Regenerative effects of RKI-1447 on human corneal endothelial cells: a comparative study

Abstract

Background

To evaluate and compare the effects of two Rho-associated protein kinase (ROCK) inhibitors (Y-27632 and RKI-1447) on human corneal endothelial cell (HCEC) proliferation and wound healing.

Methods

Using a commercial primary HCEC line, three groups of HCECs were created; control (no treatment), Y-27,632-treated, and RKI-1447-treated. The control, Y-27,632, and RKI-1447 groups were compared regarding cell proliferation (5-bromo-2-deoxyuridine [BrdU] incorporation), wound healing (ImageJ analysis), and ROCK activity (enzyme-linked immunosorbent assay [ELISA]).

Results

Both RKI-1447 (1 µM) and Y-27,632 (10 µM) significantly enhanced HCEC proliferation when compared to the control group (p < 0.05). However, RKI-1447 showed a more potent effect than Y-27,632 in terms of stimulating cell proliferation under the experimental conditions tested (p < 0.05). Furthermore, endothelial wound healing was faster in the RKI-1447 group compared to the control and Y-27,632 groups (p < 0.05). Y-27,632 was found to stimulate wound healing more than the control group only in the first 24 h (p < 0.05).

Conclusion

This pilot study demonstrated that RKI-1447 was more potent than Y-27,632 regarding HCEC proliferation, endothelial wound healing and suppressing ROCK activity. However, further in vitro and in vivo preclinical studies are required to validate the efficacy and ocular safety profile of RKI-1447 before any consideration of clinical translation.

Background

Cornea is a transparent tissue which has the highest refractive power of the eye. The innermost endothelial layer is composed of a single layer of hexagonal cells and has an important role in maintaining the corneal transparency [1]. On the other hand, human corneal endothelial cells (HCECs) have very limited ability to proliferate in vivo. These cells are arrested in the G1/S phase of the cell cycle due to high concentrations of inhibitory factors in the aqueous fluid, and intercellular contact inhibition [2–4]. Number of HCECs decreases over time due to physiologic causes such as the natural aging process or pathologic causes such as surgery, trauma, infection, inflammation and endothelial dystrophies. When the HCEC count falls below 500 cells/mm², persistent corneal edema occurs [1–5].

Currently, lamellar or full-thickness keratoplasty is the only available treatment for corneal endothelial failure. Despite the recent advances in lamellar keratoplasty techniques, the need for corneal donor still remains [6, 7]. According to recent estimations, around one corneal tissue is available for every 70 patients worldwide, which is a significant global indicator of the donor shortage [8, 9]. These factors have encouraged investigations into enhancing the number of surviving endothelial cells in vivo or implantation of the cultivated HCECs into the anterior chamber to treat endothelial failure [10, 11]. Rho kinase (ROCK) is a serine/threonine protein kinase activated by Ras homolog family member A (RhoA), a small guanosine triphosphatase (GTPase) protein of the Rho family. These kinases are involved in smooth muscle contraction, cell apoptosis, adhesion, migration and motility, and are also involved in the cell cycle. Abnormal functioning of the ROCK and Rho-GTPase pathway has been associated with many pathological conditions, and selective inhibition of this signaling pathway has been used or is being studied for the treatment of various diseases. Although some ROCK inhibitors have been approved for the treatment of glaucoma, no ROCK inhibitor has been approved for the treatment of corneal endothelial failure [12–14].

The aim of this study is to investigate the comparative effects of Y-27,632 and RKI-1447 ROCK inhibitors on HCEC proliferation and wound healing. To the best of our knowledge, this is the first study to investigate the effects of RKI-1447 on proliferation and wound healing in commercially available primary effects of RKI-1447 on proliferation and wound healing in commercially available primary HCECs. Thus, it is aimed to fill a gap in the literature and to take new steps towards the development of cell-based therapies in corneal endothelial failure.

Methods

Human corneal endothelial primary cell line and reagents

The human corneal endothelial primary cell line was supplied as viable cells in a T25 culture flask (Celprogen, 36081-13-T25; Passage 2). Cells were received at Passage 2 (P2) and all experiments were performed using cells from a single batch at Passage 3 (P3). The cell population consisted of approximately 95% adherent and 5% suspended cells, and the culture medium was refreshed every 24–48 h.

The following media, reagents, and materials were used throughout the study: serum-containing HCEC medium (Celprogen, M36081-13 S), serum-free HCEC medium (Celprogen, M36081-13), extracellular matrix (ECM)-coated culture flasks (Celprogen, E36081-13-T25), ECM-coated 6-well plates (Celprogen, E36081-13-6), ECM-coated 96-well plates (Celprogen, E36081-13-96), freezing medium (Celprogen, M36081-13FM), 1X phosphate-buffered saline (PBS) solution containing ethylenediaminetetraacetic acid (EDTA) (Celprogen, P1408-013), and 1X trypsin-EDTA solution (Celprogen, T1509-014). The ROCK inhibitors Y-27,632 2HCl (Selleckchem, S1049) and RKI-1447 (Selleckchem, S7195) were used as treatment agents.

Cell culture, passaging, thawing, and storage

HCECs were cultured in sterile medium at 37 °C in a humidified incubator containing 5% carbon dioxide (CO₂) and 95% air. When the cells reached approximately 60–70% confluence, they were detached from the culture surface and passaged into new flasks.

For passaging in T25 flasks, the culture medium was first aspirated, and the cells were washed with 2 mL of 1X phosphate-buffered saline (PBS) for 2–3 min, after which the PBS was removed. Subsequently, 2 mL of trypsin was added dropwise, and the flask was incubated for 3–4 min to allow cell detachment. Cell detachment was confirmed using inverted phase-contrast microscopy (Olympus CKX41SF). Trypsin activity was neutralized by adding an equal volume (2 mL) of serum-containing medium, followed by gentle pipetting. The resulting cell suspension was transferred into 15 mL sterile conical tubes and centrifuged at 100 × g for 7 min. After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in 500 µL of fresh serum-containing medium. The cells were then re-seeded into T25 flasks with 5 mL of fresh medium.

For thawing, cryopreserved cells stored at − 80 °C were rapidly thawed in a 37 °C water bath by gentle agitation of the cryotube until completely thawed.

For long-term storage, cells at 70–80% confluence were first washed with PBS following removal of the culture medium. Cells were then detached using trypsin in T75 flasks by incubation for approximately 2 min. Complete detachment was confirmed by inverted microscopy, after which 4 mL of fresh serum-containing medium was added to neutralize trypsin. Cells were subsequently collected and prepared at a target density of 5 × 10⁵ to 1 × 10⁶ cells per cryotube in freezing medium. The cryotubes were placed in a controlled-rate freezing container containing isopropyl alcohol and stored at − 20 °C for at least 4 h before transfer to − 80 °C for long-term preservation.

Cell counting and proliferation experiments

Cell counting was performed using cells that had been trypsinized and neutralized as described above. Briefly, 0.5 mL of the homogenized cell suspension was transferred into an Eppendorf tube, and a 10 µL aliquot was loaded onto a Neubauer hemocytometer for counting. Cells within the designated grid areas, including those on the left and upper boundaries, were counted according to standard counting rules. The total number of cells (N) was calculated as the sum of the counts obtained from four quadrants (N = α + β + γ + δ), and the cell concentration was determined using the formula (N/4) × 10⁴ cells/mm³.

For proliferation experiments, cells were divided into ten experimental groups. The control group received no treatment, while the remaining groups were treated with different concentrations of ROCK inhibitors. Specifically, Y-27,632 was applied at concentrations of 1, 3, 10, and 30 µM, and RKI-1447 was applied at concentrations of 0.1, 0.5, 1, 10, and 30 µM.

BrdU (5-bromo-2-deoxyuridine) proliferation assay

Cell proliferation was assessed using a colorimetric BrdU (5-bromo-2-deoxyuridine) incorporation assay. Briefly, cells were seeded into 96-well plates at a density of 20,000 cells per well, with each experimental condition performed in triplicate. Upon reaching confluence, the cells were incubated with the designated treatment agents in serum-free medium for 24 and 48 h at the specified concentrations. Proliferation was then quantified using the BrdU Cell Proliferation ELISA Kit (ab126556) according to the manufacturer’s instructions.

Reagent preparation

All reagents were equilibrated to room temperature (18–25 °C) for 4 h prior to use. The 1X BrdU working solution was prepared by adding 6 µL of BrdU stock solution to 3 mL of cell culture medium. The 1X plate wash buffer was obtained by a 1:50 dilution of the 50X stock solution. The peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) reagent was prepared by diluting 6 µL of conjugate into 12 mL of conjugate diluent, resulting in a 1:2000 dilution, followed by filtration through a 0.22 μm membrane filter.

Experimental procedure

Cells were seeded into 96-well plates at a density of 2 × 10⁴ cells per well. Following seeding, treatment agents were applied at a final volume of 100 µL per well at predetermined concentrations established through preliminary optimization experiments. BrdU labeling solution (1X) was subsequently added at 20 µL per well, and the cells were incubated for 2–24 h to allow incorporation.

After incubation, the culture medium was removed, and 200 µL of fixation solution was added to each well, followed by incubation for 30 min at room temperature. The plates were then washed three times with 1X wash buffer. Subsequently, 100 µL of anti-BrdU monoclonal detector antibody was added to each well and incubated for 1 h at room temperature, followed by three additional wash steps.

Thereafter, 100 µL of peroxidase-conjugated goat anti-mouse IgG was added to each well and incubated, followed by three further washes with 1X wash buffer. After the final wash, residual liquid was removed and the plates were briefly rinsed with distilled water before being air-dried.

Finally, 100 µL of tetramethylbenzidine (TMB) peroxidase substrate was added to each well and incubated for 30 min in the dark at room temperature. The reaction was terminated by adding 100 µL of stop solution, and optical density was measured at 450 nm using a microplate reader.

Preparation of treatment agents

The concentrations of all treatment agents were calculated using the formula C₁V₁ = C₂V₂. The ROCK inhibitor Y-27,632 was prepared by dissolving 5 mg of compound in 357 µL of distilled water to obtain a stock solution of 14 mg/mL. The working solution was then prepared at a final concentration of 10 µM with a total volume of 1500 µL.

Similarly, RKI-1447 was prepared by dissolving 10 mg of compound in 154 µL of dimethyl sulfoxide (DMSO) to obtain a stock solution of 200,000 µM. The working solution was subsequently diluted to a final concentration of 1 µM with a total volume of 1500 µL. The final DMSO concentration in the culture medium was 0.0005% (v/v), which is well below the threshold associated with cytotoxic or proliferative effects in cell culture (≥ 0.1%).

Treatment groups

Cells were assigned to three experimental groups. The control group received culture medium only without any treatment. The Y-27,632 group was treated with 10 µM Y-27,632 dihydrochloride (Selleckchem, S1049). The RKI-1447 group received 1 µM RKI-1447 (Selleckchem, S7195) as the treatment agent.

Determination of ROCK activity

ROCK activity was measured using a commercially available 96-well ROCK Activity Assay Kit (Cell Biolabs, STA-416), which is based on an enzyme immunoassay detecting phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at threonine 696 (Thr696). The assay utilizes recombinant MYPT1-coated microplates to provide a sensitive and non-isotopic method for quantifying ROCK activity using a physiological substrate.

For reagent preparation, a 10X kinase reaction buffer containing dithiothreitol (DTT) and adenosine triphosphate (ATP) was prepared by adding 10 µL of 1 M DTT and 20 µL of 100 mM ATP to 970 µL of 10X kinase buffer. The active ROCK-2 positive control was prepared by mixing 8 µL of active ROCK-2 with 20 µL of 10X kinase buffer and 172 µL of deionized water. The 1X wash buffer was obtained by diluting the 10X concentrate with deionized water. Anti-phospho-MYPT1 (Thr696) antibody and horseradish peroxidase (HRP)-conjugated secondary antibody were diluted 1:1000 in the recommended test diluent prior to use.

For the assay procedure, cell lysate samples were analyzed in duplicate. A total of 90 µL of diluted active ROCK-2 control or samples were added to each well of the recombinant MYPT1-coated 96-well plate, followed by 10 µL of 10X kinase reaction buffer containing DTT and ATP. The plate was incubated at 30 °C for 60 min. The reaction was terminated by removing the liquid from each well and adding 50 µL of 0.5 M EDTA (pH 8.0). Wells were then washed three times with 250 µL of 1X wash buffer.

Subsequently, 100 µL of diluted anti-phospho-MYPT1 (Thr696) antibody was added to each well and incubated for 1 h at room temperature on an orbital shaker, followed by three additional wash steps. Next, 100 µL of HRP-conjugated secondary antibody was added and incubated for 1 h under the same conditions, followed by washing. Thereafter, 100 µL of substrate solution was added and incubated for 20 min on an orbital shaker. The reaction was stopped by adding 100 µL of stop solution, and optical density was measured at 450 nm using a spectrophotometer.

Wound healing experiment

Treatment agents were administered to HCECs at the determined doses and times. Afterwards, a wound area was drawn with a 200 µL pipette tip for wound healing experiments. Following scratching, wells were washed once with 1× PBS to remove detached cells and debris before fresh treatment medium was applied. The area within the drawn wound area was calculated with the ImageJ program and the wound healing time was monitored. Wound healing ratio was defined as the percentage of the original scratch area re-covered by cells at each time point, calculated as: (baseline wound area − residual wound area) / baseline wound area × 100.

Statistical analysis

The SPSS version 25.0 package (IBM Corp., Armonk, NY, USA) was used for the data analysis. Continuous variables were expressed as mean ± standard error. Shapiro-Wilk test was applied to test normality and Levene’s test was used to assess homogeneity of variances; both tests confirmed that assumptions were met for all outcome variables (p > 0.05 for all). One-way analysis of variance (ANOVA) was used to test statistically significant differences among the control, Y-27,632, and RKI-1447 groups. Significant differences were evaluated using the Tukey’s honestly significant difference (HSD) post hoc test. A p value < 0.05 was considered as statistical significance at 95% of confidence level.

Results

BrdU cell proliferation assay

Determination of the effect of different doses of Y-27,632 on HCEC proliferation

HCECs was incubated with the following doses of Y-27,632; 1, 3, 10, and 30 µM for 24 and 48 h. Three independent Y-27,632 experiments were performed and averaged for the analysis. 10 µM Y-27,632 significantly increased cell proliferation after 24 and 48 h of incubation more than the control, 1, 3, and 30 µM groups (p < 0.05 for all). Therefore, 10 µM Y-27,632 was selected as the effective dose and 48 h as the effective time for incubation.

Determination of the effect of different doses of RKI-1447 on HCEC proliferation

HCECs were incubated for 24 and 48 h with the following doses of RKI-1447; 0.1, 0.5, 1, 10, and 30 µM. The average values of the three independent experiments were used for the analysis. After several comparisons, 1 µM RKI-1447 was found to increase cell proliferation at 24 and 48 h of incubation more than the control, 0.1, 0.5, 10, and 30 µM groups (p < 0.05 for all). Hence, 1 µM RKI-1447 was selected as the effective dose and 48 h as the effective incubation period for further experiments.

Comparison of the effects of effective doses of Y-27,632 and RKI-1447 on HCEC proliferation

The BrdU-incorporated cell ratios were 116 ± 0.5% vs. 140 ± 3.9% at 24 h; and 138 ± 2.7% vs. 171 ± 1.1% at 48 h in the Y-27,632 (10 µM) and RKI-1447 (1 µM) groups in order (Fig. 1). These values were significantly higher when compared to the untreated control group (100 ± 0.8% at 24 h, 100 ± 0.7% at 48 h) (p < 0.05 for all) (Fig. 1). Moreover, RKI-1447 provided significantly higher HCEC proliferation ratio when compared to the Y-27,632 at both 24 and 48 h (p = 0.0001 for all) (Fig. 1).

Fig. 1.

Human corneal endothelial cell (HCEC) proliferation (ratio of BrdU incorporation) in the control (no treatment), Y-27,632 (10 µM) and RKI-1447 (1 µM) groups after 24-hours and 48-hours of incubation. (Values were expressed as mean ± standard error of the mean (SEM); n = 3 independent experiments, each performed in triplicate. BrdU data normalized to the within-experiment untreated control (set to 100%). ᵃp = 0.004, Y-27632 vs. Control at 24 h; ᵇp = 0.0001, Y-27632 vs. Control at 48 h; ᶜp < 0.0001, RKI-1447 vs. Control at 24 h; ᵈp < 0.0001, RKI-1447 vs. Control at 48 h; ᵉp = 0.0001, RKI-1447 vs. Y-27632 at 24 h; ᶠp = 0.0001, RKI-1447 vs. Y-27632 at 48 h)

ROCK activity

In the Y-27,632 and RKI-1447 groups, ROCK activity was significantly decreased by 4.49 ± 0.04% and 9.77 ± 0.06% (respectively) when compared to the untreated control group (p = 0.004 and p = 0.001, respectively) (Fig. 2). Comparing Y-27,632 and RKI-1447 groups regarding ROCK activity suppression, the difference was statistically significant (p = 0.002) (Fig. 2).

Fig. 2.

Change in ROCK activity after 48 h of incubation in the control, Y-27,632 and RKI-1447 human corneal endothelial cell (HCEC) groups. (Values were expressed as mean ± standard error of the mean (SEM); n = 3 independent experiments, each performed in duplicate. ^a p = 0.004, Y-27632 vs. Control; ^b p = 0.001, RKI-1447 vs. Control; ^c p = 0.002, RKI-1447 vs. Y-27632)

HCEC wound healing experiments

The control, Y-27,632 (10 µM) and RKI-1447 (1 µM) groups were evaluated after 24 and 48 h of incubation. The wound healing ratio (healed area/baseline wound area x100) was 17% at 24 h and 47% at 48 h in the Y-27,632 group, compared to 10% at 24 h and 47% at 48 h in the control group (Fig. 3). In the RKI-1447 group, the wound area healed by 60% at 24 h and 78% at 48 h. In terms of wound healing ratio, RKI-1447 showed more potent effects than the control (p < 0.0001 at 24 and 48 h) and Y-27,632 (p < 0.0001 at 24 and 48 h) groups under the experimental conditions tested (Fig. 3). However, in the Y-27,632 group, the wound healing rate was statistically higher than in the control group at 24 h (p = 0.028) but it lost significance at 48 h (p > 0.05) (Fig. 3).

Fig. 3.

Wound healing progress in the control, Y-27,632 and RKI-1447 groups after 24 and 48 h of incubation. (Wound healing ratio: Control group: 10% at 24 h and 47% at 48 h; Y-27632 group: 17% at 24 h^* and 47% at 48 h^*; RKI-1447 group: 60% at 24 h^** and 78% at 48 h**). *Y-27,632 vs. Control group comparisons, p = 0.028 at 24 h, p > 0.05 at 48 h. **RKI-1447 vs. Y-27,632 and control groups comparisons, p < 0.0001 for all at 24 and 48 h

Discussion

The current study investigated the effects of RKI-1447 on HCEC regeneration and contrasted it with Y-27,632, a widely studied ROCK inhibitor which was found to induce HCEC proliferation. The present study demonstrated that both Y-27,632 and RKI-1447 promoted HCEC proliferation and wound healing, while RKI-1447 was more potent than Y-27,632 under the experimental conditions tested.

Y-27,632 is a selective inhibitor of Rho-associated coiled-coil forming protein serine/threonine kinase. It inhibits both Rho-associated protein kinase 1 (ROCK1) and Rho-associated protein kinase 2 (ROCK2), which are involved in various cellular processes including smooth muscle contraction, cell motility, and proliferation [12–16]. On the other hand, RKI-1447 is a potent and selective inhibitor of ROCK1 and ROCK2, similar to Y-27,632, but it has shown higher efficacy in certain contexts. It specifically inhibits ROCK-mediated stress fiber formation and the phosphorylation of key substrates involved in cell contraction and motility, such as myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) [17, 18].

Looking at the literature, most studies focused on the effects of Y-27,632 on HCECs. The majority of the studies demonstrated that Y-27,632 increased HCEC proliferation and viability [3, 19–21]. On the other hand, there are limited studies of RKI-1447 on ocular hypertension and some cancer types. For example, Dang et al. [18] demonstrated that RKI-1447 significantly decreased the intraocular pressure compared to the control group by suppressing the stress fibers and increasing the phagocytosis effect without causing significant micro-macroscopic histological changes in the trabecular meshwork structure. In a transgenic mouse-breast cancer model study by Patel et al. [17], RKI-1447 reduced tumor size by 87% and suppressed tumor activity and invasion. Similarly, Li et al. [22] suggested that inhibition of the ROCK signaling pathway by RKI-1447 can suppress colorectal cancer through cytoskeleton-related mitochondrial dysfunction and cellular bioenergetic impairment. It should be noted that a recent small-molecule screening study tested RKI-1447 at 50 µM in the immortalized B4G12 corneal endothelial cell line [23]; however, this concentration is 50-fold higher than the effective dose identified in the present study (1 µM) and was identified as cytotoxic rather than regenerative in that screening context. To the best of our knowledge, the present study is therefore the first to investigate the effects of RKI-1447 on proliferation and wound healing in commercially available primary HCECs at a physiologically plausible concentration. In addition, recent studies have systematically compared multiple ROCK inhibitors for corneal endothelial applications. Peh et al. [24] demonstrated that both AR-13,324 (Netarsudil) and its active metabolite AR-13,503 showed improved cellular adherence compared to Y-27,632. While AR-13,324 exhibited comparable proliferative capacity to Y-27,632, AR-13,503 demonstrated significantly superior pro-proliferative effects, and wound healing capacity of AR-13,324 was comparable to Y-27,632. The same screening study [23] also identified Chroman-1 as highly potent at nanomolar concentrations, highlighting the broader potential of ROCK inhibitor repurposing for corneal endothelial regeneration. The present findings for RKI-1447 are complementary to this growing landscape and identify a further candidate warranting systematic evaluation.

In the current study, Y-27,632 and RKI-1447 were both found to stimulate HCEC proliferation. However, the effects of these ROCK inhibitors on endothelial wound healing are also important. For this purpose, in the present study, HCEC wound healing experiments were performed at 24 and 48 h of incubation in the control, Y-27,632 and RKI-1447 groups. In the Y-27,632 group, the wound healing ratio was 17% at 24 h and 47% at 48 h, while it was 10% and 47% at 48 h in the control group. Although the difference between the control and Y-27,632 groups regarding wound healing ratios at 24 h was statistically significant, it lost significance at 48 h. On the other hand, the RKI-1447 group experienced a 60% healing rate at 24 h and a 78% healing rate at 48 h. RKI-1447 significantly outperformed the control and Y-27,632 groups in terms of wound healing ratio.

Although there are no corneal endothelial disease studies in the literature comparing RKI-1447 and Y-27,632, some studies have examined the effects of Y-27,632 on HCEC wound healing [16, 19, 23]. For example, in an HCEC wound healing study, a faster healing was observed in the Y-27,632-added medium compared to the control group [16, 25]. It should be acknowledged that scratch wound healing assays reflect both cell migration and cell proliferation, and the present study does not distinguish between these two processes. ROCK inhibition is known to promote cell motility through cytoskeletal relaxation and lamellipodia formation, as well as proliferation, as previously demonstrated for Y-27,632 in HCECs [16]. Distinguishing the relative contributions of migration and proliferation would require the use of anti-mitotic agents and represents an important objective for follow-up studies.

The ability of Y-27,632 and RKI-1447 to suppress ROCK activity was further investigated in our study, and ROCK activity was shown to be significantly lower in both Y-27,632 and RKI-1447 groups than in the control group. However, RKI-1447 was more effective than Y-27,632 in reducing ROCK activity. This finding might be related to the greater potency of RKI-1447 over Y-27,632 regarding HCEC regeneration and endothelial wound healing under the experimental conditions tested. ROCK activity was assessed using MYPT1-Thr696 phosphorylation as a surrogate readout, the most widely used approach in aqueous humor dynamics research [18]. While this provides a validated indicator of ROCK inhibition, it represents a single downstream target and does not capture the full breadth of pathway modulation. Additional readouts such as MLC2-Ser19 phosphorylation are planned for follow-up mechanistic studies. Regarding clinical translation, it should be noted that the ophthalmic safety profile of RKI-1447 has yet to be established in clinical settings. This represents a critical step that must be addressed before any consideration of clinical application.

Despite the promising findings presented here, this study has limitations that warrant further investigation. First, all experiments were conducted using a single commercially available primary HCEC batch, and findings may not be directly generalizable to cells from different donors, ages, or health backgrounds; multi-donor validation is required. Second, the study does not include mechanistic investigation of cytoskeletal changes, cell cycle dynamics, or apoptosis, which would be necessary to explain the differential potency of RKI-1447 over Y-27,632. Third, endothelial phenotype preservation was not formally assessed via ZO-1 or Na+/K+-ATPase markers. Fourth, as noted above, the scratch assay does not distinguish migration from proliferation. Fifth, in vivo validation has not been performed. Further studies would address these limitations by integrating mechanistic, phenotypic, and in vivo analyses.

Conclusions

This pilot study showed that RKI-1447 was more potent than Y-27,632 in terms of HCEC proliferation, endothelial wound healing and suppression of ROCK activity. However, further in vitro and in vivo preclinical studies are needed to validate the efficacy and ocular safety profile of RKI-1447 before any consideration of clinical translation.

Abbreviations

HCEC

Human Corneal Endothelial Cell

ROCK

Rho-associated protein kinase

BrdU

5-bromo-2-deoxyuridine

ELISA

Enzyme-linked immunosorbent assay

MYPT1

Myosin phosphatase target subunit 1

MLC

Myosin light chain

PBS

Phosphate-buffered saline

EDTA

Ethylenediaminetetraacetic acid

DMSO

Dimethyl sulfoxide

DTT

Dithiothreitol

ATP

Adenosine triphosphate

HRP

Horseradish peroxidase

TMB

Tetramethylbenzidine

ECM

Extracellular matrix

CO₂

Carbon dioxide

GTPase

Guanosine triphosphatase

RhoA

Ras homolog family member A

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by EKT, AC, HSA, OP and IT. The first draft of the manuscript was written by IT and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. The authors also appreciate Hande Senol, PhD, from the Department of Biostatistics for statistical assistance.

Funding

This study was supported by Pamukkale University Scientific Research Projects Coordination Unit (2020TIPF026, 2020HZDP019, 2020HZDP020 and 2020HZDP022).

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable. This study was conducted using a commercially available human corneal endothelial primary cell line (Cat# 36081-13-T25, Cellprogen Inc., Torrance, CA, USA). As the cells were obtained from a commercial source and the research involved strictly in vitro experiments on an established cell line, institutional ethics committee approval and informed consent were not required.

Consent for publication

Not applicable. This study did not involve human participants, patient data, or any identifiable personal information.

Competing interests

The authors declare no competing interests.