In the present study we investigated the potential use of the peptide AZX100 for the prevention of excessive scarring and fibrotic disorders. AZX100 decreased TGF-β1-induced expression of CTGF and type I collagen in cultured keloid fibroblasts (). As CTGF appears to be involved in inducing collagen expression (Duncan et al., 1999
), the effect of AZX100 on collagen expression likely results from its ability to decrease the expression of CTGF. AZX100 did not alter TGF-β1-induced SMAD signaling (), hence the effects of AZX100 appear to be SMAD independent.
The reduction in CTGF expression caused by AZX100 could be attributed to its effect on the actin cytoskeleton. AZX100 displaces the actin accessory protein cofilin from its binding site on the 14-3-3 adapter protein. Such displacement leads to dephosphorylation and activation of cofilin, which, in turn, mediates actin depolymerization and stress fiber disruption (Gohla and Bokoch, 2002
; Dreiza et al., 2005
). In a previous study (Dreiza et al., 2005
), we showed that cofilin displacement occurs in the presence of 10 and 25 µm
of AZX100; in this study, the concentration of AZX100 was higher (50 µm
), and thus, enough to prevent the binding of cofilin to 14-3-3. Cofilin dephosphorylation by AZX100 was confirmed in this study in TGF-β1-stimulated cells (). It seems, therefore that SMAD-independent cytoskeleton changes may be a major determinant of TGF-β1- induced CTGF production. Indeed, several studies have shown that disruption of the cytoskeleton by cytochalasin D reduces the stimulated expression of CTGF in renal fibroblasts and rat mesangial cells (Hahn et al., 2000
; Heusinger-Ribeiro et al., 2001
). Recently, Muehlich et al. (2007)
have proposed a molecular mechanism for such a phenomenon. Using techniques that involve coexpression of mutant actins and different CTGF promoter constructs, these authors observed that the CTGF gene promoter contains an actin-sensitive site, which may be activated by monomeric actin. Their data indicate an inverse relationship between CTGF expression and depolymerized actin.
Another important observation of the present study is that AZX100 reduces the expression of α-SMA (). This protein is a hallmark of the differentiation of fibroblasts into myofibroblasts, a process that appears to contribute substantially to the development of fibrosis (Desmouliere et al., 2005
). Although the precise mechanism by which AZX100 reduces α-SMA expression in keloid fibroblasts remains to be determined, it should be mentioned that α-SMA expression increases with force tension (Wang et al., 2003
). Thus, the ability of AZX100 to reduce α-SMA expression may also be related to the cytoskeleton-disrupting activity of AZX100.
Besides TGF-β, a multitude of cytokines, growth factors, and proteins are released at the wound-healing site. Many of these compounds stimulate profibrotic activity, such as LPA and ET (Hahn et al., 2000
; Shi-Wen et al., 2004
). Thus, it is important for a potential antifibrotic drug to be able to inhibit the effects of multiple profibrotic mediators. AZX100 was also able to reduce CTGF expression induced by LPA and ET (). As the signaling pathways used by these molecules are diverse, the reduction of ET- and LPA-induced CTGF expression by AZX100 suggests that AZX100 is not specific for a certain pathway, but instead affects CTGF expression induced by the activation of several pathways, presumably because its mechanism of action is downstream and related to the cytoskeleton.
One of the fundamental problems associated with protein-based therapeutics is that large molecules such as proteins and peptides do not cross cell membranes. By using a PTD as a “carrier”, the attached active peptide can be transported across cell membranes (Schwarze et al., 1999
; Flynn et al., 2003
; Snyder and Dowdy, 2004
). The PTD used in this study is an optimized version of TAT (Ho et al., 2001
). We observed that without the PTD, the free HSP20 phosphopeptide had no effect on TGF-β1-stimulated expression of CTGF and collagen, demonstrating that the PTD is necessary for intracellular delivery of this active moiety of HSP20 ().
Consistent with our in vitro
observations regarding the molecular mechanisms of AZX100, in vivo
data demonstrate a significant impact on collagen fiber orientation, density, and maturity (). This improvement from a single injection in a rodent model of dermal scarring is comparable to another molecule currently under development to improve clinical scar appearance, TGF-β3 (Shah et al., 1995
). Although TGF-β3 has been shown to have in vivo
efficacy in dermal scarring with multiple injections in a rodent model, the molecular mechanism(s) of action are not clearly known. TGF-β3 is believed to promote epidermal homeostasis by preventing keratinocyte apoptosis in culture and reducing phorbol acetate-induced c-Jun N-terminal kinase activity (Lee et al., 1999
). In addition, TGF-β3 has been shown to promote palatal shelf adhesion by inducing chondroitin sulfate proteoglycan expression in mice (Gato et al., 2002
). Clinical trials using recombinant TGF-β3 (Justiva) in various dermal scarring indications have shown a nominal but significant improvement in scar appearance (see Renovo Annual Report, http://www.renovo.com/documents.asp?c_id=52
), although the underlying molecular mechanisms are not clear.
Although many different pharmacologic agents have been studied to improve adult scarring, several of them have failed to show clinical efficacy despite in vitro
and in vivo
preclinical efficacy. For example, IFN-γ has been shown to decrease collagen synthesis in a wide variety of cells, including human dermal fibroblasts, human chondrocytes, and rat myofibroblasts (Lee et al., 1999
; Phan et al., 2002
; Ragoowansi et al., 2003
; Amadeu et al., 2004
; Liu et al., 2004
; Wong, 2005
; Al-Attar et al., 2006
; Davison et al., 2006
; Uysal, 2006
). These results were confirmed using murine models of fibrosis (Sun et al., 1993
; Katzung, 1996
), but mixed results were obtained in the clinic (Berman and Flores, 1998
; Davison et al., 2006
To the best of our knowledge, therapeutic modalities like AZX100, in which a relevant phosphoprotein motif is directly delivered inside the cell and modulates downstream events that regulate scar formation, are previously unreported. Exploiting posttranslational (for example, phosphorylated) targets in this pathway may lead to finer, more specific control of collagen deposition than other pharmacologic agents that act through receptor-based modulation of signaling cascades, which typically amplify multiple enzymatic activities. The AZX100 molecule bypasses upstream events, thus potentially offering a treatment for abnormal wound-healing processes that respond to multiple upstream events. Taken together, these findings suggest that AZX100 may present a molecularly targeted, proteomic therapy for improving dermal scarring.