We hypothesized that
HGF gene transfer in combination with
BMP7 locally into the opaque cornea could reverse the fibrotic events in vivo in a preclinical rabbit model of corneal fibrosis. The results of this study demonstrate that delivery of
BMP7+
HGF genes into stromal fibroblasts/keratocytes via nanoparticle (PEI2-GNP) significantly reduced corneal opacity by 3 weeks post injury in a preclinical rabbit model of corneal fibrosis (
Figs. 1–
4). Importantly, since the
BMP7+
HGF therapy was administered 1 day after injury, the findings suggest that resolution of corneal opacity and vision restoration is achievable even in a significantly damaged cornea in vivo. Furthermore, administration of
BMP7+
HGF gene therapy was associated with a significant reduction in
α-SMA, a molecular marker for myofibroblasts, and a concomitant decrease in profibrotic genes (
Figs. 5 and
6). To corroborate the selective apoptosis observed in rabbit myofibroblasts was driven by
HGF, we performed in vitro studies of the effects of r
HGF on human corneal myofibroblasts and fibroblasts, which demonstrated that r
HGF induces apoptosis in human corneal myofibroblasts but not in HCFs (
Figs. 11 and
12). This finding aligns with earlier reports of
HGF function in pulmonary and liver fibrosis models, which suggests that
HGF-induced myofibroblast cell death is a key event in the healing process.
53 TGF-β signaling has been shown to increase the expression of c-Met, the receptor for
HGF,
54 suggesting that in a fibrotic microenvironment the
HGF activity may be more pronounced in myofibroblasts than in quiescent stromal keratinocytes. The c-Met receptor, a proto-oncogene, has multiple functions, including cell survival via sequestration of the Fas receptor and inhibition of death-domain–induced signaling.
55 However, in presence of
HGF, the binding of c-Met with Fas receptors would be dampened due to c-Met activation by phosphorylation, and the cells can then respond to Fas ligand–dependent death signals.
56 In addition,
HGF also induces increased caspase-3 activity via intracellular signaling.
57 Caspase-3–dependent cleavage of the c-Met receptor converts it into a 40-kDa proapoptotic signal.
58 Therefore, the selective death of myofibroblasts observed by
HGF treatment could be the result of a combination of three factors: increased c-Met receptors on myofibroblasts, elevated caspase activity, and the generation of proapoptotic c-Met fragments.
HGF has also been shown in lung fibrosis models to increase matrix metalloproteinase expression, leading to cell death and a concomitant reduction in ECM.
39 Hence, increased matrix metalloproteinase levels could be a mechanism to prevent corneal scar formation and aberrant rearrangement of the ECM in the presence of
HGF.