For more than 3 decades, the conventional wisdom has been that serum factors — collectively called growth factors — represent the primary force in Mother Nature’s design for wound healing (6
). These often cell-type–specific growth factors either appear only when tissue is wounded or rise significantly from their basal concentrations in response to injury, such as TGF-α and KGF (FGF7) for HKs, PDGF-BB for HDFs, and VEGF-A for HDMECs. Since the mid 1970s, more than 30 growth factors have been subjected to extensive in vitro, preclinical, and clinical studies alone or in combinations (5
). Despite enormous efforts, in vivo functions for many of these growth factors remained unconfirmed, and their efficacy in human trials fell short of providing significant clinical benefits (4
). These rather unexpected statistics argue against the long-standing paradigm that growth factors are the critical driving force of wound closure. We speculated that there must be fundamental reasons underlining the ineffectiveness of conventional growth factors in wound healing. We have since undertaken 2 mutually complementary approaches to (a) examine the physiological barriers for conventional growth factor actions and (b) identify a new generation of wound healing factors. In this study, we have provided several lines of evidence for why conventional growth factor therapies, such becaplermin/PDGF-BB, could not have been as effective as they were hoped to be. First, not all skin cell types express a common receptor for a given growth factor. For example, HKs and HDMECs completely lack the PDGFR and, therefore, do not respond to becaplermin. Second, the abundant presence of the TGF-β family cytokines throughout the early phase of wound healing blocks any growth factor-stimulated migration of the dermal cells (note, not epidermal cells; see ref. 28
) and, therefore, their recruitment into the wound bed (28
). Third, additional pathophysiological conditions, such as hyperglycemia in diabetes, add layers that block the effectiveness of growth factors in diabetic wounds (44
). More importantly, we have identified a more effective wound healing agent, F-5, a fragment from secreted Hsp90α. In contrast to conventional growth factors, F-5 equally promotes migration of all 3 types of human skin cells that are essential for wound healing; F-5 overrides the inhibition of human dermal cell migration by TGF-β; and F-5 resists hyperglycemia to promote cell migration. Topical treatment of acute and diabetic wounds with F-5 greatly accelerates wound closure through increased reepithelialization. Based on these findings, we propose a new paradigm for what drives epidermal and dermal cell migration to close the wound, as schematically shown in Figure . Prior to injury, cell motility remains undetected in intact skin (Figure , step 1). Within hours after skin injury, HKs start to migrate laterally across the wound (possibly induced by hypoxia-driven Hsp90α autocrine signaling or TGF-α; see ref. 44
) and to secrete Hsp90α. At the same time, however, HDFs and HDMECs at the wound edge are not able to immediately move into the wound bed due to the presence of TGF-β3 (Figure , step 2). Once the secreted Hsp90α reaches the threshold concentration of 100 nM (29
), it triggers the dermal cells to migrate into the wound bed from the surrounding wound edge, even in the presence of TGF-β3 (Figure , step 3). Finally, the migrating HKs completely close the wound, and the newly moved-in HDFs start to remodel the wounded tissue and HDMECs to rebuild new blood vessels. We propose here that injury-induced secretion of Hsp90α, instead of the conventional growth factors, is the initial driving force of wound closure. After the initial wound closure, the dermal remodeling neovascularization processes would take many months to complete. Many other factors, including conventional growth factors, may play roles in the later events of wound healing, when the TGF-β levels decrease (54
A model of how released Hsp90α, but not conventional growth factors, promotes reepithelialization and recruits dermal cells into the wound during wound healing.
The capability of F-5 to strongly accelerate diabetic wound closure is consistent with previous studies on a recognized cause for diabetic wounds, hyperglycemia. One of the critical environmental stimuli for wound healing is relative hypoxia (54
). HIF-1α is a master transcription factor that regulates tissue adaptive responses to environmental hypoxia (58
) and is expressed throughout the multistage processes of acute wound healing. Impaired response, i.e., lack of HIF-1α accumulation in the cells, to hypoxia in diabetic ulcers is a known contributor to the delayed wound healing (45
). In vivo, lower levels of HIF-1α protein were reported in foot ulcer biopsies in patients with diabetes (59
). In vitro studies showed that hyperglycemia impairs HIF-1α protein stability and function via the von Hippel-lindau pathway (45
). Botusan et al. have demonstrated that forced stabilization of HIF-1α was necessary and sufficient to resume diabetic wound healing (45
). In parallel, we have previously shown that HIF-1α is a key upstream regulator of Hsp90α secretion. The secreted Hsp90α in turn promotes human epidermal and dermal cell migration via a novel “HIF-1α > Hsp90α secretion > LRP-1” signaling pathway (29
). Results of these 2 previously unrelated studies together point out the possibility that hyperglycemia destabilizes HIF-1α, blocks hypoxia-driven Hsp90α secretion and delays diabetic wound healing. The addition of F-5 bypasses the hyperglycemia-caused damage at HIF-1α and jump-starts migration of the cells that otherwise cannot respond to the environmental hypoxia.
Our data indicated that F-5 is more effective than the full-length Hsp90α in vivo, but requires higher concentrations to maintain that effect. Our current understanding of this phenomenon is largely at the level of speculation. It is conceivable that without possibly steric interferences by the 235-aa N-terminal domain and the 381-aa C-terminal domain, F-5 can fully reveal its effect of promoting cell motility. On the other hand, without the N-terminal and C-terminal domains, the shorter peptide may compromise on binding affinity and even stability and, therefore, show the requirement for higher concentration to maintain an equivalent promotility activity as the full-length protein. Our experiments show that even a single application of F-5 could lead to a remarkable acceleration of the wound closure in db/db
mice. If both such efficacy and duration of the F-5 action could translate into humans, it may significantly improve patient life and help to reduce the overall cost of diabetic wound clinic as well. The high cost of the currently available care mostly comes from home visits by physicians with various specialties and daily passive assistance of nurses, due to unavailability of effective treatments (51
). On the other hand, we expect that multiple treatments with F-5 should result in more prominent healing effects. Becaplermin gel, for instance, is recommended for daily applications to achieve its clinical effect. In the current study, we focused on a single treatment in our animal experiments for 2 technical reasons. First, for experiments that involve a large number of mice, it is hard to ensure that the procedures on all wounds are performed universally. Second, frequent opening and closing a healing wound for new treatments will risk damaging the on-going healing tissue (the newly generated epidermal layer in particular) and add extra stress and discomfort to the animals. Nonetheless, there have been reported options to deal with these technical limitations. Covering the wound with Tegaderm and multiple applications of the tested agent by injecting it through the Tegaderm with a gauge needle was reported as a way to minimize these technical concerns (63
The fact that extracellular Hsp90α is a motogen but not a mitogen (i.e., it does not stimulate cell proliferation) makes physiological sense (29
). First, keratinocyte migration occurs almost immediately after skin injury and plays a critical role in closing the wound. After the initial epidermal closure, completion of the subsequent dermal neovascularization and remodeling processes would take many months. Second, when a cell is migrating toward the wound area, it cannot proliferate at the same time. In addition, growth factor-stimulated proliferation of both epidermal and dermal cells would be inhibited by TGF-β that appears in the injured skin (28
). Third, cell migration precedes cell proliferation during wound healing. While the cells at the wound edge are moving toward the wound bed, they leave behind “empty space” between themselves and the cells behind them. The cells that are located behind the migrating cells start to proliferate after losing contact inhibition with the front moving cells. The stimuli of the cell proliferation likely come from plasma growth factors in the surrounding unwounded blood vessels, in which TGF-β levels are low or undetectable. Thus, cell proliferation appears to refill the space generated by the front-migrating cells. The role of secreted Hsp90α appears to promote the initial wound closure as quickly as possible.
Finally, proof of the relevance of animal model research to humans is the ultimate standard, especially considering the fact that many animal models for human diseases do not exactly reflect the genetic setting in humans. Many believe that this is the main reason for the majority of the therapeutic agents in the past, which show great promise in animal studies, to have ultimately failed in humans. For instance, human diabetes is a polygenic disease, whereas the db/db mouse is a monogenic (i.e., mutation in a single gene) diabetic model. Therefore, whether or not F-5 has similar effect on human diabetic wounds remains to be seen.