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

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067100-10
Application #
8515447
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Somers, Scott D
Project Start
2003-01-01
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
10
Fiscal Year
2013
Total Cost
$316,520
Indirect Cost
$123,520
Name
University of Southern California
Department
Dermatology
Type
Schools of Medicine
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
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
Cheng, Chieh-Fang; Sahu, Divya; Tsen, Fred et al. (2011) A fragment of secreted Hsp90? carries properties that enable it to accelerate effectively both acute and diabetic wound healing in mice. J Clin Invest 121:4348-61
Li, Wei; Fan, Jianhua; Chen, Mei et al. (2004) Mechanism of human dermal fibroblast migration driven by type I collagen and platelet-derived growth factor-BB. Mol Biol Cell 15:294-309