ROCK Inhibitors as an Alternative Therapy for Corneal Grafting: A Systematic Review

Abstract

Currently, corneal blindness is affecting >10 million individuals worldwide, and there is a significant unmet medical need because only 1.5% of transplantation needs are met globally due to a lack of high-quality grafts. In light of this global health disaster, researchers are developing corneal substitutes that can resemble the human cornea in vivo and replace human donor tissue. Thus, this review examines ROCK (Rho-associated coiled-coil containing protein kinases) inhibitors as a potential corneal wound-healing (CWH) therapy by reviewing the existing clinical and nonclinical findings. The systematic review was done from PubMed, Scopus, Web of Science, and Google Scholar for CWH, corneal injury, corneal endothelial wound healing, ROCK inhibitors, Fasudil, Netarsudil, Ripasudil, Y-27632, clinical trial, clinical study, case series, case reports, preclinical study, in vivo, and in vitro studies. After removing duplicates, all downloaded articles were examined. The literature search included the data till January 2023. This review summarized the results of ROCK inhibitors in clinical and preclinical trials. In a clinical trial, various ROCK inhibitors improved CWH in individuals with open-angle glaucoma, cataract, iris cyst, ocular hypertension, and other ocular diseases. ROCK inhibitors also improved ocular wound healing by increasing cell adhesion, migration, and proliferation in vitro and in vivo. ROCK inhibitors have antifibrotic, antiangiogenic, anti-inflammatory, and antiapoptotic characteristics in CWH, according to the existing research. ROCK inhibitors were effective topical treatments for corneal infections. Ripasudil, Y-27632, H-1152, Y-39983, and AMA0526 are a few new ROCK inhibitors that may help CWH and replace human donor tissue.

Introduction

Corneal wound healing (CWH) is a complicated mechanism that involves cell death, proliferation, differentiation, migration, and extracellular matrix (ECM) repairing. The cornea must heal quickly and completely, but in some patients, such as those with neurotrophic keratopathy, the healing is disrupted after damage, leading to persistent epithelial abnormalities.¹ In addition, the cornea of diabetic individuals can have a delayed wound-healing response that causes scarring or abnormalities on the eye surface, impairing vision.² The effects of corneal blindness extend beyond the patients' typical quality of living to include their mental health.

In accordance with the studies, only one-third of people with severe visual impairments report having a good quality of life. In addition, a mild disability can significantly affect a child's quality of life.^3,4 Given that there are so few lasting therapeutic treatments for many corneal disorders, corneal transplantation is frequently required. Owing to the scarcity of high-quality grafts, there is now a significant unmet medical need, with <1.5% of transplantation demands being met globally.⁵

Although corneal grafting is the most popular and successful type of human tissue transplantation, the demand for it is rising, and the supply of human corneal donor tissue to meet this rising need is not certain globally.^5,6 However, corneal graft failure has been considered to be a big worry because it raises the demand for corneal tissue even more.⁷ In addition, each graft failure that necessitates regrafting raises each person's chance of rejection and graft failure in the future.⁸

Thus, CWH not only interests basic research but also represents an unmet medical problem that must be addressed. Despite being exterior to the eye, cornea seems to be a tissue that is readily accessible to drugs, but because of its structure, which is made up of various barriers, efficient drug delivery of biologics is challenging.⁹ There must be alternatives to the pointless blinding. In this regard, substantial research is being done on corneal bioengineering (ie, 3D-printed corneas and scaffolds).^10,11 Currently, corneal blindness touched the mark of >10 million individuals globally.¹² Considering this widespread health issue, researchers are now centering their efforts on creating corneal substitutes that can match the features of the human cornea in vivo and so serve as an alternative to human donor tissue.

Numerous crucial physiological processes, including cell shape, secretion, proliferation, motility, and gene expression, are mediated by the ROCKs (Fig. 1).^13,14 These have already been attributed to the controlling of oxidative stress, inflammation, and vascular tone.¹³ Both ROCK1 and ROCK2 are conventional serine–threonine kinases of 160 kDa belonging to the AGC (protein kinase A, G, and C) family, and both are highly expressed in tissues of both embryos and adults.

FIG. 1.

The role of ROCK inhibitors in the ROCK molecular pathway. Activation of the ROCK pathway leads to the phosphorylation of MLC, resulting in the promotion of actomyosin contractility. The MLC pathway and the LIMK pathway are both involved in regulating actin cytoskeleton dynamics and cell contractility. LIMK directly phosphorylates and inactivates cofilin, an actin-binding protein that facilitates actin filament disassembly. By inhibiting cofilin, LIMK indirectly influences actin turnover and stabilization, thereby affecting MLC-mediated contractility. The ERM pathway is another cellular signaling pathway that plays a role in modulating actin cytoskeleton dynamics, cell membrane interactions, and cell shape changes. ERM proteins act as linkers between the plasma membrane and the actin cytoskeleton. Shared regulation by Rho GTPases allows for potential coordination between ERM-mediated actin organization and MLC-mediated contractility, thus influencing cell adhesion and migration processes. In addition, the FAK pathway can interact with the PI3K/Akt pathway, which regulates cell survival, growth, and migration. FAK can activate PI3K, leading to the generation of PIP3, which in turn recruits Akt to the plasma membrane. Akt activation downstream of FAK promotes cell survival and regulates cytoskeletal dynamics, including actin remodeling. Conversely, Akt activation can also phosphorylate and modulate FAK activity. MLC, myosin light chain; MLCK, MLC kinase; LIMK, LIM kinase; ERM, Ezrin, Radixin, Moesin; FAK, focal adhesion kinase; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PI3K, phosphoinositide-3-kinase–protein kinase; Akt, protein kinase B; ROCK, Rho-associated protein kinase; IOP, intraocular pressure.

Although ROCK2 is largely expressed in the heart, brain, skeletal muscle, and lungs, ROCK1 is more abundant in the liver, spleen, kidney, and testes.^15–17 ROCKs are also distributed widely in ocular tissues such as retina, iris, ciliary muscles, and trabecular meshwork. It has been found that ROCK antagonism has potential therapeutic value for several illnesses. Therefore, ROCK inhibitors (ROCKIs) may be used therapeutically to manage an array of pathological disorders, such as glaucoma, cancer, osteoporosis, insulin resistance, neuronal degeneration, erectile dysfunction, kidney failure, and asthma.^14,18,19

The ROCKs, which are components of the serine/threonine kinase family, are downstream effectors of the small GTPase Rho (Rho A, Rho B, Rho C, and Rho E).^14,15 It has 2 isoforms, ROCK1 (ROKβ/rho kinase β/ROCK I/p160 ROCK) and ROCK2 (ROKα/rho kinase α/ROCK II), the gene of which is placed on chromosomes 18 (18q11.1) and 2 (2p24), respectively.^13,20 However, the fundamental amino acid sequence of the 2 ROCK isoforms is only 64% same, whereas the kinase domain is significantly more homologous (92%) and the coiled-coil domains are more varied.^13,17

ROCK isoforms have a Ser/Thr kinase domain at the N-terminal end, which is accompanied by a core coiled-coil structure and a rho-binding domain. The C-terminal domain functions as an autoinhibitory area that may bind to the N-terminal kinase region on its own, which lowers their catalytic activity. A split pleckstrin homology (PH) domain with an internal cysteine-rich C1 conserved region is present in this area (73% identity).^16,17,21

Controlling the dynamics of the actin cytoskeleton and generating actin–myosin contractility are ROCKs' main functions.^22,23 Myosin light chain 2 (MLC2) and the myosin-binding subunit of myosin phosphatase are the principal ROCK substrates in the modulation of actin–myosin contraction.²⁴

Molecular Pathways of CWH

Several molecular pathways are taking part in the CWH process including but are not limited to nuclear factor κB (NF-κB),²⁵ phosphatidylinositol 3-kinase (PI3K)–Akt,²⁶ extracellular signal-regulated kinase (ERK1/2),²⁷ p38 mitogen-activated protein (MAP) kinase,²⁸ c-Jun N-terminal kinase (JNK),^28,29 Janus kinase (JAK),³⁰ and ROCK (Rho-associated coiled-coil containing protein kinases)^13,31,32 pathways. Among these, a recent breakthrough is the inhibition of the ROCK pathway, which is mainly involved in regulating cell movement through actin stress fibers and focal adhesions (Fig. 2).^23,33

FIG. 2.

Molecular pathways in corneal wound healing. ROCK, Rho-associated coiled-coil containing protein kinases; EGFR-Akt, epidermal growth factor receptor-Ak strain transforming; PI3K-Akt, phosphatidylinositol 3-kinase-Ak strain transforming; JAK, janus kinase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Numerous functions of corneal cells, including epithelial differentiation, cell adhesion, proliferation, cytoskeleton reorganization, and cell–matrix interactions, are controlled by ROCKs.^31,34–36 The formation and regulation of barrier integrity, along with cell–cell adhesion mediated by β-catenin and E-cadherin, are all mediated by ROCKs.³¹ Experiments using cardiac cells, where ROCK is activated by transforming growth factor-beta (TGF-β) and suppressed by bone morphogenetic protein (BMP)-2 expression, may offer a clue as to how ROCKIs affect wound healing. BMP-2 can also impact cell migration by triggering few ECM components.^37,38

Inhibiting ROCK may change the signaling from profibrotic TGF-β to promigratory BMP, promoting the cell migration required for wound healing. Therefore, the main purpose of this review is to explore the present knowledge on ROCKIs in the process of CWH by reviewing the existing literature on clinical and nonclinical studies.

Marketed ROCK Inhibitors

Fasudil has been licensed for usage in Japan and China since 1995, but it has not yet received approval from the EMA or the USFDA.^13,39 It is recommended for the therapeutic management of cerebral vasospasm, and its application to hypertension and other cardiovascular diseases is now under consideration.^18,40 The pharmacology of Fasudil is being studied in several clinical studies for diseases such as amyotrophic lateral sclerosis, myocardial ischemia, diabetic macular edema, and Raynaud's phenomenon.^41–45

For the therapeutic benefit of glaucoma and ocular hypertension, Ripasudil was initially licensed in Japan in 2014 with a low incidence of adverse effects in phase I and phase II studies.^46,47 Even when adverse effects (such as conjunctival hyperemia) were noticed, the impact was very temporary, demonstrating a good safety profile.^48,49 Likewise, it has also been examined as a possible therapy for Fuchs corneal dystrophy, and a phase II clinical research is being conducted for the control of diabetic retinopathy.⁵⁰ In comparison with other Rho kinase inhibitors, Ripasudil is regarded as an advanced selective Rho kinase inhibitor with higher potency.

Netarsudil is another Rho kinase inhibitor of both ROCK1 and ROCK2. In 2019, the FDA authorized its use for treating ocular hypertension and open-angle glaucoma patients who have increased intraocular pressure (IOP).^51–53 In addition, Netarsudil decreased IOP in rabbits and monkeys during preclinical studies by causing actin stress fibers and focal adhesions in trabecular meshwork cells to break down. It also prevented TGF-β2 profibrotic's actions.⁵⁴

The U.S. FDA has approved Rocklatan (Netarsudil (0.02%) and Latanoprost (0.005%) ocular solution), a fixed dosage combination of the Rho kinase inhibitor Netarsudil (Rhopressa) and the prostaglandin F2α analog Latanoprost (Xalatan).⁵⁵ It was developed by Aerie Pharmaceuticals, Inc., and is intended to lower high IOP in people with ocular hypertension or open-angle glaucoma.⁵⁶ Different ROCKIs and their structure, mode of action, specificity, and average half-life are described in Table 1.

Table 1.

Different ROCK Inhibitors: Structure, Mode of Action, Specificity, and Average Half-Life

S.no.	Drug	Mode of action	Specificity	Average half life	References
1.	Ripasudil	Inhibiting the enzyme Rho kinase	Relaxes the smooth muscle cells in the trabecular meshwork, increasing the outflow of fluid and reducing IOP	0.4–0.8 h	^57,58
2.	Y-27632	Inhibitor of the Rho-associated protein kinase	Affect various cellular processes, including cell adhesion, migration, and contraction	12–16 h	^59,60
3.	H-1152	Inhibitor of the Rho-associated protein kinase, PKC and MLCK	Play a role in the regulation of cell shape, movement, and division	Limited pharmacokinetic data	^61,62
4.	Y-39983	Inhibitor of the Rho-associated protein kinase	Decrease IOP by increasing the outflow of aqueous humor from the eye	Limited pharmacokinetic data	^63,64
5.	Fasudil	Inhibiting the enzyme Rho kinase	Relax smooth muscle cells in blood vessels, and other tissues can help reduce IOP and improve blood flow in the eye.	<0.5 h	^65,66
6.	Netarsudil	Inhibit Rho kinase and norepinephrine transporter	Lower IOP in patients with open-angle glaucoma or ocular hypertension, decrease the production of aqueous humor in the eye	12–16 h	^67–70

IOP, intraocular pressure; MLCK, myosin light chain kinase; PKC, protein kinase C; ROCK, Rho-associated coiled-coil containing protein kinase.

Methods

The systematic review was done from the bibliographic databases (PubMed, Scopus, Web of Science, and Google Scholar) by searching the following keywords alone or in combination: CWH, corneal injury, corneal endothelial wound healing (CEWH), ROCK inhibitors, Fasudil, Netarsudil, Ripasudil, Y-27632, clinical trial, clinical study, case series, case reports, preclinical study, in vivo and in vitro studies. In addition, the references of selected articles were searched for the relevant articles. All the downloaded articles were further filtered to remove the duplicates and analyzed accordingly. Articles that are available in the English language are included. The literature search was conducted by including the data till January 2023. Studies presenting information exclusively about the role of ROCKIs in CWH are included in this review.

Results

Clinical studies

Case series

In a study by Okumura et al., 8 patients with corneal endothelial dysfunction (4 with central and 4 with diffuse edema) were given Y-27632 ophthalmic drops 6 times a day for 7 days, after trans-corneal freezing. Best corrected visual acuity (BCVA) improved from logMAR 0.7 to 0.18 at 6 months after therapy. In all patients, neither trans-corneal freezing nor ROCKI ocular drops caused intraocular or systemic problems.⁷¹

Case reports

Case report I

A 70-year-old man with fuchs endothelial corneal dystrophy (FECD) and NS3 grade cataract underwent intraocular lens implantation (IOL) and femtosecond laser-assisted cataract surgery (FLACS). Six weeks later, therapeutic contacts were removed and at 15 weeks postoperation, the right eye's BCVA was 20/20 and endothelial guttae with mild stromal edema were detected.

The patient exhibited ocular pain, inferior paracentral epithelial bullae, stromal edema, and Descemet's folds at 17 weeks postoperatively and subjected to Ripasudil eye drops 4 times daily in each eye. The ROCKI treatment increased BCVA to 20/20, and the center of the cornea was clear. In addition, enhanced hexagonal shape and bigger endothelial cells were observed.⁷²

Case report II

A 65-year-old lady with C2NS2-grade cataract and FECD II started using Ripasudil 4 times a day in each eye. Three months later, the patient had right eye FLACS, then left eye a week later. The patient's BCVA was improved from 20/40 to 20/20 in both eyes 1 month after the treatment without any corneal decompensation.⁷²

Case report III

A 77-year-old glaucoma patient with FECD stage II and cataracts of grades C1NS3 in the right eye and C2NS2 in the left underwent FLACS. Before surgery, Ripasudil eye drops were advised 4 times a day for 3 months. One week after surgery, a clinical examination indicated central corneal stromal edema, which decreased and led to a 20/20 BCVA from 20/40 thirteen weeks after surgery. Nasal thinnest point, posterior height, pachymetry map, and central corneal thickness were stable.⁷²

Case report IV

An 84-year-old woman had cataract surgery using phacoemulsification. Descemet's membrane spontaneously separated from the top incision tunnel during the procedure, and more than two-thirds of it was aspirated. The capsular bag was implanted with a foldable IOL. The patient received Y-27632 ophthalmic drops (1 mM) 4 times daily for 2 months and 6 times daily for 4 months.

The cornea had returned to clarity at 2 weeks, and by 3 months the patient's visual acuity had reached 20/20. Twelve weeks after Descemet's membrane removal, corneal endothelium grew directly onto the stroma. The unaffected area's cell density (2,732 cells/mm²) did not change after cataract surgery, but the regenerated corneal endothelium had a lower cell density than the undamaged area, indicating that wound healing involved both proliferation and migration.⁷³

Case report V

An eye trauma that occurred ∼50 years ago produced the diagnosis of an iris cyst in a 71-year-old individual. The cystic iris was separated from the cornea after the IOL was implanted because the victim was experiencing visual disturbances brought on by cataract advancement. After the procedure, corneal edema from corneal endothelium damage was noticed, and the patient's vision was 20/63.

The patient received 1 mM Y-27632 eye drops 4 times per day for 3 months, during which time the cornea cleared up, and the patient's vision improved to 20/25. After surgery, the central corneal thickness was reduced to 611 μm, and at 3 months it was down to 503 μm. After cataract surgery, corneal edema prevented the core corneal endothelial cell density from being seen, but after using ROCKI ocular drops it became more visible at a density of ∼500 cells/mm².⁷³

Case report VI

Keratoplasty was suggested for a 52-year-old Japanese man with late-onset Fuchs corneal dystrophy. The left eye's BCVA was 20/63, while the right eye's was 20/20. In addition, both eyes had guttae. The left cornea exhibited considerable central edema, whereas the right cornea was clear. After prepupillary corneal endothelium denudation, the patient received topical Y-27632 for a week.

A 24-month follow-up is reported. The patient's eyesight was 20/20 fourteen days after the operation. Vision was 20/16 after 6 months, and the central corneal thickness was 568 μm, a reduction from pretreatment. Even 2 years after the commencement of the therapy, endothelial function and eyesight have been maintained to a satisfactory level.⁷⁴ The results from different studies involving human subjects are described in Table 2.

Table 2.

Summary of Results from Different Studies Involving Human Subjects

S.no.	Type of study	Treatment	No. of subjects	Outcome	Reference
1	Case series	Y-27632	8	Y-27632 eye drops significantly reduced the central corneal edema but not diffuse corneal edema in patients with corneal endothelial dysfunction.	⁷¹
2	Case report	Ripasudil	1	After 8 weeks of therapy, the BCVA had improved to 20/20, the central cornea was clear, and the inferior quadrants still had low-grade edema with persisting endothelial guttae. The clinical status was stable at week 40, although there had been no notable alterations in ECD.	⁷²
3	Case report	Ripasudil	1	After 1 month of treatment, the patient's corneal edema had greatly diminished, and both eyes' BCVAs were 20/20 without any signs of corneal decompensation.	⁷²
4	Case report	Ripasudil	1	Central corneal stromal edema was resolved within 1 week, achieving a BCVA of 20/20 at 13 weeks after surgery.	⁷²
5	Case report	Y-27632	1	The cornea had returned to clarity at 2 weeks, and by 3 months the patient's visual acuity had reached 20/20. In addition, the corneal endothelium was directly grown on the stroma.	⁷³
6	Case report	Y-27632	1	At 13 weeks postsurgery, the cornea became clear, with a visual acuity of 20/25. The central corneal thickness was also reduced.	⁷³
7	Case report	Y-27632	1	Even 24 months after the therapy, endothelial function and eyesight have been kept in good condition.	⁷⁴

BCVA, best corrected visual acuity; ECD, endothelial corneal dystrophy.

Non/preclinical studies

In vitro models

Human corneal endothelial cells

There is a plethora of research showing the protective role of ROCKIs in CEWH. Many studies involving ROCKIs have revealed their wound-healing effect on human corneal endothelial cells (HCECs). The development of Ripasudil as eye drops for the treatment of acute corneal endothelial damage resulting from eye surgeries was supported by a study by Okumura et al., which showed that the HCECs exposed to the drug had promoted CEWH. In this work, the HCECs were treated for 48 h with the ROCKIs Y-27632 (10 μM), Fasudil (10 μM), and Ripasudil (0.3–100 μM).

The findings revealed that ROCKI exposure caused cells to proliferate in greater numbers. Especially, Ripasudil at concentrations between 0.3 and 30 μM greatly improved cell proliferation, suggesting that repurposing Ripasudil to manage corneal endothelial diseases would be a great way to deliver ROCKI ophthalmic drops in the clinical setting.⁷⁵ In another study by Pipparelli et al., toxicity, cell morphometry, proliferation, adhesion, CEC density, apoptosis, and wound-healing effect of Y-27632 were evaluated.

This work demonstrates that the Y-27632 affects cellular behavior but has no influence on the capability of HCECs to proliferate. It causes cell shape alterations, improves cell adhesion, and speeds up wound healing.⁷⁶ Several factors contribute to the inhibition of cell proliferation, including the presence of TGF-β2 in the aqueous humor and a robust contact inhibition mechanism within the corneal endothelial mosaic, which is facilitated by the cyclin kinase inhibitor p27Kip1.

On the contrary, Y-27632 dramatically enhanced the attachment and proliferation of primary HCECs, according to a research by Peh et al.⁷⁷ The increased proliferation observed may be attributed to the activation of the PI-3-kinase signaling cascade by Y-27632, resulting in the upregulation of cyclin D and the downregulation of p27. These alterations play a crucial role in promoting the proliferation of CECs. Schlötzer-Schrehardt et al. conducted a study to examine how Ripasudil affects the CECs of patients with FECD using both ex vivo tissue and in vitro cellular models.

In the ex vivo model, a single dose of 30 μM Ripasudil resulted in significant upregulation of genes and proteins associated with cell cycle progression, cell–matrix adhesion and migration, endothelial barrier and pump function for up to 72 h, regardless of the patients' age. The in vitro experiment demonstrated that Ripasudil induced changes in the expression of functional signature genes in the FECD cell line, suggesting that even the central endothelial cells of FECD patients retain some regenerative potential and are capable of restoring improved functionality in situ.⁷⁸

Human umbilical vein endothelial cells

Sijnave et al. investigated the effect of AMA0526 (a specific ROCKI) on cell viability, proliferation, and migration of human umbilical vein endothelial cells (HUVECs). Addition of AMA0526 significantly decreased EC proliferation in a dose-dependent manner, and the equivalent vehicle concentrations did not have any effect on the ECs. Migration of ECs stimulated by a serum and growth factor gradient was dose dependently inhibited by AMA0526. Also, unstimulated migration of ECs was significantly reduced by AMA0526 as compared with untreated ECs. Overall, these data revealed that AMA0526 might be able to reduce new blood vessel formation by inhibiting EC proliferation and migration.⁷⁹

Human corneal epithelial cells

The Y-27632 was examined for its capacity to promote corneal epithelial wound healing in a research by Yin J and Yu FSX. This study concluded that ROCK activities mediate corneal epithelial wound healing by enhancing cell proliferation, promoting epithelial differentiation, and adversely modifying cell migration and adhesion.³¹ In addition, Y-27632's ability to induce limbal epithelial cell proliferation was examined by Sun et al. In this work, ex vivo limbal extension was accelerated by Y-27632, particularly in cells that resembled epithelial cells.

Even after 30 h of the therapy, Y-27632 dose dependently encouraged the proliferation of in vitro-cultured human limbal epithelial cells. In addition, it improved the number of p63/Ki67-positive cells and S-phase proliferating cells, confirming limbal epithelial cell growth both ex vivo and in vitro.⁸⁰ The specific mechanisms through which Y-27632 stimulates the proliferation of corneal epithelial cells are still not fully understood. However, Y-27632 stimulates cell proliferation in CECs by affecting cyclin D and the cell cycle inhibitor p27kip through the PI3-K signaling pathway. This indicates that alterations in cell cycle regulatory proteins might contribute to subsequent signaling events. However, additional research is required to establish whether this mechanism is also applicable to Y-27632–treated limbal epithelial cells.

Monkey corneal endothelial cells

Okumura et al. investigated whether Y-27632 can enhance the culture efficacy of monkey corneal endothelial cells (MCECs). MCECs cultured with Y-27632 showed the presence of a larger number of Ki67-positive cells, colony area, and also a significantly greater number of BrdU-positive cells, demonstrating that Y-27632 plays a central role in the proliferation of MCECs. Furthermore, MCECs treated with 10 μM Y-27632 showed a less wound distance than the control, indicating that Y-27632 promoted wound healing in the in vitro model.^81,82

In another study by the same authors, the inhibition of Rho signaling by Y-27632 enhanced the adhesion of MCECs, inhibited apoptosis, and increased the quantity of proliferating cells.⁸³ The enhanced adhesion observed with Y-27632 may be attributed to its capability to promote actin reconstitution, leading to increased membrane protrusion. In addition, Y-27632 may enhance cell-to-cell adhesion through its interaction with cadherin. The increased proliferative activity of the MCECs may be a result of inhibiting ROCK, which leads to the blockade of sustained ERK/MAPK activity. This, in turn, triggers the rapid induction of cyclin-D1 through the activation of Rac1 and Cdc42.

Porcine corneal endothelial cells

Two ROCKIs (Y-27632 and H-1152) were tested on porcine corneal endothelial cells (PCECs) in a research by Meekins et al. to see how well they inhibited ROCK ability to encourage CEC proliferation and migration. This investigation treated confluent CECs with 10-μM Y-27632 or 2.5-μM H-1152 for 2, 24, and 48 h in serum-free media, and determined a complete collapse of actin cytoskeletal architecture with noticeably less actin stress fibers and tight junctions after 2 h of drug delivery. Within 48 h of the therapy, cell morphology was substantially recovered, indicating that Y-27632 and H-1152 significantly improved CEC proliferation and migration.⁸⁴

Bovine corneal endothelial cells

According to Li et al., 10 μM Y-27632 considerably escalated the proliferation of cultivated bovine corneal endothelial cells (BCECs) and had the morphological alterations like those of fibroblasts. This research indicated that the adhesion and migration of BCECs were also shown to be greatly improved by Y-27632, and it has the capability to be very effective in the proliferation of cultivated BCECs.⁸⁵ It inhibits ROCK, leading to decreased phosphorylation of MLC and relaxation of actin cytoskeletal tension. This causes changes in cell shape, adhesion, and contractility. Furthermore, Y-27632 promotes BCECs survival by suppressing apoptotic signaling and facilitating cell cycle progression.

Murine peripheral corneal nerves

In a research by Mertsch et al., Y-27632 was used to promote corneal nerve regeneration to treat neurotrophic keratopathy. According to this research, ROCKI has a beneficial impact on corneal nerve regeneration by increasing the branching and fiber length after injury in vitro. In this work, the researchers developed a collagen I-based 3D model with neurons inside, and demonstrated that Y-27632 has significant effect on corneal nerve regeneration.⁸⁶

The inhibition of ROCK resulted in a notable improvement in fiber outgrowth, along with increased branching and length of fibers in unmyelinated peripheral nerves. These findings suggest a direct influence of ROCK inhibition on the growth of nerve fibers. The results from different in vitro studies are described in Table 3.

Table 3.

Summary of Results from Different In Vitro Studies

S.no	Type of cell line	Treatment	Outcome	Reference
1	HCECs	Ripasudil (0.3–100 μM)	Ripasudil (0.3–30 μM)-treated HCECs showed a substantial increase in BrdU-positive cells compared with untreated HCECs.	⁷⁵
2	HCECs	Y-27632 (10 μM)	It did not affect the proliferative abilities of HCECs, but instead caused alterations in cell morphology, boosted cell adhesion, and speeded up wound healing.	⁷⁶
3	HCECs	Y-27632 (10 μM)	Significantly enhanced the attachment and proliferation of primary HCECs.	⁷⁷
4	HCECs	Ripasudil (30 μM)	Enhanced the pump/barrier function, cell migration, and cycle progression in central corneal endothelial cells of FECD patients.	⁷⁸
5	HUVECs	AMA0526 (0.1, 1, and 10 μM)	ROCKI dose dependently inhibited vascular endothelial cell proliferation and migration.	⁷⁹
6	HCEPCs	Y-27632 (10 μM)	Enhanced cell proliferation, promoted epithelial differentiation, but negatively modulated cell migration and cell adhesion.	³¹
7	HCEPCs	Y-27632 (0–20 μM)	Promoted the proliferation of limbal epithelial cells.	⁸⁰
8	MCECs	Y-27632 (10 μM)	Promoted cell adhesion, inhibited apoptosis, and increased cell proliferation.	^81–83
9	PCECs	Y-27632 (10 μM) H-1152 (2.5 μM)	Proliferation of PCECs was significantly higher with administration of 2.5-μM H-1152 than the 10 μM Y-27632 and control.	⁸⁴
10	BCECs	Y-27632 (10 μM)	Significantly enhanced the adhesion, migration, and proliferation of BCECs.	⁸⁵
11	MPCNs	Y-27632 (100 & 200 μM)	Showed a great enhancement in fiber growth.	⁸⁶

BCEC, bovine corneal endothelial cell; FECD, fuchs endothelial corneal dystrophy; HCEC, human corneal endothelial cell; HUVEC, human umbilical vein endothelial cell; MCEC, monkey corneal endothelial cell; MPCN, murine peripheral corneal nerve; PCEC, porcine corneal endothelial cell.

In vivo models

Transcorneal cryogenic injury model

Rabbits

In a research by Okumura et al., rabbits were used to create a corneal endothelial wound using trans-corneal cryogenic injury to see if Y-27632 would improve CEWH in an in vivo model. The injured rabbits were treated with Y-27632 at a concentration of 10 mM 6 times per day, and slit lamp examination revealed that the corneal transparency was higher, and the corneal thickness and mean wound area were much lower in the Y-27632–treated group than in the control.⁸²

In another study, Y-39983 was tested for CEC proliferation. In brief, rabbit corneas were subjected to trans-corneal freezing, and the wound areas were measured 2 days after the topical treatment with 0.095 mM (0.003%), 0.32 mM (0.01%), or 0.95 mM (0.03%) of Y-39983. After Y-39983 treatment at 0.095 mM, the wound area of the corneal endothelium was much smaller (by 43%) than that in the control eye. The reduction in wound area might be because of the increased proliferation of CECs confirmed by the existence of Ki67-positive cells (20% in control and 35% in Y-39983 treated).⁸⁷

In addition, trans-corneal freezed rabbits given ocular drops containing either 0.4% or 0.8% of Ripasudil showed decreased haze, wound area in their corneas when compared with control rabbits. The Ki67 staining also indicated that the Ripasudil treatment significantly promoted the cell proliferation.⁷⁵

Monkeys

Okumura et al. made an effort to prove that Y-27632 can stimulate wound healing of monkey corneal endothelium. To test this, they created partial corneal endothelial wounds by trans-corneal freezing, and representative slit-lamp images showed that both Y-27632 treated and untreated corneas were cloudy before regaining their clarity 4 weeks later. The Y-27632 enhanced CEC density recovery and wound healing in terms of both form and function.⁷¹

Upon application of Y-27632 eye drops, the corneal endothelium in the central damaged area underwent regeneration. Notably, it regained its characteristic hexagonal cell shape, restored adhesion profiles (ZO-1 and Na⁺/K⁺-ATPase), and re-established the actin cytoskeleton. Furthermore, the use of Y-27632 eye drops effectively normalized the cell density, demonstrating its positive impact on corneal tissue healing.

Dogs

After cryoinjury and treatment with Y-27632, transient corneal opacity, corneal vascularization, conjunctival congestion, conjunctival chemosis, and mild anterior uveitis were observed in a study by Miyagi et al. In this work, CEC regeneration was reported in all dogs at day 28 after cryoinjury. Until day 21 post-cryoinjury, endothelial cells could not be clearly visualized in the central cornea by IVCM because of severe corneal edema.

At days 28 to 56 post-cryoinjury, small, irregularly shaped endothelial cells were seen. The ECD was significantly higher in dogs that received Y-27632 treatment. In addition, administration of Y-27632 decreased IOP and corneal thickness as compared with PBS therapy, illuminating the drug's protective impact on CEWH.⁸⁸

Mechanical scrapping model

Rabbits

Okumura et al. found that the administration of 10 mM Y-27632 ocular drops to the rabbits exposed to mechanical scrapping model (MSM) showed significantly increased Ki67-positive cells (66%) than the control (13%). After 2 weeks, the corneas of the eyes receiving Y-27632 treatment were clear with mild edema and improved hexagonal endothelial monolayer.

Nearly all cells in the Y-27632–treated eyes exhibited N-cadherin, a marker of adhesion junctions, and Na⁺/K⁺-ATPase, a hallmark of pump activity, indicating that the ROCKI increased functional and morphological recovery.⁷³ In a different investigation by Okumura et al., the CECs were transplanted together with Y-27632 by employing a MSM in rabbits.

The corneal transparency was achieved without any side effects. When ZO-1 and Na^+/K⁺-ATPase were expressed normally during restoration of the corneal endothelium, Y-27632 showed a monolayer hexagonal cell shape, but reconstruction without Y-27632 showed a stratified fibroblastic phenotype without the expression of markers. Together, these results showed that Rho-ROCK signaling activity adjustment enhances cell engraftment for cell-based regenerative medicine, and that the specific ROCKI Y-27632 facilitates CEC-based therapy.⁸⁹

Meekins et al. have demonstrated that 1-mM H-1152 eye drops administered daily for 10 days improved corneal opacification with less corneal edema. Trypan blue and alizarin red stains showed that the H-1152–treated corneas displayed regions of wound closure in comparison with the control and expressed multinucleate CECs with varied shape.⁸⁴

The improved function of the Na⁺-K⁺ ATPase pump is likely a result of the enhanced migration and proliferation of CECs observed in vitro upon administration of a ROCKI. However, further investigations are necessary to determine the relative significance of cell migration and cell proliferation in the process of CEWH. In addition, Okumura et al. investigated the effect of 0.4% Ripasudil (3 times per day) in a MSM of rabbits, and found that Ripasudil-treated corneas were clear with decreased edema all over the cornea. Further, Ripasudil-treated eyes had smaller central corneal thicknesses and increased Ki67-positive cells than vehicle-treated eyes.⁷⁵

Monkeys

In a preclinical study for cell-based therapy using a primate model, the corneal endothelium was demonstrated to regenerate when cultivated MCECs were injected into the anterior chamber together with the Rho kinase (ROCK) inhibitor, Y-27632. This study showed that activation of ROCK/MLC signaling cascade impaired the CEC engraftment by limited substrate adhesion, due to actomyosin contraction. By inhibiting the activation of actomyosin and promoting the formation of the focal adhesion complex, ROCKI enhances the effectiveness of CEC engraftment. This advancement contributes to the potential therapeutic utility of cell-based therapy as a treatment for corneal endothelial dysfunction.⁹⁰

Rats

Sun et al. tested the effect of Y-27632 on corneal epithelial wound healing in a rat model. Y-27632–treated rats had nearly cured corneal epithelial lesions by day 4. It was also affirmed by less residual defect area and more Ki67-positive cells, showing that proliferation was increased in contrast to control animals.⁸⁰ The researchers also verified that the wound-healing activity was demonstrated through the inhibition of the conventional ROCK/MLC molecular pathway.

Mice corneal micropocket neovascularization assay

To endorse the antiangiogenic capacities of AMA0526 in vivo, the corneal micropocket neovascularization assay was used by Sijnave et al. Several mouse groups were given topical treatment of the vehicle, bevacizumab, or AMA0526. The vessel length and number of clock hours were remarkably decreased by AMA0526 and Bevacizumab compared with vehicle-treated eyes.

Both AMA0526- and bevacizumab-treated corneas showed a significantly reduced vessel area and density compared with vehicle-treated mice, but were not different from each other. Inflammation was also reduced after AMA0526 treatment, whereas the vascular endothelial growth factor (VEGF) antibody showed no anti-inflammatory effect. Together, these results suggest that ROCKI AMA0526 has clear antiangiogenic and potential anti-inflammatory effects.⁷⁹ The Rho/ROCK pathway has been recognized to have a significant involvement in various inflammatory processes, including TNF-α signaling, activation of NF-kb for cytokine transcription, and more.

Mice alkali burn model

In addition, AMA0526 effect was tested in alkali burn mouse model that displays the various processes of pathological CWH, including inflammation, angiogenesis, and fibrosis. A significant increase in the ROCK began in the burned corneas on day 1 compared with naive corneas. Chemical cauterization of the cornea resulted in corneal opacification and neovascularization, but topical administration of the AMA0526 significantly decreased these clinical parameters.

In comparison with the vehicle-treated eyes, both dexamethasone and AMA0526 significantly reduced CD45⁺-infiltrated leukocytes and blood vessel density. A notable decline in collagen III was observed after topical AMA0526 treatment, whereas dexamethasone did not diminish collagen III deposition, indicating that AMA0526 has more efficacy as an antifibrotic agent than does dexamethasone.

Overall, these data revealed that AMA0526 has both anti-inflammatory and antiangiogenic effects in the alkali burn model (ABM) comparable with steroid treatment.⁷⁹ Activation of the RhoA/ROCK pathway in response to the signaling molecule TGF-β is recognized for its ability to activate the nuclear factor-kb. NF-kb, along with its regulators such as ROS, plays a partial role in facilitating the processes involved in wound healing.

Mice allogeneic corneal transplantation model

The impact of topical Ripasudil treatment on corneal transplant survival was studied by Inomata et al. After receiving an allogeneic corneal transplant, mice were given Ripasudil (0.4% & 2%) 3 times each day. With reduced graft opacity and neovascularization scores and lower mRNA levels of angiogenic and proinflammatory markers in the treated transplanted corneas, graft survival was noticeably improved in the both 0.4% and 2.0% Ripasudil-treated groups.

Furthermore, Ripasudil treatment lowered the amount of Cd11b and Cd11c mRNA, as well as the IFN-γ and IL-17 producing CD4⁺ T cells, and CD45⁺-infiltrated leukocytes in the recipients' dLNs. The results of this study showed that the Ripasudil increased graft survival by reducing neovascularization and inflammatory factors while encouraging corneal re-epithelization, indicating that Ripasudil may be helpful for reducing immunological rejection in corneal transplantation.⁹¹

Mice trephined corneal model

In a study conducted by Mertsch et al., Y-27632 tested in vivo by cutting the corneal nerves of C57BL/6 mice (70–80 μm depth). The mice received Y-27632 ocular drops or the vehicle twice daily and at day 7, a significant enhanced fiber length was found for the Y-27632 group with 5.94 ± 0.17 mm/HPF versus 4.45 ± 0.29 mm/HPF, indicating a higher regrowth rate under ROCKI.

The ROCKI caused a considerable increase in fiber length over time to that of control, which peaked at day 21 and stopped increasing between days 21 and 28 (7.34 ± 0.27 mm/HPF at day 14, 8.59 ± 0.26 mm/HPF at 21 days, and 8.59 ± 0.20 mm/HPF at day 28). The researchers had also measured the fiber length in whole mount stained corneas, and confirmed that the Y-27632–treated group displayed a significant enhancement in fiber length compared with control.⁸⁶ The results from different in vivo studies are described in Table 4.

Table 4.

Summary of Results from Different In Vivo Studies

S.no	Animal model	Treatment	Outcome	Reference
1	Rabbit TCCIM	Y-27632 (10 μM)	The mean wound area of the Y-27632–treated group was significantly lower than that of control.	⁸²
2	Rabbit TCCIM	Y-39983 0.095, 0.32, 0.95 mM	The Y-39983 treatment dramatically reduced the wound area of the corneal endothelium.	⁸⁷
3	Rabbit TCCIM	Ripasudil (0.4% & 0.8%)	Ripasudil treatment effectively enhanced the cell proliferation and promoted wound healing.	⁷⁵
4	Monkey TCCIM	Y-27632	Promoted wound healing with regard to both form and function, as well as the recovery of ocular endothelial cell density.	⁷¹
5	Dog TCCIM	Y-27632 (10 mM)	During days 28 and 42 after cryoinjury, there was a significant enhancement in endothelial cell density and a reduction in corneal thickness during the acute phase.	⁸⁸
6	Rabbit MSM	Y-27632 (10 mM)	Both morphological and functional recovery were accelerated.	⁷³
7	Rabbit MSM	Y-27632	The corneal transparency restored with the transplantation of CECs together with Y-27632.	⁸⁹
8	Rabbit MSM	H-1152 (1-mM)	The ocular edema was significantly reduced by H-1152, and the CECs developed many nuclei.	⁸⁴
9	Rabbit MSM	Ripasudil (0.4%)	Compared with control eyes, eyes treated with Ripasudil had considerably more Ki67-positive cells and had undergone regeneration. Na⁺/K⁺-ATPase and N-cadherin expression was increased in almost all CECs in eyes treated with Ripasudil but reduced in eyes under control.	⁷⁵
10	Monkey MSM	Y-27632 (100 μM)	Improved efficiency of engraftment of CECs and enabled cell-based therapy for managing corneal endothelial dysfunction.	⁹⁰
11	Mice CMNVA	AMA0526 (1 mg/mL)	Inhibition of ROCK by AMA0526 has clear antiangiogenic and potential anti-inflammatory effects, whereas inhibition of VEGF only reduces the formation of blood vessels.	⁷⁹
12	Mice ABM	AMA0526 (1 mg/mL)	AMA0526 exhibited both anti-inflammatory and antiangiogenic effects, comparable with steroid treatment. Importantly, the antifibrotic activity of the ROCKI was significantly larger than that of dexamethasone.	⁷⁹
13	Mice ACTM	Ripasudil (0.4% & 2%)	Neovascularization and inflammatory factors are downregulated, while corneal re-epithelization is encouraged, prolonging transplant life.	⁹¹
14	Mice TCM	Y-27632 (5 nM)	The ROCKI leads to a significant enhancement in fiber length.	⁸⁶

ABM, alkali burn model; ACTM, allogeneic corneal transplantation model; CMNVA, corneal micropocket neovascularization assay; MSM, mechanical scrapping model; TCCIM, transcorneal cryogenic injury model; TCM, trephined corneal model.

Applications of ROCK Inhibitors in Different Ocular Diseases

Glaucoma

The increase in IOP due to aqueous humor drainage channel being blocked by the trabecular meshwork is the most important risk factor for glaucoma. ROCKIs have been observed to modify the cell form in the trabecular meshwork, allowing one to enhance the aqueous humor outflow and thereby decreasing the IOP.⁹² The cell structure undergoes several morphological changes when the ROCK pathway is blocked, including the rounding of cell bodies and an interruption in the actin synthesis. These 2 changes enable higher aqueous humor outflow through the trabecular meshwork, which eventually lowers IOP.⁹³

Fuchs dystrophy

A study conducted by Macsai and Shiloach in the year 2018 to evaluate the effect of topical ROCK inhibitor, Ripasudil, on patients with Descemet stripping revealed that the drug was able to clear the cornea and improve the visual resolution within 10 days after treatment. After Descemet stripping automated endothelial keratoplasty, it is possible to rebuild the corneal endothelium without a donor, which may lessen the higher order aberrations and light dispersion that are known to decrease visual acuity.⁹⁴

After receiving Ripasudil therapy, some morphological alterations in CECs were improved, and the cells went back to their normal stage. These modifications included fuzzy cell boundaries, pseudoguttae, and corneal edema.⁹⁵ In addition, ROCK inhibitors were able to increase endothelial cell proliferation quickly and with little or no adverse effects.⁵⁰

Diabetic retinopathy

ROCKIs have been successfully used to treat diabetic retinopathy for a variety of reasons, including their ability to lower IOP, vasodilation, influence regular aqueous humor outflow, reduce contractility and fibrotic activity, reduce microvasculopathy, and increase the production of ECM. This improves retinal blood flow, in turn preventing the neurons from various stresses, and thus controls the wound-healing mechanism.⁹⁶ Some of the potential rock inhibitors evaluated for the treatment of diabetic retinopathy are Fasudil, Ripasudil, AMA0428 along with few others.⁹⁷

Diabetic macular edema

Diabetic macular edema (DME) is an extremely prevalent cause of vision loss in patients with diabetes. It has a diverse and confusing pathogenesis; the rupture of the blood retinal barrier being the primary one.⁹⁸ The first line of treatment considered is anti-VEGF therapy. However, anatomical and esthetic outcomes are unsatisfactory. Ahmadieh et al. in 2013 reported the combinational efficacy of Fasudil and Bevacizumab in the treatment of DME.⁹⁹

After 40 days of administration through intravitreal route, the combined outcome of both ROCKIs could substantially improve the BCVA and central macular thickness without having any undesirable impacts on retinal function. Other drugs such as Ripasudil, Netarsudil, and AR-13503 along with anti-VEGF remedy have shown potential results in the treatment of DME by improving retinal pigment epithelial cells and retinal vascular endothelial cells.¹⁰⁰

Ischemic optic neuropathy

Various researchers across the globe are currently analyzing studies on optic nerve disorders to understand the possible mechanism involved, and hence suggest a prospective line of treatment. Elderly patients aged >50 having high blood sugar levels, cholesterol, and high blood pressures are more susceptible to this visual abnormality.¹⁰¹

The Journal of Clinical Pharmacology published a research in 2015, which demonstrated that Fasudil, a calcium channel-based rock inhibitor, when administered intravitreally could improve the visual acuity in patients with nonarteritic ischemic optic neuropathy (NAION) within 1 month of treatment.¹⁰² The possible mechanism might be due to the already proven anti-inflammatory and vasodilatory effects of Fasudil along with improved optic blood flow and reduction in ganglionic cell mortality in retina.^66,103

Proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a complication of rhegmatogenous retinal detachment (RRD) that is marked by the increased number and movement of various cells, including retinal pigment epithelial and glial cells, among others.¹⁰³ PVR risk is elevated in patients with an extensive history of untreated RRD. Herein, the primary cytokine involved in etiology is the TGF-β, which activates the Rho/ROCK pathway to modulate cell migration, cell death, and fibrous tissue cramping.¹⁰⁴

A group of young scientists from Japan demonstrated that ROCKIs were able to suppress the TGF-β–induced phosphorylation of myosin light chain kinase along with collagen gel contraction. This suggests the critical role of selective ROCKIs such as Fasudil, in potentially blocking TGF-β without significantly influencing retinal cell viability.¹⁰⁵ Very recently, Dai et al. in their extensive review also supported the previous findings and reported the role of Fasudil in the treatment of PVR.¹⁰⁴ Thus, Rho kinase inhibition can serve as a beneficial target for patients suffering from PVR.

Corneal fibrosis

Corneal fibrosis is one of the fundamental causes of blindness caused by trauma, infection, or refractive surgery worldwide, mostly identified by an accumulation of corneal myofibroblasts and abnormal deposition of ECM over the corneal stroma. A report submitted by Fink et al. at the annual meeting of ARVO held in May 2016 suggested that ROCK-I HA1077 (Fasudil) was able to potentially reduce corneal fibrosis in a rabbit model developed by the application of alkali topically.^106,107 Microscopic study revealed ∼3-fold decreases in fibrosis within 10 days of treatment as compared with the control group of animals treated with a balanced salt solution.

During immunofluorescence analysis, the levels of primary markers of fibrosis, such as vascular actin, F-actin, and fibronectin, dropped significantly as compared with the control animals.^107,108 Ibrahim et al. in 2019 further suggested that ROCK-I, Y-27632, could restrict fibrosis by suppressing the transformation of tenon fibroblastic cells into myofibroblasts and blocking the signaling process of TGF-β after surgery by regulating MAPK pathway. Thus, ROCKIs can be served as a viable option for treating corneal fibrosis resulting from the overactivation of healing cells in cornea.¹⁰⁹

Retinal angiogenesis

Through antiangiogenic and vascular normalization actions, Ripasudil eye drop has shown therapeutic promise for the treatment of retinal angiogenesis.¹¹⁰ Few Japanese scholars designed this study to stimulate the human retinal microvascular endothelial cells in vitro with VEGF, followed by its treatment with Ripasudil. On the contrary, several concentrations of Ripasudil were compared with normal saline to check the efficacy in vivo.

The overall efficacy was matched by measuring the hypoxic area in the retina with a standard hypoxic sensitive drug pimonidazole using immunohistochemistry. The study garnered significant information, and proposed that Ripasudil inhibited VEGF-induced cell proliferation and increased retinal perfusion, which can in turn help manage retinal angiogenesis.^110,111

Corneal neovascularization

Among the many intriguing areas of study based on corneal wound, the effect of ROCKIs in corneal neovascularization holds great promise. Vision loss may result from corneal neovascularization, which is defined by the development of aberrant blood vessels in the cornea.¹¹² In preclinical and clinical investigations, ROCKIs such as Y-27632 and Fasudil have shown significant potential in reducing angiogenesis and preventing vessel development.¹¹³

As far as the mechanism is concerned, ROCKIs work by interfering with the cytoskeletal reorganization, limiting actomyosin contractions and impeding endothelial cell migration. They also prevent the synthesis of proangiogenic substances that are linked to corneal neovascularization, including VEGF and matrix metalloproteinases alongside providing anti-inflammatory effects.^107,114,115 Table 5 describes the role of ROCKIs in different ocular diseases.

Table 5.

Effect of ROCK Inhibitors in Response to Various Ocular Diseases

S.no.	Purpose	Drugs	Mechanism	References
1	Glaucoma	Ripasudil, Netarsudil	Enhanced aqueous humor outflow and decreased intraocular pressure.	^92,93
2	Fuchs dystrophy	Ripasudil	Accelerated endothelial cell proliferation.	^50,94,95
3	Diabetic retinopathy	Fasudil, Ripasudil, AMA0428	Reduction in contractility, microvasculopathy and fibrotic activity along with vasodilation to improve the production of extracellular matrix.	^96,97
4	Diabetic macular edema	Fasudil+ Bevacizumab, Ripasudil, Netarsudil, and AR-13503 along with anti VEGF drugs	ROCKIs in synergy upregulate VEGF and improve microvascular complications.	^98,100
5	Ischemic optic neuropathy	Fasudil	Anti-inflammatory response and vasodilation.	^66,101–103
6	Proliferative vitreoretinopathy	Fasudil	Inhibition of TGF-β phosphorylation.	^103–105
7	Corneal fibrosis	Fasudil, Y-27632	MAPK pathway inhibition.	^106–109
8	Retinal angiogenesis	Ripasudil	Anti-VEGF effects of cell proliferation and vascular normalization.	^110,111
9	Corneal neovascularization	Y-276332, AMA0526, Fasudil	Regulation of endothelial cell behavior, antiangiogenesis, improvement in vascular stability, and reduction of inflammation.	^{107,112–115}

MAPK, mitogen-activated protein kinase; TGF-β, transforming growth factor-beta; VEGF, vascular endothelial growth factor.

Conclusion and Future Perspectives

Research interest in creating corneal substitutes that could imitate human cornea features in vivo and serve as an alternative to human donor tissue has hiked in the recent decade considering corneal blindness. Given the massive burden of glaucoma, which is currently affecting 64 million individuals globally and is expected to reach 112 million by 2040, it is crucial to fully characterize the safety implications of glaucoma medication. Besides glaucoma, the prevalence of many other visual illnesses, such as cataracts, ocular hypertension, and Fuchs corneal dystrophy, is rapidly rising.

Results of preclinical and clinical studies demonstrate that ROCKIs promote CEC adhesion, proliferation, and migration; protect against apoptosis, fibrosis, inflammation; and improve endothelial cell wound healing. These outcomes raise the likelihood that ROCKIs may have an impact on corneal health. Despite the availability of therapeutic contact lenses, conjunctival flap, collagen crosslinking, phototherapeutic keratectomy, and topical hyperosmotic medicines, corneal transplantation remains the sole effective treatment for the most of eye illnesses.

After all pharmacological drugs including EGF, FGF-2, PDGF, and small interfering RNA of Connexin-43 have been proven to increase the proliferative potential of CECs, no clinically useful therapy has yet been created. Therefore, the discovery that a pharmaceutical agent is effectively treating corneal endothelium dysfunction encourages researchers to continue developing ROCKIs as a budding therapeutic for certain types of corneal endothelial dysfunction.

The ROCKIs such as Ripasudil, Fasudil, Y-27632, Y-39983, H-1152, and AMA0526 have the potential to be chosen as an alternative strategy for corneal grafting as they have significantly improved the therapeutic outcomes in corneal disorders. Further research is required to fully understand the mechanisms and function of ROCKIs, and develop them as a promising approach in the CWH process.

Footnotes

Acknowledgments

One of the authors, Mr. Subramanyam Polopalli, is grateful to Defence Research Laboratory (DRL), Tezpur, Assam, India under Defence Research and Development Organisation (DRDO), Ministry of Defence, Govt. of India, and also to the Department of Chemical Technology, University of Calcutta, Kolkata, India, for providing the necessary support. All the authors extend their gratitude to the members of the Division of Pharmaceutical Technology, Defence Research Laboratory, Tezpur, Assam, India. Furthermore, they are thankful to the anonymous internal reviewer, for their timely suggestions. All the figures available in this review are generated through ChemBioDraw Ultra 14.0 software.

Authors' Contributions

S.P. contributed to conceptualization and writing—original draft. A.S. assisted with supervision, writing—review and editing. P.N. provided the methodology. M.K. and P.D. performed visualization. D.V.K. provided validation. P.C. contributed to supervision, validation, and writing—review and editing.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

Supplementary Material

References

Sacchetti

, Lambiase

Diagnosis and management of neurotrophic keratitis. Clin Ophthalmol, 2014; 8:571–579.

, Shih

, Kwok

, et al. Experimental modeling of cornea wound healing in diabetes: Clinical applications and beyond. BMJ Open Diabetes Res Care, 2019; 7:e000779.

Nyman

, Gosney

, Victor

. Emotional well-being in people with sight loss: Lessons from the grey literature. Br J Vis Impairment, 2010; 28:175–203.

Tadić

, Robertson

, Cortina-Borja

, et al. An age- and stage-appropriate patient-reported outcome measure of vision-related quality of life of children and young people with visual impairment. Ophthalmology, 2020; 127:249–260.

Gain

, Jullienne

, He

, et al. Global survey of corneal transplantation and eye banking. JAMA Ophthalmol, 2016; 134(2):167–173; doi: 10.1001/JAMAOPHTHALMOL.2015.4776

Alió del Barrio

, Arnalich-Montiel

, De Miguel

, et al. Corneal stroma regeneration: Preclinical studies. Exp Eye Res, 2021; 202:108314.

Alio

, Montesel

, El Sayyad

, et al. Corneal graft failure: An update. Br J Ophthalmol, 2021; 105(8):1049–1058; doi: 10.1136/BJOPHTHALMOL-2020-316705

Williams

, Lowe

, Bartlett

, et al. Risk factors for human corneal graft failure within the Australian corneal graft registry. Transplantation, 2008; 86:1720–1724.

Willcox

MDP

, Argüeso

, Georgiev

, et al. TFOS DEWS II tear film report. Ocul Surf, 2017; 15:366–403.

10.

Zhang

, Xue

, Li

, et al. 3D bioprinting for artificial cornea: Challenges and perspectives. Med Eng Phys, 2019; 71:68–78.

11.

Lin

, Jin

The development of tissue engineering corneal scaffold: Which one the history will choose?. Ann Eye Sci, 2018; 3; doi: 10.21037/AES.2018.01.01

12.

Barrientez

, Nicholas

, Whelchel

, et al. Corneal injury: Clinical and molecular aspects. Exp Eye Res, 2019; 186:107709.

13.

Liao

, Seto

, Noma

Rho kinase (ROCK) inhibitors. J Cardiovasc Pharmacol, 2007; 50:17–24.

14.

Shahbazi

, Baradaran

, Khordadmehr

, et al. Targeting ROCK signaling in health, malignant and non-malignant diseases. Immunol Lett, 2020; 219:15–26.

15.

Sharma

, Roy

ROCK-2-selective targeting and its therapeutic outcomes. Drug Discov Today, 2020; 25:446–455.

16.

Shah

, Savjani

A review on ROCK-II inhibitors: From molecular modelling to synthesis. Bioorgan Med Chem Lett, 2016; 26:2383–2391.

17.

de Sousa

, Vieira

, das Chagas

, et al. Should we keep rocking? Portraits from targeting Rho kinases in cancer. Pharmacol Res, 2020; 160:105093.

18.

Feng

, Lograsso

, Defert

, et al. Rho kinase (ROCK) inhibitors and their therapeutic potential. J Med Chem, 2016; 59:2269–2300.

19.

Pan

, Shen

, Yu

, et al. Advances in the development of Rho-associated protein kinase (ROCK) inhibitors. Drug Discov Today, 2013; 18:1323–1333.

20.

Koch

, Tatenhorst

, Roser

, et al. ROCK inhibition in models of neurodegeneration and its potential for clinical translation. Pharmacol Ther, 2018; 189:1–21.

21.

Olson

MF.

Applications for ROCK kinase inhibition. Curr Opin Cell Biol, 2008; 20:242–248.

22.

Abedi

, Hayes

, Reiter

, et al. Acute lung injury: The therapeutic role of Rho kinase inhibitors. Pharmacol Res, 2020; 155:104736.

23.

Narumiya

, Tanji

, Ishizaki

Rho signaling, ROCK and mDia1, in transformation, metastasis and invasion. Cancer Metastasis Rev, 2009; 28:65–76.

24.

Yoneda

, Multhaupt

HAB

, Couchman

. The Rho kinases I and II regulate different aspects of myosin II activity. J Cell Biol, 2005; 170:443–453.

25.

Wang

, Wu

, Shi

, et al. Epidermal growth factor (EGF)-induced corneal epithelial wound healing through nuclear factor κB subtype-regulated CCCTC binding factor (CTCF) activation. J Biol Chem, 2013; 288:24363.

26.

, Yu

FSX

. Impaired epithelial wound healing and EGFR signaling pathways in the corneas of diabetic rats. Invest Ophthalmol Vis Sci, 2011; 52:3301–3308.

27.

Sharma

, He

, Bazan

HEP

. p38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: Evidence of cross-talk activation between map kinase cascades. J Biol Chem, 2003; 278:21989–21997.

28.

Saika

, Okada

, Miyamoto

, et al. Role of p38 MAP kinase in regulation of cell migration and proliferation in healing corneal epithelium. Invest Ophthalmol Vis Sci, 2004; 45:100–109.

29.

Terai

, Call

, Liu

, et al. Crosstalk between TGF-β and MAPK signaling during corneal wound healing. Invest Ophthalmol Vis Sci, 2011; 52:8208.

30.

Sugaya

, Sakimoto

, Shoji

, et al. Regulation of soluble interleukin-6 (IL-6) receptor release from corneal epithelial cells and its role in the ocular surface. Jpn J Ophthalmol, 2011; 55:277–282.

31.

Yin

, Yu

FSX

. Rho kinases regulate corneal epithelial wound healing. Am J Physiol Cell Physiol, 2008; 295(2):C378–C387; doi: 10.1152/AJPCELL.90624.2007

32.

Ljubimov

, Saghizadeh

Progress in corneal wound healing. Prog Retin Eye Res, 2015; 49:17–45.

33.

Leung

, Manser

, Tan

, et al. A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J Biol Chem, 1995; 270:29051–29054.

34.

Kim

, Lakshman

, Petroll

. Quantitative assessment of local collagen matrix remodeling in 3-D culture: The role of rho kinase. Exp Cell Res, 2006; 312:3683.

35.

Kim

, Matthew Petroll

. Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture. Exp Eye Res, 2007; 85:546.

36.

Yin

, Lu

, Yu

FSX

. Role of Small GTPase rho in regulating corneal epithelial wound healing. Invest Ophthalmol Vis Sci, 2008; 49:900.

37.

Wang

, Sun

, Li

, et al. Up-regulation of BMP-2 antagonizes TGF-β1/ROCK-enhanced cardiac fibrotic signalling through activation of Smurf1/Smad6 complex. J Cell Mol Med, 2012; 16:2301–2310.

38.

Inai

, Burnside

, Hoffman

, et al. BMP-2 Induces Versican and Hyaluronan That Contribute to Post-EMT AV Cushion Cell Migration. PLoS One, 2013; 8:e77593.

39.

Kamei

, Oishi

, Takasu

. Evaluation of fasudil hydrochloride treatment for wandering symptoms in cerebrovascular dementia with 31P-magnetic resonance spectroscopy and Xe- computed tomography. Clin Neuropharmacol, 1996; 19(5):428–438; doi: 10.1097/00002826-199619050-00006

40.

Saito

, Ozawa

, Hibino

, et al. FilGAP, a Rho/Rho-associated protein kinase-regulated GTPase-activating protein for Rac, controls tumor cell migration. Mol Biol Cell, 2012; 23:4739–4750.

41.

Sun

, Minohara

, Kikuchi

, et al. The selective Rho-kinase inhibitor Fasudil is protective and therapeutic in experimental autoimmune encephalomyelitis. J Neuroimmunol, 2006; 180:126–134.

42.

Zang-Edou

, Bisvigou

, Taoufiq

, et al. Inhibition of plasmodium falciparum field isolates-mediated endothelial cell apoptosis by fasudil: Therapeutic implications for severe Malaria. PLoS One, 2010; 5:e13221.

43.

Lee

, Moon

, Pak

, et al. HA1077 displays synergistic activity with daclatasvir against hepatitis C virus and suppresses the emergence of NS5A resistance-associated substitutions in mice. Sci Reports, 2018; 8:1–13.

44.

Shapiro

, Kietzman

, Guo

, et al. Rho-kinase inhibition has antidepressant-like efficacy and expedites dendritic spine pruning in adolescent mice. Neurobiol Dis, 2019; 124:520–530.

45.

Toshima

, Satoh

, Ikegaki

, et al. A new model of cerebral microthrombosis in rats and the neuroprotective effect of a rho-kinase inhibitor. Stroke, 2000; 31:2245–2250.

46.

Tanihara

, Inoue

, Yamamoto

, et al. Phase 2 randomized clinical Study of a rho kinase inhibitor, K-115, in primary open-angle glaucoma and ocular hypertension. Am J Ophthalmol, 2013; 156:731–736.e2.

47.

Tanihara

, Inoue

, Yamamoto

, et al. Phase 1 clinical trials of a selective rho kinase inhibitor, K-115. JAMA Ophthalmol, 2013; 131:1288–1295.

48.

Sakamoto

, Ishida

, Sumi

, et al. Evaluation of offset of conjunctival hyperemia induced by a Rho-kinase inhibitor; 0.4% Ripasudil ophthalmic solution clinical trial. Sci Rep, 2019; 9(1):3755.

49.

Komizo

, Ono

, Yagi

, et al. Additive intraocular pressure-lowering effects of the Rho kinase inhibitor ripasudil in Japanese patients with various subtypes of glaucoma. Jpn J Ophthalmol, 2018; 63(1):40–45.

50.

Macsai

, Shiloach

Use of topical rho kinase inhibitors in the treatment of fuchs dystrophy after descemet stripping only. Cornea, 2019; 38:529–534.

51.

Ren

, Li

, Le

, et al. Netarsudil increases outflow facility in human eyes through multiple mechanisms. Invest Ophthalmol Vis Sci, 2016; 57:6197–6209.

52.

Kahook

, Serle

, Mah

, et al. Long-term safety and ocular hypotensive efficacy evaluation of netarsudil ophthalmic solution: Rho kinase elevated IOP treatment trial (ROCKET-2). Am J Ophthalmol, 2019; 200:130–137.

53.

Khouri

, Serle

, Bacharach

, et al. Once-Daily netarsudil versus twice-daily timolol in patients with elevated intraocular pressure: The randomized phase 3 ROCKET-4 Study. Am J Ophthalmol, 2019; 204:97–104.

54.

Lin

, Sherman

, Moore

, et al. Discovery and preclinical development of netarsudil, a novel ocular hypotensive agent for the treatment of glaucoma. J Ocul Pharmacol Ther, 2018; 34:40–51.

55.

Rocklatan (netarsudil and latanoprost) FDA Approval History - Drugs.com. Available from: https://www.drugs.com/history/rocklatan.html [Last accessed: 26 July, 2022].

56.

Drug Approval Package: ROCKLATAN OPHTHALMIC SOLUTION. Available from: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2019/208259Orig1s000TOC.cfm [Last accessed: 26 July, 2022].

57.

Tanihara

, Kakuda

, Sano

, et al. Long-term intraocular pressure-lowering effects and adverse events of ripasudil in patients with glaucoma or ocular hypertension over 24 months. Adv Ther, 2022; 39:1659–1677.

58.

Tanihara

, Kakuda

, Sano

, et al. Safety and efficacy of ripasudil in Japanese patients with glaucoma or ocular hypertension: 12-month interim analysis of ROCK-J, a post-marketing surveillance study. BMC Ophthalmol, 2020; 20:275.

59.

Zhang

X-H

, Sun

N-X

, Feng

Z-H

, et al. Interference of Y-27632 on the signal transduction of transforming growth factor beta type 1 in ocular Tenon capsule fibroblasts. Int J Ophthalmol, 2012; 5:576–581.

60.

Zhang

, Shao

, Yu

, et al. Y-27632 promotes the repair effect of umbilical cord blood-derived endothelial progenitor cells on corneal endothelial wound healing. Cornea, 2021; 40:203–214.

61.

, Chen

, Wang

, et al. H-1152 effects on intraocular pressure and trabecular meshwork morphology of rat eyes. J Ocul Pharmacol Ther, 2008; 24:373–379.

62.

Nishio

, Fukunaga

, Sugimoto

, et al. The effect of the H-1152P, a potent Rho-associated coiled coil-formed protein kinase inhibitor, in rabbit normal and ocular hypertensive eyes. Curr Eye Res, 2009; 34:282–286.

63.

Tokushige

, Inatani

, Nemoto

, et al. Effects of topical administration of y-39983, a selective rho-associated protein kinase inhibitor, on ocular tissues in rabbits and monkeys. Invest Ophthalmol Vis Sci, 2007; 48:3216–3222.

64.

Watabe

, Abe

, Yoshitomi

Effects of Rho-associated protein kinase inhibitors Y-27632 and Y-39983 on isolated rabbit ciliary arteries. Jpn J Ophthalmol, 2011; 55:411–417.

65.

Pakravan

, Beni

, Ghahari

, et al. The ocular hypotensive efficacy of topical fasudil, a rho-associated protein kinase inhibitor, in patients with end-stage glaucoma. Am J Ther, 2017; 24:e676–e680.

66.

Sugiyama

, Shibata

, Kajiura

, et al. Effects of fasudil, a Rho-associated protein kinase inhibitor, on optic nerve head blood flow in rabbits. Invest Ophthalmol Vis Sci, 2011; 52:64–69.

67.

Batra

, Gupta

, Nair

, et al. Netarsudil: A new ophthalmic drug in the treatment of chronic primary open angle glaucoma and ocular hypertension. Eur J Ophthalmol, 2021; 31:2237–2244.

68.

Prager

, Tang

, Pleet

, et al. Effectiveness and tolerability of netarsudil in combination with other ocular hypotensive agents. Ophthalmol Glaucoma, 2021; 4:597–603.

69.

Aref

, Geyman

, Zakieh

A-R

, et al. Netarsudil and latanoprost ophthalmic solution for the reduction of intraocular pressure in open-angle glaucoma or ocular hypertension. Expert Rev Clin Pharmacol, 2019; 12:1073–1079.

70.

Sit

, Gupta

, Kazemi

, et al. Netarsudil improves trabecular outflow facility in patients with primary open angle glaucoma or ocular hypertension: A Phase 2 Study. Am J Ophthalmol, 2021; 226:262–269.

71.

Okumura

, Koizumi

, Kay

EDP

, et al. The ROCK inhibitor eye drop accelerates corneal endothelium wound healing. Invest Ophthalmol Vis Sci, 2013; 54:2439–2502.

72.

Antonini

, Coassin

, Gaudenzi

, et al. Rho-associated kinase inhibitor eye drops in challenging cataract surgery. Am J Ophthalmol Case Rep, 2022; 25:101245; doi: 10.1016/J.AJOC.2021.101245

73.

Okumura

, Inoue

, Okazaki

, et al. Effect of the Rho Kinase inhibitor Y-27632 on corneal endothelial wound healing. Invest Ophthalmol Vis Sci, 2015; 56:6067–6074.

74.

Koizumi

, Okumura

, Ueno

, et al. Rho-associated kinase inhibitor eye drop treatment as a possible medical treatment for fuchs corneal dystrophy. Cornea, 2013; 32:1167–1170.

75.

Okumura

, Okazaki

, Inoue

, et al. Effect of the rho-associated kinase inhibitor eye drop (Ripasudil) on corneal endothelial wound healing. Invest Ophthalmol Vis Sci, 2016; 57:1284–1292.

76.

Pipparelli

, Arsenijevic

, Thuret

, et al. ROCK inhibitor enhances adhesion and wound healing of human corneal endothelial cells. PLoS One, 2013; 8:1–19.

77.

Peh

GSL

, Adnan

, George

, et al. The effects of rho-associated kinase inhibitor Y-27632 on primary human corneal endothelial cells propagated using a dual media approach. Sci Rep, 2015; 5:1–10.

78.

Schlötzer-Schrehardt

, Zenkel

, Strunz

, et al. Potential functional restoration of corneal endothelial cells in fuchs endothelial corneal dystrophy by ROCK inhibitor (Ripasudil). Am J Ophthalmol, 2021; 224:185–199.

79.

Sijnave

, Van Bergen

, Castermans

, et al. Inhibition of rho-associated kinase prevents pathological wound healing and neovascularization after corneal trauma. Cornea, 2015; 34:1120–1129.

80.

Sun

, Chiu

, Lin

, et al. Y-27632, a ROCK inhibitor, promoted limbal epithelial cell proliferation and corneal wound healing. PLoS One, 2015; 10:1–18.

81.

Okumura

, Koizumi

, Ueno

, et al. The new therapeutic concept of using a rho kinase inhibitor for the treatment of corneal endothelial dysfunction. Cornea, 2011; 30:54–59.

82.

Okumura

, Koizumi

, Ueno

, et al. Enhancement of corneal endothelium wound healing by Rho-associated kinase (ROCK) inhibitor eye drops. Br J Ophthalmol, 2011; 95:1006–1009.

83.

Okumura

, Ueno

, Koizumi

, et al. Enhancement on primate corneal endothelial cell survival in vitro by a rock inhibitor. Invest Ophthalmol Vis Sci, 2009; 50:3680–3687.

84.

Meekins

, Rosado-Adames

, Maddala

, et al. Corneal endothelial cell migration and proliferation enhanced by rho kinase (ROCK) inhibitors in in vitro and in vivo models. Invest Ophthalmol Vis Sci, 2016; 57:6731–6738.

85.

, Wang

, Dai

, et al. The stimulatory effect of ROCK inhibitor on bovine corneal endothelial cells. Tissue Cell, 2013; 45:387–396.

86.

Mertsch

, Neumann

, Rose

, et al. The effect of Rho Kinase inhibition on corneal nerve regeneration in vitro and in vivo. Ocul Surf, 2021; 22:213–223.

87.

Okumura

, Nakano

, Kay

EDP

, et al. Involvement of cyclin D and p27 in cell proliferation mediated by ROCK inhibitors Y-27632 and Y-39983 during corneal endothelium wound healing. Invest Ophthalmol Vis Sci, 2014; 55:318–329.

88.

Miyagi

, Kim

, Li

, et al. Topical rho-associated kinase inhibitor, Y27632, accelerates corneal endothelial regeneration in a canine cryoinjury model. Cornea, 2019; 38:352–359.

89.

Okumura

, Koizumi

, Ueno

, et al. ROCK inhibitor converts corneal endothelial cells into a phenotype capable of regenerating in vivo endothelial tissue. Am J Pathol, 2012; 181:268–277.

90.

Okumura

, Sakamoto

, Fujii

, et al. Rho kinase inhibitor enables cell-based therapy for corneal endothelial dysfunction. Sci Rep, 2016; 6:1–11.

91.

Inomata

, Fujimoto

, Okumura

, et al. Novel immunotherapeutic effects of topically administered ripasudil (K-115) on corneal allograft survival. Sci Rep, 2020; 10:1–11.

92.

Moshirfar

, Parker

, Birdsong

, et al. Use of rho kinase inhibitors in ophthalmology: A review of the literature. Med Hypothesis Discov Innov Ophthalmol, 2018; 7(3):101–111.

93.

Honjo

, Tanihara

, Inatani

, et al. Effects of Rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility. Invest Ophthalmol Vis Sci, 2001; 42:137–144.

94.

Fujimoto

, Kiryu

Efficacy of the rho-kinase inhibitor ripasudil for fuchs' endothelial corneal dystrophy. J Ophthalmol Res, 2021; 04:231–243.

95.

Carnahan

, Goldstein

. Ocular complications of topical, peri-ocular, and systemic corticosteroids. Curr Opin Ophthalmol, 2000; 11:478–483.

96.

Honjo

, Tanihara

Impact of the clinical use of ROCK inhibitor on the pathogenesis and treatment of glaucoma. Jpn J Ophthalmol, 2018; 62:109–126.

97.

Shahsuvaryan

ML.

Diabetic Retinopathy: The Promise of New Druggable Target. EC Ophthalmology SI.02 (2020): 04-09.

98.

Miller

, Fortun

. Diabetic macular edema: Current understanding, pharmacologic treatment options, and developing therapies. Asia-Pacific J Ophthalmol, 2018; 7:28–35.

99.

Ahmadieh

, Nourinia

, Hafezi-Moghadam

, et al. Intravitreal injection of a Rho-kinase inhibitor (fasudil) combined with bevacizumab versus bevacizumab monotherapy for diabetic macular oedema: a pilot randomised clinical trial. Br J Ophthalmol, 2019; 103:922–927.

100.

Mateos-Olivares

, García-Onrubia

, Valentín-Bravo

, et al. Rho-kinase inhibitors for the treatment of refractory diabetic macular oedema. Cells, 2021; 10:1–23.

101.

Hayreh

SS.

Management of ischemic optic neuropathies. Indian J Ophthalmol, 2011; 59:123–136.

102.

Sanjari

, Pakravan

, Nourinia

, et al. Intravitreal injection of a Rho-kinase inhibitor (Fasudil) for recent-onset nonarteritic anterior ischemic optic neuropathy. J Clin Pharmacol, 2016; 56:749–753.

103.

Nourinia

, Nakao

, Zandi

, et al. ROCK inhibitors for the treatment of ocular diseases. Br J Ophthalmol, 2018; 102:1–5.

104.

Dai

, Dai

, Sun

Inflammatory mediators of proliferative vitreoretinopathy: Hypothesis and review. Int Ophthalmol, 2020; 40:1587–1601.

105.

Kita

, Hata

, Arita

, et al. Role of TGF-β in proliferative vitreoretinal diseases and ROCK as a therapeutic target. Proc Natl Acad Sci U S A, 2008; 105:17504–17509.

106.

Fink

, Gupta

, Ebers

, et al. ROCK Inhibitor HA1077: Potently Inhibits Corneal Fibrosis and Neovascularization. Invest Ophthalmol Vis Sci, 2016; 57(12).

107.

Singh

, Singh

Rho-kinase inhibitors in ocular diseases: A translational research journey. J Curr Glaucoma Pract, 2023; 17:44–48.

108.

Karamichos

, Guo

, Hutcheon

AEK

, et al. Human corneal fibrosis: An in vitro model. Invest Ophthalmol Vis Sci, 2010; 51:1382–1388.

109.

Ibrahim

, Ko

, Iwata

, et al. An in vitro study of scarring formation mediated by human Tenon fibroblasts: Effect of Y-27632, a Rho kinase inhibitor. Cell Biochem Funct, 2019; 37:113–124.

110.

Yamaguchi

, Nakao

, Arita

, et al. Vascular normalization by ROCK inhibitor: Therapeutic potential of ripasudil (K-115) eye drop in retinal angiogenesis and hypoxia. Invest Ophthalmol Vis Sci, 2016; 57:2264–2276.

111.

Bryan

, Dennstedt

, Mitchell

, et al. RhoA/ROCK signaling is essential for multiple aspects of VEGF-mediated angiogenesis. FASEB J, 2010; 24:3186–3195.

112.

Roshandel

, Eslani

, Baradaran-Rafii

, et al. Current and emerging therapies for corneal neovascularization. Ocul Surf, 2018; 16:398–414.

113.

Zeng

, Pi

, Li

, et al. Fasudil hydrochloride, a potent ROCK inhibitor, inhibits corneal neovascularization after alkali burns in mice. Mol Vis, 2015; 21:688–698.

114.

Shoshani

, Pe'er

, Doviner

, et al. Increased expression of inflammatory cytokines and matrix metalloproteinases in pseudophakic corneal edema. Invest Ophthalmol Vis Sci, 2005; 46:1940–1947.

115.

Chang

, Huang

, Cunningham

, et al. Matrix metalloproteinase 14 modulates signal transduction and angiogenesis in the cornea. Surv Ophthalmol, 2016; 61:478–497.