Corneal fibrosis therapies and investigations in the canine corna in vitro

PHASE 1: ABSTRACT Purpose: To evaluate the safety and anti-fibrotic effects of the drugs, angiotensin-converting enzyme inhibitor (ACE-I) lisinopril and rho-kinase inhibitor (ROCK-I) fasudil, alone and in combination, on the canine cornea using an established in vitro model. Methods: To test the safety and efficacy of lisinopril and fasudil, primary canine corneal fibroblasts (CCFs) generated from donor corneas of healthy dogs (n=20) were used. A series of dose-dependent and time-dependent assays with lisinopril (1-50 µM) and fasudil (1-10 nM) were performed. qRT-PCR, immunofluorescence (IF) staining, cell viability assay, cell proliferation assay, LIVE/DEAD Viability/Cytotoxicity assay, TUNEL assay, and total cell count were performed. Results: A 25 µM lisinopril and 3 nM fasudil dose were safe, non-toxic, and optimal for therapeutic evaluations in vitro. Treatments of lisinopril or fasudil, alone or in-combination, to CCFs grown in the presence of TGF-[beta]1 (5ng/ml) showed inhibition of myofibroblast formation based on phase-contrast microscopy. The qRT-PCR and IF studies showed a significant decrease in expression of profibrotic markers, including [alpha]-smooth muscle actin ([alpha]-SMA; p<0.0001), Fibronectin (FN; p = 0.0002), Tenascin C (TNC; p<0.0001), Collagen I (Col-I; p<0.0001), Collagen IIIA1 (Co-IIIA1; p<0.0001), and Collagen IV (Co-lV; p<0.0001). Conclusion: An ophthalmic formulation consisting of lisinopril and fasudil may offer a safe and effective method to treat canine corneal fibrosis. Additional studies evaluating safety and efficacy of this formulation in vivo are warranted. PHASE II: ABSTRACT Purpose: To investigated the modulation of autophagy-related genes (LC3, Beclin1, Sqstm1/p62, and Lamp1) in healthy and injured corneal stromal fibroblasts. Methods: Primary cultures of corneal stromal fibroblasts (CSFs) were generated from healthy donor canine corneas and grown in minimum essential medium (MEM) containing 10 percent fetal bovine serum up to ~60 percent confluence. Following incubation, the cultures were exposed to nitrogen mustard (NM) to induce cellular insult and subjected to an autophagy activator, rapamycin (R), or vehicle treatment. Phase-contrast microscopy, qRT-PCR, and immunofluorescence staining were used to study the role of autophagy genes in canine corneal wound healing in vitro. Results: Phase-contrast microscopy showed that NM exposure led to morphological changes with stress fibers in CSFs, which was noticeably decreased by rapamycin treatment. Treatment of CSFs with rapamycin alone showed fibroblast hypertrophy while vehicle-treated population of CSFs exhibited typical spindle morphology. The qRT-PCR showed increased expression of LC3, Beclin1, and Lamp1 mRNA when treated with NM, NM+R and R in comparison to vehicle treated CSFs. Sqstm1/p62 expression was upregulated in the NM and NM+R treatment groups but was reduced in the R treated group. Immunofluorescence showed similar results of the protein levels. Conclusions: This study suggests that autophagy is an essential component in corneal healing post injury. Further, results suggest that targeting autophagy may offer an attractive treatment option to reestablish corneal clarity following ocular insult. Additional studies are warranted.