Topical Rho-Associated Protein Kinase Inhibitor HA1077 Reduces Rabbit Corneal Fibrosis and Neovascularization <i>in Vivo</i>

<b><i>Purpose:</i></b> Rho-associated kinase (ROCK) regulates fibrosis and angiogenesis. This study evaluated the effects of the topical ROCK inhibitor HA1077 (fasudil) to attenuate corneal fibrosis and corneal neovascularization (CNV) <i>in vivo</i>. <b><i>Methods:</i></b> Primary human corneal stromal fibroblasts (hCSF) and New Zealand White rabbits (n = 12) were used. Corneal fibrosis and CNV <i>in vivo</i> were provoked by alkali injury. Immediately post-injury, eyes received topical balanced salt solution (BSS) or HA1077 (3 nM twice/day for 3 days). Clinical slit-lamp biomicroscopy and stereomicroscopy gauged corneal haze/fibrosis (Fante's score) and CNV (morphometric score) in live rabbits. Post-euthanasia, H&E, immunofluorescence, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays analyzed α-smooth muscle actin (α-SMA), filamentous actin (F-actin), endoglin, CD11b, and apoptotic cells. <b><i>Results:</i></b> HA1077 treatment to hCSF did not alter viability, morphology, and proliferation ability at 3 nM or lower doses. Topical HA1077 (3 nM) treatment significantly reduced corneal haze/fibrosis (<i>P</i> < 0.0001) and CNV (<i>P</i> < 0.0001) <i>in vivo</i> at days 7 and 14 compared with the corresponding controls. Histological H&E analysis revealed retrieval of overall corneal health with a remarkable decrease in fibrotic (myofibroblasts), angiogenic (neovessels), and immune cell infiltration in rabbit corneas treated with HA1077 than the control corneas. HA1077 therapy significantly reduced α-SMA⁺, F-actin⁺, and endoglin⁺ cells, markers of fibrosis and CNV. Intraocular pressure (IOP) remained within the normal physiological range of eyes with HA1077. TUNEL assay revealed the tolerability of the examined HA1077 regimen <i>in vivo</i>. <b><i>Conclusions:</i></b> The tested HA1077 dosage regimen is effective and tolerable to rabbit eyes in abrogating corneal fibrosis and CNV triggered by alkali injury <i>in vivo</i> without major adverse effects. ROCK inhibition represents a promising therapeutic strategy for vascularized and fibrotic corneal wounds but warrants additional dose-response <i>in vivo</i> studies.