Mitochondria as key therapeutic targets for the treatment of corneal fibrosis
Over the past decade, our laboratory developed a cat model of corneal wound healing after photorefractive keratectomy (PRK). Recently, we used this model in a combination of in vivo and in vitro approaches to show that myofibroblasts directly inhibit nerve regeneration in the wounded stroma, sub-basal plexus and epithelium. In vitro, two distinct phases to this inhibition have been observed: (1) initial slowing of neurite elongation via a releasable factor, and (2) neurite contact inhibition by myofibroblasts. For phase 1, transforming growth factor beta 1 (TGF-?1) was shown to be the soluble, anti-neuritogenic factor released by myofibroblasts, and the specific signaling cascade it triggered to slow neurite elongation was identified. Ultimately, preventing myofibroblast differentiation suppressed both phase 1 and phase 2 inhibition, and resulted in faster nerve regeneration. However, in most cases of accidental injury, patients present after myofibroblast differentiation/fibrosis have occurred. The issue then is no longer preventing fibrosis, but rather overcoming its effects. We recently discovered that the synthetic PPAR? ligand and Thiazolidinedione (TZD) drug, Troglitazone, stimulates myofibroblast de-differentiation in vitro and in vivo through a PPAR? independent pathway. We now show that this TDZ's ability to promote myofibroblast de-differentiation can be mimicked by UK-5099, a compound that spares PPAR?, instead inhibiting a recently identified alternative target of TZDs: the mitochondrial pyruvate carrier (MPC). The recent literature and our preliminary results thus form a strong premise for refocusing our efforts on mitochondria, and critically testing the hypothesis that inhibiting the MPC facilitates myofibroblast de-differentiation, which together with metabolic remodeling in regenerating nerves can treat both fibrosis and overcome the blocked reinnervation inherent in its context. Work is ongoing to understand the metabolic changes that underlie the ability of TDZs to facilitate wound healing though mito-centric regulation of both myofibroblasts and corneal nerves. Here, we present the results of an RNA sequencing experiment designed to examine changes to the global transcriptome that occur as a result of TGF-ß1-induced activation of corneal fibroblasts, and of TDZ- or UK5099-stimulated myofibroblast de-differentiation. Overall design: Cat corneal fibroblasts in culture (d0FB) were treated for two days with TGFb1 to induce differentiation to myofibroblasts (d2TGFb). Myofibroblasts were then treated for three days with either Troglitazone (d5Trog), Troglitazone and GW9662 (d5T-G), or UK-5099 (d5UK). Five independent replicates of cells from each of these five conditions were harvested for RNA sequencing.