woodley et al. 2009, Cheng et al. 2011). Latest preliminary studies show that topical recombinant Hsp90? bypasses the "damaging point" of hyperglycemia, i.e. hypoxia-inducible factor-1 (HIF-1), in diabetic wounds and dramatically accelerates diabetic wound healing in db/db mice (Progress Report). These findings lead to the new and exciting direction of this continuation application: Secreted Hsp90? is a critical physiological driving force of wound healing and its secretion (but not action) is suppressed in diabetic wounds. Therefore, we are presented with an exciting possibility that supplementation of a therapeutic Hsp90? peptide, called F-5, to diabetic wounds should resume diabetic wound healing. If this research direction is approved by the referees, we will 1) investigate how Hsp90? secretion is affected in diabetic dermal fibroblasts isolated from db/db mice and from diabetic human skin specimens and 2) use db/db mouse model and "human diabetic skin grafted onto mouse" model to establish whether topical supplementation of the F-5 peptide of Hsp90? overrides hyperglycemia to promote dermal (as well as epidermal) cell migration and diabetic wound healing. We believe that elucidation of Hsp90?'s function in wound healing and its mechanism of action will change the conventional paradigms for therapeutic intervention of human chronic wounds, whose current annual healthcare costs exceed $25 billion in the US.

Public Health Relevance

Skin ulcers and non-healing wounds are a major healthcare problem due to the lack of effective therapy. For the past two decades, the conventional wisdom is that growth factors are the driving force for wound healing. Despite of studies and clinical trials on more than a dozen of growth factors, only recombinant PDGF-BB has ultimately received the FDA approval (RegranexTM) for treatment of diabetic limb ulcers. However, its initial efficacy from trials of randomized samples did not translate well to the clinic and its higher risk of causing cancer received a black box from the FDA in 2008. During the previous funding cycle, our laboratory identified two reasons for why Regranex could not have been as effective and, more importantly, discovered that hypoxia induces dermal fibroblasts (and keratinocytes) to secrete the intracellular chaperone protein called heat shock protein alpha (Hsp901). We found that secreted Hsp90? overcomes the two hurdles facing Regranex and it heals wound three times faster than Regranex. This proposal aims at establishing secreted Hsp90? as a potentially new wound healing agent for diabetic wounds

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Surgery, Anesthesiology and Trauma Study Section (SAT)
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Somers, Scott D
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University of Southern California
Schools of Medicine
Los Angeles
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Zou, M; Bhatia, A; Dong, H et al. (2016) Evolutionarily conserved dual lysine motif determines the non-chaperone function of secreted Hsp90alpha in tumour progression. Oncogene :
Bhatia, Ayesha; O'Brien, Kathryn; Chen, Mei et al. (2016) Dual therapeutic functions of F-5 fragment in burn wounds: preventing wound progression and promoting wound healing in pigs. Mol Ther Methods Clin Dev 3:16041
Dong, Hangming; Zou, Mengchen; Bhatia, Ayesha et al. (2016) Breast Cancer MDA-MB-231 Cells Use Secreted Heat Shock Protein-90alpha (Hsp90α) to Survive a Hostile Hypoxic Environment. Sci Rep 6:20605
Jayaprakash, Priyamvada; Dong, Hangming; Zou, Mengchen et al. (2015) Hsp90α and Hsp90β together operate a hypoxia and nutrient paucity stress-response mechanism during wound healing. J Cell Sci 128:1475-80
O'Brien, Kathryn; Bhatia, Ayesha; Tsen, Fred et al. (2014) Identification of the critical therapeutic entity in secreted Hsp90α that promotes wound healing in newly re-standardized healthy and diabetic pig models. PLoS One 9:e113956
Tsen, Fred; Bhatia, Ayesha; O'Brien, Kathryn et al. (2013) Extracellular heat shock protein 90 signals through subdomain II and the NPVY motif of LRP-1 receptor to Akt1 and Akt2: a circuit essential for promoting skin cell migration in vitro and wound healing in vivo. Mol Cell Biol 33:4947-59
Li, Wei; Tsen, Fred; Sahu, Divya et al. (2013) Extracellular Hsp90 (eHsp90) as the actual target in clinical trials: intentionally or unintentionally. Int Rev Cell Mol Biol 303:203-35
Sahu, Divya; Zhao, Zhengwei; Tsen, Fred et al. (2012) A potentially common peptide target in secreted heat shock protein-90ýý for hypoxia-inducible factor-1ýý-positive tumors. Mol Biol Cell 23:602-13
Li, Wei; Sahu, Divya; Tsen, Fred (2012) Secreted heat shock protein-90 (Hsp90) in wound healing and cancer. Biochim Biophys Acta 1823:730-41
Cheng, Chieh-Fang; Sahu, Divya; Tsen, Fred et al. (2011) A fragment of secreted Hsp90α carries properties that enable it to accelerate effectively both acute and diabetic wound healing in mice. J Clin Invest 121:4348-61

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