Anti-fibrotic effects of lisinopril (ACE inhibitor) and fasudil (ROCK inhibitor) in combination for canine corneal fibrosis in vitro.

BACKGROUND

Corneal fibrosis is a leading cause of blindness in mammalian species and may result in compromised performance in sports and daily functions. This study evaluated the safety and anti-fibrotic effects of the FDA-approved 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, nontoxic, and optimal for therapeutic evaluations in vitro. Treatments of lisinopril or fasudil, alone or in-combination, to CCFs grown in the presence of TGF-β1 (5 ng/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 α-smooth muscle actin (α-SMA; p < .0001), fibronectin (FN; p = .0002), tenascin C (TNC; p < .0001), Collagen I (Col-I; p < .0001), Collagen IIIA1 (Co-IIIA1; p < .0001), and Collagen IV (Co-lV; p < .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.