Novel immunotherapeutic effects of topically administered ripasudil (K-115) on corneal allograft survival

Corneal allograft survival is mediated by the variety of immunological reactions and wound healing process. Our aim was to explore the effects of topical administration of ripasudil, a selective Rho-associated coiled-coil protein kinase inhibitor, on corneal allograft survival. Ripasudil was administered to mice thrice a day after allogeneic corneal transplantation. Corneal graft survival, opacity, neovascularization, re-epithelization, immune cell infiltration, and mRNA levels of angiogenic and pro-inflammatory factors in the grafted cornea and draining lymph nodes (dLNs) were evaluated with slit-lamp microscopy, immunohistochemistry, flow cytometry, and polymerase chain reaction. Graft survival was significantly prolonged with lower graft opacity and neovascularization scores in 0.4% and 2.0% ripasudil-treated groups, and mRNA levels of angiogenic and pro-inflammatory factors in ripasudil-treated grafted corneas were reduced. Moreover, 0.4% and 2.0% ripasudil reduced CD45+-infiltrated leukocyte frequency, Cd11b and Cd11c mRNA levels, and the frequencies of mature dendritic cells, IFNγ-, and IL-17- producing CD4+T cells in the dLNs of recipients. Re-epithelization rate of the grafted cornea was significantly higher in the 0.4% and 2.0% ripasudil groups than in the control. Topically applied ripasudil prolonged graft survival by downregulating neovascularization and inflammation factors, while promoting corneal re-epithelization, suggesting that ripasudil may be useful for suppressing immunological rejection in corneal transplantation.


Results
Topical administration of ripasudil inhibited graft cornea angiogenesis and lymphangiogenesis. Figure 1A shows a representative image of the grafted cornea at day 14 post-transplantation. Topical administration of 0.4% and 2.0% ripasudil significantly reduced the CNV score compared to that in the control group on day 14 post-transplantation (Fig. 1B p = 0.022 and p = 0.037, respectively). Figure 1C shows the results of immunofluorescence staining for blood (CD31 + ) in the grafted corneas. Upper row shows the whole grafted cornea. Bottom row shows the center of the grafted cornea at higher magnification. Quantification of the vascularized area in corneal whole mounts revealed that topical administration of 0.4% and 2.0% ripasudil significantly reduced the de novo generation of CD31 + blood vessels (p = 1.000, 0.008, and 0.001 for 0.04%, 0.4%, 2.0% ripasudil versus control, respectively; n = 5, Fig. 1D). Figure 1E shows the results of immunofluorescence staining for lymphatic vessels (lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) + ) in the grafted corneas. Upper row shows the whole grafted cornea. Bottom row shows the center of the grafted cornea at higher magnification. Quantification of the vascularized area in corneal whole mounts revealed that topical administration of 0.4% and 2.0% ripasudil significantly reduced the de novo generation of LYVE-1 + lymphatic vessels (p = 1.000, 0.012, and 0.009 vs. control, respectively; n = 5, Fig. 1F).

Topical administration of ripasudil downregulated angiogenic and lymphangiogenic factors in grafted corneas.
To investigate the effects of the topical administration of ripasudil on angiogenic signals in grafted corneas, we analyzed the mRNA levels of angiogenic and lymphangiogenic genes in the grafted cornea through reverse transcription-quantitative PCR (RT-qPCR) on day 14 post-transplantation (Fig. 2). Pecam1 (Cd31), Vegfa, Vegfc, Lyve1, and Vegf receptor (Vegfr)1, 2, 3 mRNA were significantly downregulated in the ripasudil groups than in the control group ( Fig. 2A,B; n = 3).

Topical administration of ripasudil decreased leukocyte infiltration and inflammation-related mRNA expression in grafted corneas.
To determine the effect of ripasudil on the leukocyte infiltration of allografts, we examined CD45 + leukocytes in the grafted corneas through flow cytometry. CD45 + leukocytes in the grafted corneas were enumerated through flow cytometry on day 14 post-transplantation (Fig. 3A). The frequency of occurrence of CD45 + cells was significantly lower in the grafted corneas in the 0.4% and 2.0% ripasudil groups than in the control group (Fig. 3B, n = 3, control vs. 0.4% ripasudil, p = 0.031; control vs. 2.0% ripasudil, p = 0.039). The expression levels of Cd11b and Cd11c, surface markers of leukocytes such as neutrophils, macrophages, and dendritic cells (DCs) [21][22][23] , were significantly lower in the 0.4% and 2.0% ripasudil groups than in the control group (Fig. 3C, Fig. 3D, n = 3). The expression levels of inflammatory cytokines in the grafted corneas were assessed through RT-qPCR on day 14 post-transplantation. Interferon (Ifn)γ, Tumor necrosis factor (Tnf)-α, Interleukin (Il)1β, Il17, Il23, and Il33 mRNA were significantly downregulated in the ripasudil groups than in the control group (Fig. 3E). Furthermore, the mRNA level of the immunoregulatory cytokine Il10 was significantly higher in the grafted corneas in the 0.4% and 2.0% ripasudil groups than in the control group (Fig. 3F, n = 3, p < 0.01 and p < 0.001, respectively).

Topical administration of ripasudil suppressed DC maturation, and IFNγ-and IL-17-expressing effector T cell generation in dLNs.
To investigate the effect of topical administration of ripasudil on dendritic cells (DCs) maturation, we determined the frequency of occurrence of CD11c + major histocompatibility complex (MHC) II + DCs and the expression of MHC II in the draining lymph nodes (dLNs) through flow cytometry 14 days after corneal transplantation ( Fig. 4A-C). The frequency of occurrence of CD11c + MHC II + DCs was significantly lower in the 0.4% and 2.0% ripasudil groups than in the control group ( Fig. 4B; n = 5; p = 0.002, p < 0.001, respectively). Furthermore, the mean fluorescence intensity of MHC II among dLNs was significantly lower in the 0.4% and 2.0% ripasudil groups than in the control group ( Fig. 4C; n = 5; p = 0.029 and p = 0.002, respectively). The frequencies of IFNγ-and IL-17-expressing T cells (Fig. 4D,E,G,H) and mean fluorescence intensities of IFNγ and IL-17 ( Fig. 4F; n = 5; p = 0.002, p = 0.001, respectively and 4I; n = 5; p = 0.007, p = 0.013, respectively) were lower in the 0.4% and 2.0% ripasudil groups than in the control.

Discussion
In this study, we investigated the efficacy of ripasudil, a ROCK inhibitor, in controlling the immune reactions and wound healing responses upon corneal transplantation when applied topically on a transplanted cornea. We found that topical administration of ripasudil mediates immunological and wound healing responses owing to its ability to be rapidly deactivated downstream of neovascularization and inflammation. These findings suggest that topical administration of ripasudil might be an effective treatment alternative for the management of corneal transplantation. After corneal transplantation, upregulation of proangiogenic and proinflammatory factors, along with epithelial and stromal damage at the local graft site, cause corneal infiltration of immune cells and induction of neovascularization and lymphangiogenesis 25,26 . The circulating immune responses of afferent (lymphatic) and efferent markers in the 0.04%, 0.4%, and 2.0% ripasudil groups compared to those in the control (n = 3, one-way ANOVA; **p < 0.01, ***p < 0.001). (B) mRNA levels of Vegfr1, 2, and 3 in the 0.04%, 0.4%, and 2.0% ripasudil groups compared to those in the control (n = 3, one-way ANOVA; **p < 0.01, ***p < 0.001). Vegf vascular endothelial growth factor, Lyve1 lymphatic vessel endothelial hyaluronan receptor-1, ANOVA analysis of variance, Vegfr vascular endothelial growth factor receptor. www.nature.com/scientificreports/ (vascular) arms contribute to the loss of immune privilege and subsequent rejection 27 . These results suggest that topical administration of ripasudil locally suppressed the upregulation of proangiogenic and proinflammatory factors and promoted graft re-epithelization, resulting in the reduction of circulating immune responses (Fig. 7). In corneal transplantation, induced local inflammation and epithelial and stromal damage lead to infiltration of inflammatory cells, primarily neutrophils and macrophages 28 . The infiltrating neutrophils and macrophages secrete VEGF. VEGFR-2 along with its ligands, VEGF-A and VEGF-C, is the principal signaling receptor for vascular endothelial cells. As ripasudil selectively inhibits the ROCK pathway and VEGF secretion in these cells, VEGF-A and VEGF-C secretion may have been reduced. We found that topical administration of ripasudil downregulated VEGFR-2 because the vascular endothelial cells themselves were reduced in the grafted cornea by the immunosuppressive function of ripasudil. Previous studies have reported that ROCK inhibitors regulate the proliferation of vascular endothelial cells by reducing VEGF-induced ROCK activation 20,29 . To determine the direct effects of ripasudil on vascular endothelial cells, further in vitro experiments are needed.
Angiogenesis is accompanied through significant inflammation 30 . After corneal transplantation, inflammatory cytokines are released, and inflammatory cells are recruited to the grafted cornea 25,31 . Thereafter, CNV is induced in the grafted cornea, delaying the epithelialization of corneal grafts, and triggering corneal rejection 32 . Topical administration of ripasudil reduced the number of CD45 + infiltrating cells and inflammatory cytokine expression in the grafted cornea. TNF-α and IL-1β were significantly downregulated in the 0.4% and 2.0% ripasudil groups compared to the 0.04% ripasudil group, indicating that their downregulation contributes to corneal graft survival. In particular, of the cytokines examined in this study, only TNF-α was not suppressed upon 0.04% ripasudil administration in corneal grafts, suggesting that TNF-α is a potential key regulator of suppressing inflammation, angiogenesis, and lymphangiogenesis upon corneal transplantation 33,34 . A previous study reported that ROCK inhibition protected the vascular endothelium by inhibiting neutrophil adhesion 35 and downregulating inflammatory cytokines including VEGF, TNF-α, matrix metalloproteinase (MMP)-2, and MMP-9 [36][37][38] . Furthermore, this study elucidates the efficacy of ripasudil in reducing the infiltration of inflammatory cells to sites in the grafted cornea after reducing the levels of the associated inflammatory cytokines at the site of injury.
Delayed corneal epithelial wound healing causes corneal infiltration of inflammatory cells and subsequent VEGF secretion 39,40 . Therefore, epithelial regeneration after corneal transplantation is important for graft survival. ROCK contributes to cell proliferation and migration 14 . The migration of recipient corneal epithelial cells  41 . Administration of 0.4% and 2.0% ripasudil promoted re-epithelization of the grafted cornea, concurrent with the a previous report on would healing 42 . As the epithelium serves as a significant barrier to graft acceptance 41 , rapid graft re-epithelization is critical for visual acuity, graft transparency, and protection of the stroma against infection and melting 43 . In particular, promotion of re-epithelization by the ROCK inhibitor may suppress the induction of inflammation, CNV, infection, and even graft rejection.
Owing to the reduction in local angiogenic factor levels and inflammation and promotion of epithelial regeneration by ripasudil administration, angiogenesis and lymphangiogenesis were suppressed, and graft survival was   8,[44][45][46] . We found that the numbers of mature DCs and IFNγ + Th1 and IL-17 + Th17 cells decreased in the dLNs, indicating that ripasudil administration inhibited angiogenesis and lymphangiogenesis and subsequently suppressed the reduction in antigen presentation in the dLNs and migration of the Th1 and Th17 cells to the grafted cornea. Furthermore, fasudil, a ROCK inhibitor, regulates the proportions of IFNγ + Th1 and IL-17 + Th17 cells, indicating that the ROCK inhibitor itself may have a local immunosuppressive effect 47 . In the efferent arm, anti-neovascularization treatments are especially important for blocking the direct and indirect migration of donor antigen-presenting cells 48 . The afferent arm is characterized by migration of the allogeneic antigens via the lymphatic tissues. This study shows that topical administration of ripasudil suppressed lymphangiogenesis by downregulating VEGFR-3 along with its ligands, VEGF-C and VEGF-D. A previous study reported the interaction between angiogenesis and lymphangiogenesis 49 , suggesting that the anti-angiogenic property of ripasudil was responsible for suppressing angiogenesis and lymphangiogenesis in the grafted cornea. There are several limitations to this study. This study identified the inhibitory effects of ripasudil for CNV using a corneal transplantation model. However, since CNV occurs in various diseases, further studies using different CNV models are necessary. Furthermore, this study did not investigate the potential immunosuppressive effect of topical administration of ripasudil with steroids. Steroids are frequently topically administered after corneal transplantation 50 . Therefore, it is necessary to investigate whether adding ripasudil to the steroid after corneal transplantation will further suppress the rejection. Recent studies have reported a protective effect of ROCK inhibitors on corneal endothelial cells 51,52 . Although the corneal endothelial cell density in corneal transplantation was not investigated in this study, we speculate that ripasudil may be effective for the long-term survival of corneal grafts because of the protective effect of ROCK inhibitors on corneal endothelial cells.
In summary, the ROCK inhibitor ripasudil inhibited CNV and inflammation and subsequently promoted graft survival. Ripasudil administration might satisfy unmet medical needs in the treatment of corneal transplantation, which require suppression of corneal neovascularization and inflammation. Allogeneic corneal transplantation. For allogeneic corneal transplantation, the corneas of C57BL/6 mice were grafted onto BALB/c host beds as described previously 53 . In brief, the central cornea (2 mm in diameter) was excised from a donor C57BL/6 mouse using scissors (Vannas-Storz Instruments, San Dimas, CA, USA). The graft bed was prepared by excising a 1.5-mm site in the central cornea of a BALB/c mouse. The donor button was then placed onto the recipient bed and secured with eight interrupted 11-0 nylon sutures. After surgery, the host eyelids were closed for 3 days, and the interrupted corneal sutures were removed 7 days after surgery.
Grafted cornea assessment. The graft's neovascularization score, opacity score, and survival rate were evaluated for 8 weeks using a slit-lamp biomicroscope. We used a standardized scoring system to assess the neovascularization score (range, 0-8) and opacity score (range, 0-5+) 53 . Corneas with an opacity score of 2+ for two consecutive examinations were rejected. Re-epithelization of the grafted cornea was assessed using 0.5% fluorescein staining under a slit-lamp biomicroscope with cobalt blue light.

Eye drop treatment. Ripasudil (K-115), a novel ROCK inhibitor, was obtained from Kowa Company
Ltd. (Nagoya, Japan) 18 . To assess the pharmacological efficacy of ripasudil in the murine cornea transplantation model, 0.04%, 0.4%, and 2.0% ripasudil (n = 5-7 mice/group) was administered thrice daily in accordance with a previous study 20,54,55 . The vehicle of ripasudil was used as the control 56 , which contained sodium dihydrogen phosphate as a buffering agent, glycerin as an isotonic agent, sodium hydroxide as a pH-adjusting agent (pH range 5-7), and benzalkonium chloride as a preservative.
Corneal whole mount and immunofluorescence staining. RNA isolation and reverse transcription-quantitative PCR (RT-qPCR). The excised corneas were immediately submerged in RNAlater solution (Ambion, Austin, TX, USA). Total RNA was isolated from five corneas per group using a NucleoSpin RNA isolation kit (Macherey-Nagal GmbH, Duren, Germany) in accordance with the manufacturer's instructions. The cDNA was reverse-transcribed from total RNA using random primers and the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan) in accordance with the manufacturer's guidelines. The qPCR primers specific for mouse mRNA are enlisted in Table S1. qPCR was performed with the ABI PRISM 7300 HT sequence detection system using the FAST-SYBR Green master mix (Life Technology Japan, Tokyo, Japan). Results were analyzed using the comparative cycle threshold method, and Gapdh mRNA expression in the same cDNA was used as the internal control.
Flow cytometry analysis. Corneas and ipsilateral dLNs were harvested, and single-cell suspensions were prepared as described previously 53,59 . To avoid non-specific staining, cells were blocked with an anti-FcR blocking antibody (eBioscience, San Diego, CA, USA). The isolated cells were stained with the respective antibodies. www.nature.com/scientificreports/ Statistical analysis. Experiments with more than two groups were analyzed using one-way or two-way analysis of variance (ANOVA) with Bonferroni's multiple comparison post-hoc test. The Mann-Whitney test was performed to compare medians between the groups. Kaplan-Meier analysis was performed to construct corneal graft re-epithelization curves and to evaluate graft survival post-corneal transplantation, and the logrank test was performed to compare corneal re-epithelization of the grafted corneas and graft survival postcorneal transplantation. Data are presented as mean ± standard error of mean values and were considered statistically significant at p < 0.05. All statistical analyses were performed using Prism version 8.0 software (GraphPad, La Jolla, CA, USA).

Data availability
All data generated or analyzed during this study are included in this published article (and its Supplementary  Information files).