Topical Rho-Associated Protein Kinase Inhibitor HA1077 Reduces Rabbit Corneal Fibrosis and Neovascularization In Vivo

Primary human corneal fibroblast cultures

Primary human corneal stromal fibroblast (hCSF) cultures were generated from donated human corneas obtained from a regional eye bank (Saving Sight, Kansas City, MO, USA). The corneas were washed twice with serum-free minimum essential medium (MEM) (Thermo Fischer Scientific, Grand Island, NY, USA). The corneal epithelium and endothelium were dissected free of the stroma with a surgical blade, and the remaining stromal sections were subdivided for explant culture. The stromal sections were placed convexly down onto a sterile 10-cm² tissue culture dish and maintained in a humidified 5% CO₂ incubator at 37°C. The cultures were incubated in MEM supplemented with essential amino acids, sodium pyruvate, MEM vitamins, penicillin/streptomycin, and 10% fetal bovine serum (MEM plus media) for approximately 21 days, and the medium was fully replaced every other day. hCSF were collected via trypsinization. In brief, 2 mL of 0.25% trypsin-EDTA was added to the plate, and the plate was kept in a CO₂ incubator for 4–5 min. Then, the plate was observed under a microscope to observe the dissociation of the cells, and two volumes of MEM plus media were immediately added for neutralization. The cell suspension was centrifuged at approximately 110 × g for 5 min (equivalent to 1,000 rpm with a rotor radius of 10 cm), and cells were seeded at a density of 7.5 × 10⁴ cells/well in six-well plates and allowed to reach 60%–70% confluence before experimental use. hCSF cultures of passage numbers 3–4 were used in this study. HA1077 (Cat# 0541) was procured from Tocris (Bio-Techne Corporation, Minneapolis, MN, USA) with purity ≥98% and used in this study.

Cytotoxicity studies

Cellular viability was evaluated via a trypan blue exclusion assay (Sigma-Aldrich, St. Louis, MO, USA). Primary human corneal fibroblasts (hCSF) were treated with HA1077 at the final concentrations of 0, 0.1, 0.5, 1, 2, 3, 4, or 5 nM. After 48 h, the cells were trypsinized and suspended in 0.4% trypan blue solution (Invitrogen, Carlsbad, CA, USA), and both nonviable (blue) and viable (unstained) cells were counted. Cellular viability (percentage) was calculated as the proportion of viable cells relative to the total cell count via an automated cell counter (Countess, Invitrogen).

Morphological changes in hCSF following HA1077 treatment were recorded via phase contrast microscopy (Leica, DMIL, Bannockburn, IL, USA). hCSF cultures treated with various concentrations of HA1077 (0–5 nM) for 48 h were examined, and representative images were captured via a digital imaging system (Leica DFC290, Bannockburn) fitted with a microscope.

The effect of HA1077 on hCSF apoptosis was examined via a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. hCSF cultures were treated with or without HA1077 (3 nM) for 48 h, washed with phosphate-buffered saline (PBS), and fixed with fresh 4% paraformaldehyde at room temperature for 20 min. The TUNEL assay was performed via a commercial ApopTag apoptosis detection kit according to the manufacturer’s instructions (S7165, ApopTag Red In Situ Apoptosis Detection Kit, Millipore Sigma, Burlington, MA, USA). In brief, the fixed cells were permeabilized with ethanol:acetic acid (2:1 ratio; at −20°C) and treated with TdT enzyme (37°C for 1 h), stop wash buffer (room temperature for 10 min), and anti-digoxigenin rhodamine conjugate (room temperature for 30 min), with assay kit reagents. Cells were mounted with Vectashield 520 containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) for the visualization of nuclei and photographed with a digital camera (SpotCam RT KE, Diagnostic Instruments, Inc., Sterling Heights, MI, USA).

In vivo rabbit model of corneal fibrosis and neovascularization

Twelve adult female New Zealand White rabbits weighing 1.8–2.2 kg (Charles River Laboratories, Wilmington, MA, USA) were used in this study. All animal work proposed for this study was approved by the Institutional Animal Care and Use Committees of the University of Missouri-Columbia and Harry S. Truman Memorial Veterans’ Hospital. All animals were treated in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. A well-established corneal alkali injury rabbit model of corneal fibrosis and CNV was utilized. ⁴³ The rabbits were anesthetized via intramuscular injection of ketamine (35–50 mg/kg) or xylazine (5–10 mg/kg). Two drops of 0.5% proparacaine hydrochloride were instilled into the eye several minutes prior to alkali injury to provide topical anesthesia. A wire eyelid speculum was used to permit the full visualization of the corneal surface. A 7-mm filter paper disc soaked in 1 N NaOH was applied to the central cornea for 1 min and then removed, after which the cornea was washed with balanced salt solution (BSS) for 30 s. This optimized protocol results in severe scarring and CNV in rabbits in vivo. ⁴⁴

Euthanasia and tissue collection

Euthanasia of the rabbits was performed via intravenous administration of pentobarbital (250 mg/kg), and the animals were placed under general anesthesia (ketamine + xylazine). Death was confirmed through cardiac auscultation, and a secondary physical means of euthanasia, bilateral pneumothrorax, was employed to ensure death. These methods of euthanasia are consistent with the standards of the American Veterinary Medical Association Guidelines for the Euthanasia of Animals: 2020 Edition. ⁴⁷

Animals were euthanized at 14 days post-alkali injury for tissue collection. This time point was chosen to capture the subacute wound-healing phase when corneal fibrosis and CNV are established and quantifiable, consistent with previous reports of alkali burn-induced corneal injury models.^48,49

The corneas were removed with forceps and sharp Westcott scissors, embedded in liquid optimal cutting temperature compound in a 24 × 24 × 5-mm mold, and snap frozen. Frozen tissue blocks were maintained at −80°C for future use. Tissue sections were cut into 7-μm-thick sections with a cryostat, placed on 25 × 75 × 1-mm microscope slides, and stored at −80°C until staining.

Immunofluorescence and TUNEL apoptosis assay

Corneal sections (7 μm) were prepared with a cryostat and were used for the immunofluorescence study. For α-SMA staining, a mouse monoclonal antibody (1:200 dilution, M0851; Dako, Carpinteria, CA, USA), for filamentous actin (F-actin) staining, Alexa 594-conjugated phallotoxin (A12381; Invitrogen, Molecular Probes, Life Technologies Corp., Eugene, OR, USA), and for endoglin (1:100 dilution, SC 18838; SantaCruz Biotechnology, Inc., Dallas, TX, USA). In brief, the cryosection slides were placed at room temperature for 5 min, washed twice with 1× PBS, followed by blocking with 2% serum (30 min at room temperature) and then with respective primary antibodies (90 min at room temperature). After primary antibody staining, the sections were exposed to Alexa Fluor-tagged IgG secondary antibody (1:500 dilution, Invitrogen Molecular Probes, Life Technologies Corp.) for 1 h in the dark, followed by subsequent three times washing with 1× PBS. Tissues were mounted with Vectashield 520 containing DAPI (Vector Laboratories) for the visualization of nuclei. Irrelevant isotype-matched primary antibody, secondary antibody alone, and tissue sections from naïve eyes were used as negative controls. Stained cells were visualized under a Leica fluorescence microscope (Leica) and photographed with a digital camera (SpotCam RT KE, Diagnostic Instruments, Inc.). For quantification and statistical analyses, six corneas were used to quantify α-SMA⁺ and F-actin⁺ cells in the tissues from each group. The α-SMA⁺ and F-actin⁺ cells in six randomly selected, nonoverlapping, full-thickness central corneal columns extending from the anterior stromal surface to the posterior stromal surface were counted at 400× magnification of the microscope field.

A TUNEL assay was performed on acetone-fixed corneal sections for apoptotic cells staining according to the manufacturer’s instructions (S7165, ApopTag Red In Situ Apoptosis Detection Kit, Millipore). In brief, the corneal tissue sections were permeabilized with ethanol:acetic acid (2:1 ratio; at −20°C) and treated with TdT enzyme (37°C for 1 h), stop wash buffer (room temperature for 10 min), and anti-digoxigenin rhodamine conjugate (room temperature for 30 min), with assay kit reagents. Tissues were mounted with Vectashield 520 containing DAPI (Vector Laboratories) for the visualization of nuclei and photographed with a digital camera (SpotCam RT KE, Diagnostic Instruments, Inc.).

Statistical analysis

The results are expressed as means ± standard errors. For clinical and molecular data statistical assessment, one-way analysis of variance (one-way ANOVA), followed by post hoc Bonferroni or Dunnett post hoc multiple comparison test, was used to compare the effect of HA1077 in naïve and alkali injury groups. A P value of <0.05 was considered significant.

HA1077 treatment is well-tolerated by human corneal fibroblasts

The tolerability of the HA1077 concentration to corneal stromal cells was evaluated via an in vitro assay. Figure 1 shows the concentration-dependent effects of HA1077 on the cellular viability of hCSF. No significant loss of cellular viability was observed at concentrations up to 3 nM HA1077 (P = 0.41 at 3 nM), whereas higher concentrations (4 and 5 nM) showed significantly reduced viability compared with control (P < 0.05 or P < 0.01). To further assess the cytotoxicity of HA1077 to hCSF, we used phase-contrast microscopy. Treatment with 3 nM HA1077 did not alter hCSF morphology at 48 h (Fig. 2A,B). In contrast, cultures treated with ≥4 nM HA1077 presented distinct morphological changes, including reduced density and loss of typical spindle-shaped appearance at the tested time points (Supplementary Fig. S1). The TUNEL apoptosis assay confirmed that 3 nM HA1077 was non-toxic to hCSFs, as there was no detectable difference in the number of TUNEL-positive cells between HA1077-treated and untreated control hCSFs (Fig. 2C,D).

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FIG. 1.

Concentration-dependent effect of HA1077 on human corneal fibroblast viability. Primary human corneal fibroblasts were treated for 48 h with HA1077 at concentrations ranging from 0 to 5 nM, and cell viability was assessed via a trypan blue assay. Viability was unaffected at concentrations ≤3 nM, whereas significant reductions were observed at 4 nM (*P < 0.05) and 5 nM (**P < 0.01) compared with untreated controls. The data represent the means ± Standard Error of the Means (SEMs) from three independent experiments.

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FIG. 2.

Effect of HA1077 (3 nM) on human corneal fibroblast morphology and apoptosis in vitro. Phase-contrast microscopy revealed no change in hCSF morphology after 48 h of HA1077 treatment (B) compared with that of the untreated controls (A). The TUNEL assay revealed no detectable difference in the number of apoptotic (TUNEL⁺, red) cells between untreated controls (C) and HA1077-treated cultures (D). Nuclei were counterstained with DAPI (blue). Scale bars: 100 µm. hCSF, human corneal stromal fibroblasts; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole.

HA1077 treatment attenuates corneal fibrosis and CNV after alkali injury in rabbit corneas

Naïve control corneas (no alkali injury, no treatment) exhibited normal transparency and morphology (Fig. 3A,D). In contrast, BSS-treated alkali-injured corneas developed marked haze and dense neovascularization (Fig. 3B,E). Compared with BSS-treated eyes, HA1077-treated corneas presented visibly reduced fibrotic and angiogenic responses on day 7 (Supplementary Fig. S2) and day 14 (Fig. 3C,F). Quantitative analysis revealed that HA1077 significantly reduced fibrosis scores at both days 7 and 14 (Fante’s score: day 7, 1.0 ± 0.2 vs 2.0 ± 0.3; day 14, 1.4 ± 0.2 vs 2.6 ± 0.3; P < 0.0001) (Fig. 3G). Similarly, CNV scores were significantly lower in HA1077-treated corneas than in BSS-treated controls at both time points (morphometric quantification score: day 7, 1.2 ± 0.2 vs 2.3 ± 0.3; day 14, 1.5 ± 0.2 vs 2.7 ± 0.3; P < 0.0001) (Fig. 3H).

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FIG. 3.

Effect of HA1077 treatment on corneal haze (fibrosis) and neovascularization after alkali injury. (A–C) Representative stereomicroscopy images of naïve (A), BSS-treated (HA1077−) (B), and HA1077-treated (HA1077+) (C) rabbit corneas 14 days after alkali injury. (D–F) Representative slit-lamp biomicroscopy images of naïve (D), BSS-treated (E), and HA1077-treated (F) rabbit corneas 14 days after alkali injury. (G) Quantification of corneal haze (fibrosis) via the Fantes scoring system at 7 and 14 days post-injury. (H) CNV quantification via the morphometric scoring system at 7 and 14 days post-injury. Compared with BSS alone, HA1077 treatment significantly reduced both fibrosis and CNV at both time points (****P < 0.0001; n = 6 per group). The data are presented as means ± SEMs. Scale bars: 3 mm. CNV, corneal neovascularization; BSS, balanced salt solution.

Periodic clinical eye examinations confirmed that the repeated topical application of HA1077 did not cause adverse effects on corneal anatomy or physiology. Across all observation points (days 1, 3, 7, and 14), there was no evidence of worsening ocular inflammation, conjunctival hyperemia, corneal or conjunctival edema, ocular discharge, or infection attributable to HA1077 treatment. The findings demonstrated that topical HA1077 significantly attenuated fibrosis and CNV after alkali injury in the rabbit cornea without apparent ocular toxicity.

HA1077 treatment does not alter normal IOP following corneal alkali injury

IOP was monitored in naïve, BSS-treated, and HA1077-treated rabbit eyes at 0, 3, 7, and 14 days post-alkali injury (Supplementary Fig. S3). Across all the groups, IOP remained within the normal physiological range for the rabbits, and no noticeable differences were detected.

Effect of HA1077 on corneal tissue architecture post-alkali injury

Histological evaluation was performed to assess cell/tissue organization in rabbit corneas at 14 days in all groups (Fig. 4). H&E-stained corneal sections from BSS-treated alkali-injured eyes showed more inflammatory cells and damaged epithelium compared with HA1077-treated corneas (Fig. 4B vs C). Naïve corneas had normal epithelium and stromal tissue organization (Fig. 4A). These data indicated that HA1077 treatment limits corneal damage post-alkali injury.

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FIG. 4.

H&E staining of naïve (A), BSS-treated (HA1077−) (B), and HA1077-treated (HA1077+) (C) rabbit corneas showed the gross morphological alteration at day 14 post-alkali injury. BSS-treated eyes exhibited marked infiltrating cells (black arrows) and epithelial disorganization (arrowheads) compared with naïve and HA1077-treated corneas. Naïve corneas displayed normal corneal architecture. Scale bars: 100 µm.

HA1077 treatment markedly attenuates myofibroblast accumulation and vessel formation following corneal alkali injury

In BSS-treated alkali-injured corneas, abundant α-SMA⁺ cells (green) and F-actin⁺ cells (red) were present throughout the anterior stroma beneath the epithelium (Fig. 5B,E), with a dense myofibroblast presence. In contrast, HA1077-treated alkali-injured corneas showed markedly reduced α-SMA⁺ and F-actin⁺ cells (Fig. 5C,F). Naïve corneas had no α-SMA- or F-actin-positive staining (Fig. 5A,D).

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FIG. 5.

Immunofluorescence detection of α-SMA and F-actin in rabbit corneas 14 days after alkali injury. (A–C) α-SMA (green) marking stromal myofibroblasts in naïve (A), BSS-treated control (HA1077−) (B), and HA1077-treated (HA1077+) (C) corneas. Numerous α-SMA⁺ cells (arrows) are present in the anterior stroma of BSS-treated eyes, fewer are observed after HA1077 treatment, and none in naïve corneas. (D–F) F-actin (red) in naïve (D), BSS-treated (E), and HA1077-treated (F) corneas. BSS-treated eyes show abundant F-actin⁺ myofibroblasts in the anterior stroma (arrowheads), whereas HA1077-treated corneas show markedly fewer positive cells; naïve corneas show no stromal staining. Nuclei: DAPI (blue). (G–H) Quantification of α-SMA⁺ and F-actin⁺ cells per 400× field (mean ± SD; ****P < 0.0001). Scale bars: 100 µm. α-SMA, α-smooth muscle actin.

The extent of CNV in rabbit eyes was assessed by immunofluorescence staining for endoglin, a marker of vascular endothelial cells. BSS-treated alkali-injured corneas presented abundant endoglin-positive endothelial cells (red) throughout the stroma (Fig. 6B), while HA1077-treated corneas had markedly reduced endoglin-positive cells in the stroma (Fig. 6C). Naïve corneas had no endoglin-positive cells (Fig. 6A). This analysis indicated that topical HA1077 suppresses CNV.

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FIG. 6.

Immunofluorescence detection of endoglin in rabbit corneas 14 days after alkali injury. Representative images of naïve (A), BSS-treated (HA1077 –) (B), and HA1077-treated (HA1077 +) (C) corneas. BSS-treated eyes presented numerous endoglin-positive endothelial cells (red, arrows) within the corneal stroma, whereas HA1077-treated corneas presented markedly fewer positive cells. Naïve corneas displayed no endoglin staining. Nuclei were counterstained with DAPI (blue). Scale bars: 100 µm.

Effect of topical HA1077 treatment on corneal epithelial and stromal apoptosis in vivo

The TUNEL assay was used to evaluate the effects of topical HA1077 on cornea (Fig. 7). Both HA1077- and BSS-treated alkali-injured corneas presented similar numbers of TUNEL-positive cells in the stroma, indicating that HA1077 is safe to corneas. Naïve corneas displayed minimal/no TUNEL staining in the stroma. These findings demonstrated that HA1077 is safe to corneal epithelial and stromal cells in vivo.

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FIG. 7.

In vivo effects of HA1077 treatment on corneal epithelial and stromal apoptosis after alkali injury. TUNEL assays were performed 14 days post-injury in naïve (A), BSS-treated (HA1077–) (B), and HA1077-treated (HA1077 +) (C) corneas. The arrows indicate TUNEL-positive apoptotic cells in the stroma; the arrowheads indicate apoptotic cells in the epithelium. Both treatment groups presented similar numbers of stromal TUNEL⁺ cells (3–5 per section) and comparable epithelial TUNEL staining, which was consistent with the normal physiological turnover. Nuclei were counterstained with DAPI (blue), and TUNEL-positive cells are shown in red. Scale bars: 100 µm.