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Purpose: The intraepithelial corneal nerves (ICNs) innervating the cornea are essential to corneal epithelial cell homeostasis. Rho-associated kinase (ROCK) inhibitors (RIs) have been reported to play roles in neuron survival after injury and in mitochondrial transfer between corneal epithelial cells. In this study, the mechanisms human corneal limbal epithelial (HCLE) cells use to control intercellular mitochondrial transfer are assessed., Methods: Mitotracker and AAV1 mitotag eGFPmCherry were used to allow us to study mitochondrial transfer between HCLE cells and neurons in co-cultures and in HCLE cultures. A mitochondrial transfer assay was developed using HCLE cells to quantify the impact of cell stress and inhibition of phagocytosis, gap junctions, and ROCK on mitochondrial transfer, cell adhesion, migration, matrix deposition, and mitochondrial content., Results: Bidirectional mitochondrial transfer occurs between HCLE cells and neurons. Mitochondrial transfer among HCLE cells is inhibited when gap junction function is reduced and enhanced by acid stress and by inhibition of either phagocytosis or ROCK. Media conditioned by RI-treated cells stimulates cell adhesion and mitochondrial transfer., Conclusions: Maximal mitochondrial transfer takes place when gap junctions are functional, when ROCK and phagocytosis are inhibited, and when cells are stressed by low pH media. Treatments that reduce mitochondrial content increase HCLE cell mitochondrial transfer. ROCK inhibition in co-cultures causes the release and adhesion of mitochondria to substrates where they can be engulfed by migrating HCLE cells and growing axons and their growth cones.

Background: Fasudil is a small molecule inhibitor of Rho-associated kinase (ROCK) and is approved for the treatment of subarachnoid haemorrhage. In preclinical studies, fasudil has been shown to attenuate neurodegeneration, modulate neuroinflammation, and foster axonal regeneration. We aimed to investigate the safety, tolerability, and efficacy of fasudil in patients with amyotrophic lateral sclerosis., Methods: ROCK-ALS was a phase 2, randomised, double-blind, placebo-controlled trial conducted at 19 amyotrophic lateral sclerosis centres in Germany, France, and Switzerland. Individuals (aged 18-80 years) with at least probable amyotrophic lateral sclerosis (as per the revised El Escorial criteria), a disease duration of 6-24 months, and a slow vital capacity greater than 65% of predicted normal were eligible for inclusion. Patients were randomly assigned (1:1:1) to receive 30 mg (15 mg twice daily) or 60 mg (30 mg twice daily) fasudil or matched placebo intravenously for 20 days over a 4-week period. Follow-up assessments were performed at 45, 90, and 180 days after treatment initiation. The co-primary endpoints were safety until day 180 (defined as the proportion without drug-related serious adverse events) and tolerability during the treatment period (defined as the proportion who did not discontinue treatment due to suspected drug-related adverse events). The primary analyses were carried out in the intention-to-treat population, which included all participants who entered the treatment phase. This trial is registered at ClinicalTrials.gov (NCT03792490) and Eudra-CT (2017-003676-31) and is now completed., Findings: Between Feb 20, 2019, and April 20, 2022, 120 participants were enrolled and randomised; two individuals assigned fasudil 30 mg withdrew consent before the baseline visit. Thus, the intention-to-treat population comprised 35 in the fasudil 30 mg group, 39 in the fasudil 60 mg group, and 44 in the placebo group. The estimated proportion without a drug-related serious adverse event was 1·00 (95% CI 0·91 to 1·00) with placebo, 1·00 (0·89 to 1·00) with fasudil 30 mg, and 1·00 (0·90 to 1·00) with fasudil 60 mg; the difference in proportions was 0·00 (95% CI -0·11 to 0·10; p>0·99) for fasudil 30 mg versus placebo and 0·00 (-0·10 to 0·10; p>0·99) for fasudil 60 mg versus placebo. Treatment tolerability (the estimated proportion who did not discontinue) was 0·93 (95% CI 0·81 to 0·99) with placebo, 1·00 (0·90 to 1·00) with fasudil 30 mg, and 0·90 (0·76 to 0·97) with fasudil 60 mg; the difference in proportions was 0·07 (95% CI -0·05 to 0·20; p=0·25) for fasudil 30 mg versus placebo, and -0·03 (-0·18 to 0·10; p=0·70) for fasudil 60 mg versus placebo. Eight deaths occurred: two in the placebo group, four in the fasudil 30 mg group, and two in the fasudil 60 mg group. The most common serious adverse events were respiratory failure (seven events), gastrostomy (five events), pneumonia (four events), and dysphagia (four events). No serious adverse events or deaths were attributed to study treatment. Adverse events, which were mainly related to disease progression, occurred in 139 participants in the placebo group, 108 in the fasudil 30 mg group, and 105 in the fasudil 60 mg group., Interpretation: Fasudil was well tolerated and safe in people with amyotrophic lateral sclerosis. The effect of fasudil on efficacy outcomes should be explored in larger clinical trials with a longer treatment duration, oral administration, and potentially higher dose of the trial drug., Funding: Framework of the E-Rare Joint Transnational Call 2016 "Clinical research for new therapeutic uses of already existing molecules (repurposing) in rare diseases"., Competing Interests: Declaration of interests JCK reports a grant from the Deutsche Gesellschaft für Muskelkranke and consulting fees from AbbVie, Biogen, Ipsen, Roche, and Zambon. RG reports grants from the Deutsche Gesellschaft für Muskelkranke, Initiative SMA, and Bundesministerium für Bildung und Forschung, as well as consulting fees from Biogen, Roche, ITF Pharma, and Zambon. DZ has received consulting fees from Novartis and Angelini Pharma and has served on an advisory board for Biogen. NB has received compensations from Mitsubishi Tanabe Pharma. PC serves on the editorial advisory boards of ALS and The Revue Neurologique; reports consultancy work or participation on advisory boards for Amylyx, Biogen, Cytokinetics, Ferrer, Mitsubishi Tanabe Pharma, VectorY, and Zambon; serves on the drug safety monitoring board for Quralis, and has received a research grant from Biogen. EDLC has received travel grants from Biogen and EFFIK. PW reports grants from the Boris Canessa Foundation and the Bundesministerium für Bildung and Forschung; and has received consulting fees from ITF Pharma, Zambon, Novartis, Biogen, and Roche. TM reports institutional grants from Cytokinetics, Ferrer, AL-S Pharma, Sanofi, Amylyx, Mitsubishi Tanabe, and Apellis Pharmaceuticals, as well as personal fees from Biogen, Amylyx, and ITF Pharma; and is co-founder and shareholder of the Ambulanzpartner Soziotechnologie APST. JG has received personal fees from UCB, Alexion, Amylyx, Roche, and Zambon; and is a member of advisory boards of the European Network to Cure ALS, Neuroimaging Society in amyotrophic Lateral Sclerosis, EU ALS coalition, and the World Federation of Neurology Motoneuron Disease group. M-HS received compensations for consulting from Amylyx, Zambon, EFFIK-Italfarmaco, and SOS Oxygene. M-LR reports grants from FightMND, MND Research Australia and the US National Institutes of Health. CN has received fees for non-related services for Biogen, Mitsubishi Tanabe, Roche, and Argenx. JW reports grants from the US National Institutes of Health. JS has received payments for participation on advisory boards, talks, travel, and research projects from Abcuro, Alnylam, Argenx, Biotest, CSL Behring, Grifols, Johnson & Johnson, Kezar, LFB, Lupin, Momenta, Novartis, Octapharma, and UCB, all unrelated to the present study. TF has received personal fees from Actimed, Bayer, Bristol Myers Squibb, Cardior, CSLBehring, Daiichi Sankyo, Galapagos, Immunic, KyowaKirin, LivaNova, Minoryx, Novartis, RECARDIO, Relaxera, Roche, Servier, Viatris, Vifor, Fresenius Kabi, PINK gegen Brustkrebs, Aslan, BionsenseWebster, Enanta, VICO Therapeutics, Pharmaceutical Product Development, and IQVIA, as well as institutional grants from Deutsche Forschungsgemeinschaft, Gemeinsamer Bundesausschuss, and the European Commission. MB reports grants from the US National Institutes of Health, the Muscular Dystrophy Association, and the ALS Association; as well as consulting fees for Alector, Alexion, Annexon, Arrowhead, Biogen, Cartesian, Denali, Eli Lilly, Horizon, Immunovant, Novartis, Roche, Sanofi, Takeda, UCB, and uniQure; the University of Miami has licensed intellectual property to Biogen to support the design of the ATLAS trial (NCT04856982), for which MB is academic lead. PL reports grants from the Bundesministerium für Bildung und Forschung and the Deutsche Forschungsgemeinschaft; consulting fees from AbbVie, Amylyx, Bial, Desitin, ITF Pharma, Novartis, Stadapharm, Raya Therapeutic, Woolsey Pharmaceuticals, and Zambon; and is co-inventor on a patent for the use of fasudil in amyotrophic lateral sclerosis (EP 2825175 B1, US 9.980,972 B2). All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Morphogenesis requires embryonic cells to generate forces and perform mechanical work to shape their tissues. Incorrect functioning of these force fields can lead to congenital malformations. Understanding these dynamic processes requires the quantification and profiling of three-dimensional mechanics during evolving vertebrate morphogenesis. Here we describe elastic spring-like force sensors with micrometre-level resolution, fabricated by intravital three-dimensional bioprinting directly in the closing neural tubes of growing chicken embryos. Integration of calibrated sensor read-outs with computational mechanical modelling allows direct quantification of the forces and work performed by the embryonic tissues. As they displace towards the embryonic midline, the two halves of the closing neural tube reach a compression of over a hundred nano-newtons during neural fold apposition. Pharmacological inhibition of Rho-associated kinase to decrease the pro-closure force shows the existence of active anti-closure forces, which progressively widen the neural tube and must be overcome to achieve neural tube closure. Overall, our approach and findings highlight the intricate interplay between mechanical forces and tissue morphogenesis., (© 2024. The Author(s).)

Chronic non-healing wounds negatively impact quality of life and are a significant financial drain on health systems. The risk of infection that exacerbates comorbidities in patients necessitates regular application of wound care. Understanding the mechanisms underlying impaired wound healing are therefore a key priority to inform effective new-generation treatments. In this study, we demonstrate that 14-3-3-mediated suppression of signaling through ROCK is a critical mechanism that inhibits the healing of diabetic wounds. Accordingly, pharmacological inhibition of 14-3-3 by topical application of the sphingo-mimetic drug RB-11 to diabetic wounds on a mouse model of type II diabetes accelerated wound closure more than 2-fold than vehicle control, phenocopying our previous observations in 14-3-3ζ-knockout mice. We also demonstrate that accelerated closure of the wounded epidermis by 14-3-3 inhibition causes enhanced signaling through the Rho-ROCK pathway and that the underlying cellular mechanism involves the efficient recruitment of dermal fibroblasts into the wound and the rapid production of extracellular matrix proteins to re-establish the injured dermis. Our observations that the 14-3-3/ROCK inhibitory axis characterizes impaired wound healing and that its suppression facilitates fibroblast recruitment and accelerated re-epithelialization suggest new possibilities for treating diabetic wounds by pharmacologically targeting this axis., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

To form blood vessels, endothelial cells rearrange their cytoskeleton, generate traction stresses, migrate, and proliferate, all of which require energy. Despite these energetic costs, stiffening of the extracellular matrix promotes tumor angiogenesis and increases cell contractility. However, the interplay between extracellular matrix, cell contractility, and cellular energetics remains mechanistically unclear. Here, we utilized polyacrylamide substrates with various stiffnesses, a real-time biosensor of ATP, and traction force microscopy to show that endothelial cells exhibit increasing traction forces and energy usage trend as substrate stiffness increases. Inhibition of cytoskeleton reorganization via ROCK inhibition resulted in decreased cellular energy efficiency, and an opposite trend was found when cells were treated with manganese to promote integrin affinity. Altogether, our data reveal a link between matrix stiffness, cell contractility, and cell energetics, suggesting that endothelial cells on stiffer substrates can better convert intracellular energy into cellular traction forces. Given the critical role of cellular metabolism in cell function, our study also suggests that not only energy production but also the efficiency of its use plays a vital role in regulating cell behaviors and may help explain how increased matrix stiffness promotes angiogenesis., Competing Interests: Conflict of interest The authors have no conflicts to disclose., (Copyright © 2024. Published by Elsevier B.V.)

Endothelial blood-brain barrier (BBB) dysfunction is critical in the pathophysiology of brain injury. Rho-associated protein kinase (ROCK) activation disrupts BBB integrity in the injured brain. We aimed to test the efficacy of a novel ROCK2 inhibitor in preserving the BBB after acute brain injury. We characterized the molecular structure and pharmacodynamic and pharmacokinetic properties of a novel selective ROCK2 inhibitor, NRL-1049, and its first metabolite, 1-hydroxy-NRL-1049 (referred to as NRL-2017 hereon) and tested the efficacy of NRL-1049 on the BBB integrity in rodent models of acute brain injury. Our data show that NRL-1049 and NRL-2017 both inhibit ROCK activity and are 44-fold and 17-fold more selective towards ROCK2 than ROCK1, respectively. When tested in a mouse model of cortical cryoinjury, NRL-1049 significantly attenuated the increase in water content. Interestingly, 60% of the mice in the vehicle arm developed seizures within 2 hours after cryoinjury versus none in the NRL-1049 arm. In spontaneously hypertensive rats, NRL-1049 attenuated the dramatic surge in Evans Blue extravasation compared with the vehicle arm after transient middle cerebral artery occlusion. Hemorrhagic transformation was also reduced. We show that NRL-1049, a selective ROCK2 inhibitor, is a promising drug candidate to preserve the BBB after brain injury., Competing Interests: Declaration of conflicting interestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Dr. McKerracher is CEO of BioAxone BioSciences, Inc. and hold an ownership interest in the company and has a significant competing interest. The data for this article were collected while Drs. Abbinanti, Woller, Ruschel and Rosen were employed at BioAxone BioSciences, and they have a modest conflict of interest. The opinions expressed in this article are the author’s own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. Neurelis, Inc. acquired the rights to BA-1049 (now referred to as NRL-1049) from BioAxone in June 2021. Ayata is on the scientific advisory board of Neurelis, Inc. and declares sponsored research agreements with Praxis and Takeda Pharmaceutical Company. Mulder declares a past sponsored research agreement with BioAxone BioSciences, Inc. (concerning work not included in this manuscript). The other authors declare no conflict of interest. No writing assistance was utilized in the production of this manuscript.

Benign prostatic hyperplasia (BPH) is a prevalent condition affecting the male urinary system, with its molecular mechanisms of pathogenesis remaining unclear. Y-27632, a non-isoform-selective Rho kinase inhibitor, has shown therapeutic potential in various diseases but its effects on static factors and fibrosis in BPH remain unexplored. This study investigated human prostate tissues, human prostate cell lines, and BPH rat model using immunofluorescence, flow cytometry, quantitative reverse transcription polymerase chain reaction, western blotting, and cell counting kit-8. ROCK1 and ROCK2 were significantly up-regulated in BPH tissues, correlating with clinical parameters. Y-27632 targeted the inhibition of ROCK1 & ROCK2 expression and inhibited cell proliferation, fibrosis, epithelial-mesenchymal transition (EMT), while induced cell apoptosis in a dose-dependent manner. Moreover, knockdown of either ROCK isoform inhibited fibrosis and EMT, induced apoptosis, while ROCK overexpression had the opposite effects. ROCK downregulation inhibited the β-catenin signaling pathway (such as C-MYC, Snail and Survivin) and decreased β-catenin protein stability, while inhibiting TGF-β/Smad 2/3 signaling. At the in vivo level, Y-27632 reversed prostatic hyperplasia and fibrosis in BPH model rats to some extent. Our study sheds light on the therapeutic potential of Y-27632 in regulating prostate cell growth, fibrosis and EMT, and demonstrates for the first time the regulatory effect of ROCK isoforms on prostate cells, providing the basis for future research of ROCK isoform-selective inhibitors., (© 2024. The Author(s).)

Purpose: The intraepithelial corneal nerves are essential to corneal health. Rho kinase or ROCK inhibitors (RIs) have been reported to play a role in neuron survival after injury. Here we assess integrin and extracellular matrix expression in primary mouse neurons and determine whether treating cells with RI impacts neurite outgrowth in vitro and reinnervation after trephine and debridement injury in mice in vivo., Methods: Cocultures of human corneal limbal epithelial cells and E11.5 mouse trigeminal neurons and neurons alone were grown on glass coverslips. High-resolution imaging was performed to localize integrins and laminin on neurons and to determine whether RI impacts neurite outgrowth in vitro and in vivo after both 1.5-mm trephine and 1.5-mm debridement injuries., Results: Several integrin α (α3, α6, αv) chains as well as β4 integrin are expressed on neuron axons and growth cones in cocultures. RI treatment of isolated neurons, cocultures, and in conditioned media increases neurite outgrowth. In vivo, RI positively impacts sensory nerve reinnervation after trephine and debridement injury., Conclusions: These studies are the first to demonstrate expression of β4 integrin on trigeminal sensory neurons and preferential adhesion of neurons to the laminin-enriched matrices found in footprints deposited by human corneal limbal epithelial cells. In addition, we also document for the first time the positive impact of RI on neurite outgrowth in vitro and reinnervation in vivo.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that leads to respiratory decline caused by scarring and thickening of lung tissues. Multiple pathways contribute to the fibrotic process in this disease, such as inflammation, epithelial-to-mesenchymal transition, and oxidative stress. The Rho-associated coiled-coil forming protein kinase (ROCK) signaling pathway is a key regulator of profibrotic signaling, as it affects the organization of actin-myosin and the remodeling of the extracellular matrix. ROCK1/2, a downstream effector of RhoA, is overexpressed in patients with IPF and is a promising target for IPF therapy. However, because of the hypotensive side effects of ROCK1/2 inhibitors, selective ROCK2 compounds are being explored. In this study, we report the discovery of GNS-3595, a potent and selective ROCK2 inhibitor that has ∼80-fold selectivity over ROCK1 at physiological concentrations of ATP. GNS-3595 effectively inhibited ROCK2-mediated phosphorylation of myosin light chain and reduced the expression of fibrosis-related proteins (e.g., collagen, fibronectin, and α-smooth muscle actin) in various in vitro cellular models. GNS-3595 also prevented transforming growth factor β-induced fibroblast-to-myofibroblast transition. In addition, in a bleomycin-induced mouse model of pulmonary fibrosis, therapeutic exposure to GNS-3595, suppressed lung fibrosis, stabilized body weight loss, and prevented fibrosis-induced lung weight gain. Transcriptome and protein expression analysis from lung tissues showed that GNS-3595 can revert the fibrosis-related gene expression induced by bleomycin. These results indicate that GNS-3595 is a highly potent, selective, and orally active ROCK2 inhibitor with promising therapeutic efficacy against pulmonary fibrosis.

Belumosudil mesylate is a selective Rho-associated coiled-coil kinase 2 inhibitor with immunomodulatory and antifibrosis effects. This multicenter, open-label, single-arm study evaluated belumosudil 200 mg once daily as second or subsequent line of therapy (LOT) in 21 Japanese patients ≥12 years of age with steroid-dependent/steroid-resistant chronic graft-versus-host disease (cGVHD). The primary endpoint of best overall response rate (ORR) at 24 weeks after enrollment of the last patient was 85.7% (95% confidence interval [CI]: 63.7-97.0), and the lower limit of the 95% CI exceeded the pre-defined threshold of 25%. The Kaplan-Meier estimate of duration of response rate at 24 weeks was 75% (95% CI: 46-90); 13/18 responders (72.2%) had a sustained response for ≥20 weeks. The median time to response was 4.1 weeks (range 3.90-8.10); ORR was 47.6% at 4 weeks and 75.0% at 24 weeks; best ORR was 80% for joints/fascia, 66.7% for the mouth, and 54.5% for skin. Overall, 57.1% of patients had clinically meaningful symptom improvement at least once; the median duration of symptom improvement was 22.2 weeks (range 4.0-51.3). Corticosteroid dose reductions were recorded for 57.1% of patients. Median failure-free and overall survival were not reached. Treatment-emergent adverse events occurred in 85.7% of patients (most commonly diarrhea, 19.0%), of which 38.1% were drug-related. There were no drug-related discontinuations or deaths. In summary, belumosudil 200 mg once daily as second or subsequent LOT in Japanese patients with steroid-dependent/steroid-resistant cGVHD was effective, with no new safety concerns., (© 2024 The Author(s). American Journal of Hematology published by Wiley Periodicals LLC.)

Rho-associated coiled-coil kinase (ROCK) is involved in multiple cellular activities regulating the actin cytoskeleton, such as cell morphology, adhesion, and migration. The inhibition of ROCK is a feasible strategy to suppress breast cancer metastasis. Herein, based on Belumosudil, a series of pyrazolo[1,5-a]pyrimidine derivatives as selective ROCK2 inhibitors were designed and synthesized. Through systematic investigation of SARs, the piperazine analog 7u was identified with optimum ROCK2 inhibitory activity (IC 50  = 36.8 nM) and excellent selectivity over the isoform protein ROCK1 (>250-fold). Intriguingly, upon treatment with 7u, the arrangement of the MDA-MB-231 cytoskeleton was affected accompanied by the alteration of morphology. Furthermore, cell scratch and transwell assays indicated that 7u inhibited MDA-MB-231 cell migration and invasion in a dose-dependent manner. Ultimately, the binding model of 7u with ROCK2 well accounted for the superior activities of 7u as a promising ROCK2 inhibitor with the potential application in breast cancer metastasis treatment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xin Zhai reports financial support was provided by Liaoning Revitalization Talents Program., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Based on the synergistic therapeutic effect of nitric oxide (NO) and Rho-associated protein kinase (ROCK) inhibitors on glaucoma, a series of NO-donating Netarsudil derivatives were designed, synthesized, and their activities in vitro and in vivo were evaluated. Among them, ( S )- 10e released an appropriate amount of NO in aqueous humor in vitro and displayed potent ROCK inhibition. Topical administration of ( S )- 10e significantly lowered intraocular pressure in an acute ocular hypertension rabbit model and protected retinal ganglion cells in a magnetic microbead occlusion mouse model. A metabolism investigation revealed that ( S )- 10e released 7a , a metabolite after NO releasing, and 13 , an active metabolite of ( S )-Netarsudil, in rabbit eyes. Notably, introducing an NO donor moiety attenuated ROCK inhibition-induced ocular irritation in an sGC-independent manner, suggesting that the attenuated conjunctival hyperemia effect of ( S )- 10e is related to the NO-induced protein S-nitrosation of phosphodiesterase 3A (PDE3A). Overall, ( S )- 10e is a promising candidate for glaucoma treatment.

Background: Glaucoma is a leading cause of blindness, affecting retinal ganglion cells (RGCs) and their axons. By 2040, it is likely to affect 110 million people. Neuroinflammation, specifically through the release of proinflammatory cytokines by M1 microglial cells, plays a crucial role in glaucoma progression. Indeed, in post-mortem human studies, pre-clinical models, and ex-vivo models, RGC degeneration has been consistently shown to be linked to inflammation in response to cell death and tissue damage. Recently, Rho kinase inhibitors (ROCKis) have emerged as potential therapies for neuroinflammatory and neurodegenerative diseases. This study aimed to investigate the potential effects of three ROCKis (Y-27632, Y-33075, and H-1152) on retinal ganglion cell (RGC) loss and retinal neuroinflammation using an ex-vivo retinal explant model., Methods: Rat retinal explants underwent optic nerve axotomy and were treated with Y-27632, Y-33075, or H-1152. The neuroprotective effects on RGCs were evaluated using immunofluorescence and Brn3a-specific markers. Reactive glia and microglial activation were studied by GFAP, CD68, and Iba1 staining. Flow cytometry was used to quantify day ex-vivo 4 (DEV 4) microglial proliferation and M1 activation by measuring the number of CD11b + , CD68 + , and CD11b + /CD68 + cells after treatment with control solvent or Y-33075. The modulation of gene expression was measured by RNA-seq analysis on control and Y-33075-treated explants and glial and pro-inflammatory cytokine gene expression was validated by RT-qPCR., Results: Y-27632 and H-1152 did not significantly protect RGCs. By contrast, at DEV 4, 50 µM Y-33075 significantly increased RGC survival. Immunohistology showed a reduced number of Iba1 + /CD68 + cells and limited astrogliosis with Y-33075 treatment. Flow cytometry confirmed lower CD11b + , CD68 + , and CD11b + /CD68 + cell numbers in the Y-33075 group. RNA-seq showed Y-33075 inhibited the expression of M1 microglial markers (Tnfα, Il-1β, Nos2) and glial markers (Gfap, Itgam, Cd68) and to reduce apoptosis, ferroptosis, inflammasome formation, complement activation, TLR pathway activation, and P2rx7 and Gpr84 gene expression. Conversely, Y-33075 upregulated RGC-specific markers, neurofilament formation, and neurotransmitter regulator expression, consistent with its neuroprotective effects., Conclusion: Y-33075 demonstrates marked neuroprotective and anti-inflammatory effects, surpassing the other tested ROCKis (Y-27632 and H-1152) in preventing RGC death and reducing microglial inflammatory responses. These findings highlight its potential as a therapeutic option for glaucoma., (© 2024. The Author(s).)

Rationale: Tunnel nanotube (TNT)-mediated mitochondrial transport is crucial for the development and maintenance of multicellular organisms. Despite numerous studies highlighting the significance of this process in both physiological and pathological contexts, knowledge of the underlying mechanisms is still limited. This research focused on the role of the ROCK inhibitor Y-27632 in modulating TNT formation and mitochondrial transport in retinal pigment epithelial (RPE) cells. Methods: Two types of ARPE19 cells (a retinal pigment epithelial cell line) with distinct mitochondrial fluorescently labeled, were co-cultured and treated with ROCK inhibitor Y-27632. The formation of nanotubes and transport of mitochondria were assessed through cytoskeletal staining and live cell imaging. Mitochondrial dysfunction was induced by light damage to establish a model, while mitochondrial function was evaluated through measurement of oxygen consumption rate. The effects of Y-27632 on cytoskeletal and mitochondrial dynamics were further elucidated through detailed analysis. Results: Y-27632 treatment led to an increase in nanotube formation and enhanced mitochondrial transfer among ARPE19 cells, even following exposure to light-induced damage. Our analysis of cytoskeletal and mitochondrial distribution changes suggests that Y-27632 promotes nanotube-mediated mitochondrial transport by influencing cytoskeletal remodeling and mitochondrial movement. Conclusions : These results suggest that Y-27632 has the ability to enhance mitochondrial transfer via tunneling nanotubes in retinal pigment epithelium, and similarly predict that ROCK inhibitor can fulfill its therapeutic potential through promoting mitochondrial transport in the retinal pigment epithelium in the future., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

As a pivotal node in modulating various cell behaviors, Rho-associated kinase-1 (ROCK1) has attracted significant attention as a promising therapeutic target in a variety of diseases. Benzamide has been widely reported as a ROCK1 inhibitors in recent years. To better understand its pharmacological properties and to explore its potential inhibitors, a series of ROCK1 inhibitors derived from N-methyl-4-(4-pyrazolidinyl) benzamides (MPBs) were investigated by using three-dimensional quantitative structure-activity relationship (3D-QSAR) models, pharmacophore models, molecular docking, and molecular dynamics (MD) simulation. The comparative Molecular Field Analysis (CoMFA) model ( q 2 = 0.616, R 2 = 0.972, ONC = 4, and r 2 pred = 0.983) and the best Comparative Molecular Similarity Indices Analysis (CoMSIA) model ( q 2 = 0.740, R 2 = 0.982, ONC = 6, and r 2 pred = 0.824) exhibited reliable predictability with satisfactory validation parameters. In the subsequent virtual screening, VS03 and VS05 were identified to have superior predicted activities and higher docking scores, meanwhile they demonstrated to be reasonably stable in the binding pocket through MD simulations. These results provide a significant theoretical direction for the rational design and development of novel ROCK1 inhibitors.Communicated by Ramaswamy H. Sarma.

The discipline of structure-based drug design (SBDD) is several decades old and it is tempting to think that the proliferation of experimental structures for many drug targets might make computer-aided drug design (CADD) straightforward. However, this is far from true. In this review, we illustrate some of the challenges that CADD scientists face every day in their work, even now. We use Rho-associated protein kinase (ROCK), and public domain structures and data, as an example to illustrate some of the challenges we have experienced during our project targeting this protein. We hope that this will help to prevent unrealistic expectations of what CADD can accomplish and to educate non-CADD scientists regarding the challenges still facing their CADD colleagues., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hippocampal area CA2 has garnered attention in recent times owing to its significant involvement in social memory and distinctive plasticity characteristics. Research has revealed that the CA2 region demonstrates a remarkable resistance to plasticity, particularly in the Schaffer Collateral (SC)-CA2 pathway. In this study we investigated the role of Nogo-A, a well-known axon growth inhibitor and more recently discovered plasticity regulator, in modulating plasticity within the CA2 region. The findings demonstrate that blocking Nogo-A in male rat hippocampal slices facilitates the establishment of both short-term and long-term plasticity in the SC-CA2 pathway, while having no impact on the Entorhinal Cortical (EC)-CA2 pathway. Additionally, the study reveals that inhibiting Nogo-A enables association between the SC and EC pathways. Mechanistically, we confirm that Nogo-A operates through its well-known co-receptor, p75 neurotrophin receptor (p75 NTR ), and its downstream signaling factor such as Rho-associated protein kinase (ROCK), as their inhibition also allows plasticity induction in the SC-CA2 pathway. Additionally, the induction of long-term depression (LTD) in both the EC and SC-CA2 pathways led to persistent LTD, which was not affected by Nogo-A inhibition. Our study demonstrates the involvement of Nogo-A mediated signaling mechanisms in limiting synaptic plasticity within the CA2 region., (© 2024 The Author(s). Hippocampus published by Wiley Periodicals LLC.)

Rho family GTPases regulate cellular processes and promote tumour growth and metastasis; thus, RhoA is a potential target for tumour metastasis inhibition. However, limited progress has been made in the development of RhoA targeting anticancer drugs. Here, we synthesised benzo[ b ]thiophene-3-carboxylic acid 1,1-dioxide derivatives based on a covalent inhibitor of RhoA (DC-Rhoin), reported in our previous studies. The observed structure-activity relationship (contributed by carboxamide in C-3 and 1-methyl-1 H -pyrazol in C-5) enhanced the anti-proliferative activity of the derivatives. Compound b19 significantly inhibited the proliferation, migration, and invasion of MDA-MB-231 cells and promoted their apoptosis. The suppression of myosin light chain phosphorylation and the formation of stress fibres confirmed the inhibitory activity of b19 via the RhoA/ROCK pathway. b19 exhibited a different binding pattern from DC-Rhoin, as observed in molecular docking analysis. This study provides a reference for the development of anticancer agents targeting the RhoA/ROCK pathway.

Purpose: Descemet stripping only (DSO) is a promising surgical option for select patients with Fuchs endothelial dystrophy (FED). There is growing support for the use of topical Rho-associated protein kinase inhibitors (ROCKi) to optimize DSO outcomes. However, in many settings, ROCKi are either unavailable or not approved to treat corneal diseases. This study sought to characterize patient outcomes after DSO in the absence of ROCKi and potentially broaden the settings where DSO can be offered to patients., Methods: Single-center retrospective case series of 15 eyes/11 patients (66 years; 52-74) that underwent DSO, alone or combined with cataract surgery, by one surgeon between August 2020 and January 2023. Patients included in analyses had FED with central guttae, no clinical evidence of corneal edema, and a clinically healthy peripheral corneal endothelium., Results: Mean follow-up time was 14 months (2-34). Fourteen of 15 eyes achieved corneal clearance (93.3%). Mean time to clearance was 8.5 weeks (3-23). Eleven eyes (73%) achieved corrected distance visual acuity of ≤0.2 with a significant postoperative improvement at 4 to 8 months ( P < 0.05) and sustained improvements at >12 months. No significant astigmatism was introduced by the procedure. Two eyes developed cystoid macular edema postoperatively. A trend toward earlier clearance was observed in the <65 years old group., Conclusions: Despite a longer time to corneal clearance in this cohort compared with the few studies using ROCKi, the overall success rate and visual outcomes for the patients in our cohort supports the use of DSO in settings where ROCKi are not readily available., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Rho-kinase has been implicated in the development of hypertension in preclinical studies and may contribute to age-related blood pressure elevation. This study tested the hypothesis that Rho-kinase contributes to elevated systolic blood pressure (SBP) in healthy older adults. Young (18-30 years, 6F/6M) and older (60-80 years, 7F/6M) adults were enrolled in a double-blind, placebo-controlled crossover study using intravenous fasudil infusion to inhibit Rho-kinase. Fasudil lowered SBP in older adults compared to placebo (saline) (2-h post-infusion: 125 ± 4 vs. 133 ± 4 mmHg, P < 0.05), whereas fasudil had no impact on SBP in young adults. Immediately following fasudil infusion, there was a transient reduction in mean arterial pressure (MAP) in young adults that was no longer evident 1-h post-infusion. In older adults, MAP remained lower throughout the fasudil visit compared to placebo (2-h post-infusion: 93 ± 3 vs. 100 ± 3 mmHg, P < 0.05) such that age-related differences in SBP and MAP were abolished. Aortic stiffness (carotid-femoral pulse wave velocity) was not altered by fasudil when central MAP was included as a covariate in analyses. Fasudil reduced forearm vascular resistance in older (2-h post-infusion: 3.3 ± 0.4 vs. 4.8 ± 0.6 mmHg/ml/min, P < 0.05) but not young (4.0 ± 0.6 vs. 3.8 ± 0.5 mmHg/ml/min) adults, which was accompanied by an increase in brachial artery diameter only in older adults. Brachial artery flow-mediated dilation was not affected by fasudil in either group. These findings indicate that Rho-kinase inhibition reduces SBP in healthy older but not young adults, which is associated with a concomitant reduction in forearm vascular resistance., (© 2024. The Author(s), under exclusive licence to American Aging Association.)

Cisplatin is an effective chemotherapy agent against various solid malignancies; however, it is associated with irreversible bilateral sensorineural hearing loss, emphasizing the need for drug development to prevent this complication, with the current options being very limited. Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine protein kinase involved in various cellular processes, including apoptosis regulation. In this study, we used a transgenic zebrafish model (Brn3C: EGFP) in which hair cells within neuromasts are observed in green under fluorescent microscopy without the need for staining. Zebrafish larvae were exposed to cisplatin alone or in combination with various concentrations of Y-27632, a potent ROCK inhibitor. Hair cell counts, apoptosis assessments using the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay, FM1-43FX labeling assay and behavioral analyses (startle response and rheotaxis) were performed to evaluate the protective effects of Y-27632 against cisplatin-induced ototoxicity. Cisplatin treatment reduced the number of hair cells in neuromasts, induced apoptosis, and impaired zebrafish larval behaviors. Y-27632 demonstrated a dose-dependent protective effect against cisplatin-induced hair cell loss and apoptosis. These findings suggest that Y-27632, as a ROCK inhibitor, mitigates cisplatin-induced hair cell loss and associated ototoxicity in zebrafish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Osteoporosis is a devastating skeletal disease, the pathogenesis of which is related to abnormal bone metabolism, featured by the imbalance between osteoblastic bone formation and osteoclastic bone resorption. Stem cell-based therapies have been demonstrated to improve osteoporosis treatment. Previously, the linear furanocoumarin heraclenin was reported to enhance osteoblast differentiation and mineralization in mouse mesenchymal stem cells (MSCs), suggesting its potential for osteogenic differentiation and bone regeneration. Our study was designed to confirm the promotive role of heraclenin on osteogenic differentiation of human bone MSCs (BMSCs) and explore the underlying mechanisms., Methods: Human BMSCs were treated for 24, 48, and 72h with heraclenin (5, 10, 20, 40, and 80 μM), and cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. To further evaluate the cytotoxicity of heraclenin, cell suspension obtained from BMSCs treated with heraclenin (5, 10, and 20 μM) for 72h was subjected to a MUSE™ cell analyzer for cell viability and count assay. BMSCs were incubated in osteogenic induction medium for 7 days. Then, osteogenic differentiation and mineralization of BMSCs were assessed through alkaline phosphatase (ALP) and Alizarin Red S staining. The expression of osteogenesis markers including ALP, osteocalcin (OCN), osterix (OSX), and runt-related transcription factor 2 (RUNX2) was detected via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The effects of heraclenin on the RhoA/ROCK pathway were estimated through western blotting. Y-27632, the ROCK inhibitor, was used to confirm the role of the RhoA/ROCK pathway in heraclenin-mediated osteogenic differentiation of BMSCs., Results: Heraclenin (5-80 μM) was non-toxic on human BMSCs. Heraclenin treatment (5-20 μM) dose-dependently enhanced ALP activity and calcium deposition. Furthermore, heraclenin promoted ALP, OCN, OSX, and RUNX2 mRNA and protein expression. Mechanically, heraclenin treatment increased RhoA and ROCK1 mRNA expression, stimulated the translocation of ROCK from the cytosolic to the membrane fraction, and elevated the protein levels of phosphorylated cofilin (p-cofilin) and active RhoA. Additionally, treatment with Y-27632 overturned the promotion of heraclenin on ALP activity, calcium deposition, the expression of osteogenesis markers, and the RhoA/ROCK signaling pathway., Conclusion: Heraclenin facilitates the osteogenic differentiation of human BMSCs through the activation of the RhoA/ROCK pathway., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Proper hydration and the clarity of the cornea are maintained through the crucial function of the corneal endothelium. Inflammation of the corneal endothelium, known as endotheliitis, can disrupt endothelial function, resulting in alterations to vision. Corneal endotheliitis is characterised by corneal oedema, the presence of keratic precipitates, inflammation within the anterior chamber, and occasionally, limbal injection, neovascularisation, and the concurrent or overlapping presence of uveitis. The aetiology of this condition is diverse, predominantly viral, but it may also be drug-induced, result from bacterial or fungal infections, be associated with systemic diseases and procedures, or remain idiopathic with no identifiable cause. To date, no standardised protocol for the treatment of this ocular disease exists, and in severe cases, corneal transplantation may be required. A 31-year-old male was transferred to our hospital for the management of corneal endothelial decompensation resulting from corneal endotheliitis. Hormonal therapy and antiviral medications proved ineffective, rendering the patient a candidate for corneal transplantation. As a final measure, treatment with the ROCK inhibitor netarsudil was initiated. The patient demonstrated significant improvement in symptoms, and the inflammation was successfully managed after nine months. In this study, a novel approach employing ROCK inhibitor therapy was utilised for the treatment of corneal endotheliitis, leading to marked recovery during patient follow-up. This case report represents the inaugural application of the ROCK inhibitor netarsudil in managing corneal endothelial decompensation attributed to corneal endotheliitis. These findings suggest that this method warrants consideration as a potential novel treatment option for similar conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Orthodontic pain affects the physical and mental health of patients. The spinal trigeminal subnucleus caudalis (SPVC) contributes to the transmission of pain information and serves as a relay station for integrating orofacial damage information. Recently, glial cells have been found to be crucial for both acute and maintenance phases of pain. It has also been demonstrated that rho kinase (ROCK) inhibitors can manage different pain models by inhibiting glial cell activation. Here, we hypothesized that orthodontic pain is related to glial cells in the SPVC, and Fasudil, a representative rho/rock kinase inhibitor, can relieve orthodontic pain by regulating the function of glial cells and the related inflammatory factors. In this study, we constructed a rat model of tooth movement pain and used immunofluorescence staining to evaluate the activation of microglia and astrocytes. Quantitative real-time PCR was used to detect the release of related cytokines and the expression of pain-related genes in the SPVC. Simultaneously, we investigated the effect of Fasudil on the aforementioned indicators., Results: In the SPVC, the expression of c-Fos peaked on day 1 along with the expression of OX42 (related to microglial activation), CD16 (a pro-inflammatory factor), and CD206 (an anti-inflammatory factor) on day 3 after tooth movement, followed by a gradual decrease. GFAP-staining showed that the number of activated astrocytes was the highest on day 5 and that cell morphology became complex. After Fasudil treatment, the expression of these proteins showed a downward trend. The mRNA levels of pro-inflammatory factors (IL-1β and TNF-α) peaked on day 3, and the mRNA expression of the anti-inflammatory factor TGF-β was the lowest 3 days after tooth movement. Fasudil inhibited the mRNA expression of pain-related genes encoding CSF-1, t-PA, CTSS, and BDNF., Conclusion: This study shows that tooth movement can cause the activation of glial cells in SPVC, and ROCK inhibitor Fasudil can inhibit the activation of glial cells and reduce the expression of the related inflammatory factors. This study presents for the first time the potential application of Fasudil in othodontic pain., (© 2024. The Author(s).)

Descemet's Stripping Only (DSO) is a surgical technique that utilizes the peripheral corneal endothelial cell (CEnC) migration for wound closure. Ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, has shown potential in DSO treatment; however, its mechanism in promoting CEnC migration remains unclear. We observed that ripasudil-treated immortalized normal and Fuchs endothelial corneal dystrophy (FECD) cells exhibited significantly enhanced migration and wound healing, particularly effective in FECD cells. Ripasudil upregulated mRNA expression of Snail Family Transcriptional Repressor ( SNAI1/2 ) and Vimentin ( VIM ) while decreasing Cadherin ( CDH1 ), indicating endothelial-to-mesenchymal transition (EMT) activation. Ripasudil activated Rac1, driving the actin-related protein complex (ARPC2) to the leading edge, facilitating enhanced migration. Ex vivo studies on cadaveric and FECD Descemet's membrane (DM) showed increased migration and proliferation of CEnCs after ripasudil treatment. An ex vivo DSO model demonstrated enhanced migration from the DM to the stroma with ripasudil. Coating small incision lenticule extraction (SMILE) tissues with an FNC coating mix and treating the cells in conjunction with ripasudil further improved migration and resulted in a monolayer formation, as detected by the ZO-1 junctional marker, thereby leading to the reduction in EMT. In conclusion, ripasudil effectively enhanced cellular migration, particularly in a novel ex vivo DSO model, when the stromal microenvironment was modulated. This suggests ripasudil as a promising adjuvant for DSO treatment, highlighting its potential clinical significance.

Anti-partial epithelial-mesenchymal transition (pEMT) treatment of renal tubular epithelial cells (TECs) represents a promising therapeutic approach. Hyperuricemia nephropathy (HN) arises as a consequence of hyperuricemia (HUA)-induced tubulointerstitial fibrosis (TIF). Studies have suggested that the Ras homolog member A (RhoA)/Rho-associated kinase (ROCK) pathway is a crucial signaling transduction system in renal fibrosis. Fasudil, a RhoA/ROCK inhibitor, has exhibited the potential to prevent fibrosis progress. However, its impact on the pEMT of TECs in HN remains unclear. Here, an HN rat model and an uric acid (UA)-stimulated human kidney 2 (HK2) cell model were established and treated with Fasudil to explore its effects. Furthermore, the underlying mechanism of action involved in the attenuation of pEMT in TECs by Fasudil during HN was probed by using multiple molecular approaches. The HN rat model exhibited significant renal dysfunction and histopathological damage, whereas in vitro and in vivo experiments further confirmed the pEMT status accompanied by RhoA/ROCK pathway activation and oxidative stress in tubular cells exposed to UA. Notably, Fasudil ameliorated these pathological changes, and this was consistent with the trend of ROCK silencing in vitro. Mechanistically, we identified the Neh2 domain of nuclear factor erythroid 2-related factor 2 (Nrf2) as a target of Fasudil for the first time. Fasudil targets Nrf2 activation and antagonizes oxidative stress to attenuate the pEMT of TECs in HN. Our findings suggest that Fasudil attenuates oxidative stress-induced pEMT of TECs in HN by targeting Nrf2 activation. Thus, Fasudil is a potential therapeutic agent for the treatment of HN., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: To investigate if there are improvements in trabeculectomy outcomes supporting filtration bleb formation caused by Rho-associated protein kinase (ROCK) inhibitors., Methods: This prospective, multicentre, randomised, open-label clinical study examined open-angle glaucoma patients who underwent trabeculectomy or trabeculectomy combined with cataract surgery followed by 3-month postoperative ripasudil treatments. After randomly allocating patients to ripasudil-ROCK inhibitor (ripasudil) or without ripasudil (non-ripasudil) groups. Mean intraocular pressure (IOP) changes, success rate, and number of eyedrops were compared for both groups., Results: A total of 17 and 15 subjects dropped out in the ripasudil group and non-ripasudil group, respectively. At baseline, the mean IOP was 16.8±5.0 mm Hg in the ripasudil group (38 patients) and 16.2±4.4 in the non-ripasudil group (52 patients). The IOP decreased to 11.4±3.2 mm Hg, 10.9±3.9 mm Hg and 10.6±3.5 mm Hg at 12, 24 and 36 months in the ripasudil group, while it decreased to 11.2±4.1 mm Hg, 10.5±3.1 mm Hg and 10.9±3.2 mm Hg at 12, 24 and 36 months in the non-ripasudil group, respectively. There was a significant decrease in the number of IOP-lowering medications after trabeculectomy in the ripasudil group versus the non-ripasudil group at 24 (p=0.010) and 36 months (p=0.016). There was no statistically significant difference between the groups for the 3-year cumulative probability of success., Conclusion: Although ripasudil application did not increase the primary trabeculectomy success rate, it did reduce IOP-lowering medications after trabeculectomy with mitomycin C., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Colorectal cancer (CRC) is significantly contributed to global cancer mortality rates. Treating CRC is particularly challenging due to metastasis and drug resistance. There is a pressing need for new treatment strategies against metastatic CRC. Photodynamic therapy (PDT) offers a well-established, minimally invasive treatment option for cancer with limited side effects. Hypericin (HYP), a potent photosensitizer for PDT, has been documented to induce cytotoxicity and apoptosis in various types of cancers. However, there are few reports on the inhibitory effects of HYP-mediated PDT on the metastatic ability of CRC cells. Here, we evaluate the inhibitory effects of HYP-mediated PDT against metastatic CRC cells and define its underlying mechanisms. Wound-healing and Transwell assays show that HYP-mediated PDT suppresses migration and invasion of CRC cells. F-actin visualization assays indicate HYP-mediated PDT decreases F-actin formation in CRC cells. TEM assays reveal HYP-mediated PDT disrupts pseudopodia formation of CRC cells. Mechanistically, immunofluorescence and western blotting results show that HYP-mediated PDT upregulates E-cadherin and downregulates N-cadherin and Vimentin. HYP-mediated PDT also suppresses key EMT regulators, including Snail, MMP9, ZEB1 and α-SMA. Additionally, the expressions of RhoA and ROCK1 are downregulated by HYP-mediated PDT. Together, these findings suggest that HYP-mediated PDT inhibits the migration and invasion of HCT116 and SW620 cells by modulating EMT and RhoA-ROCK1 signaling pathway. Thus, HYP-mediated PDT presents a potential therapeutic option for CRC., (© 2024. The Author(s), under exclusive licence to the European Photochemistry Association, European Society for Photobiology.)

Refractory wounds are a severe complication of diabetes mellitus that often leads to amputation because of the lack of effective treatments and therapeutic targets. The pathogenesis of refractory wounds is complex, involving many types of cells. Rho-associated protein kinase-1 (ROCK1) phosphorylates a series of substrates that trigger downstream signaling pathways, affecting multiple cellular processes, including cell migration, communication, and proliferation. The present study investigated the role of ROCK1 in diabetic wound healing and molecular mechanisms. Our results showed that ROCK1 expression significantly increased in wound granulation tissues in diabetic patients, streptozotocin (STZ)-induced diabetic mice, and db/db diabetic mice. Wound healing and blood perfusion were dose-dependently improved by the ROCK1 inhibitor fasudil in diabetic mice. In endothelial cells, fasudil and ROCK1 siRNA significantly elevated the phosphorylation of adenosine monophosphate-activated protein kinase at Thr172 (pThr 172 -AMPKα), the activity of endothelial nitric oxide synthase (eNOS), and suppressed the levels of mitochondrial reactive oxygen species (mtROS) and nitrotyrosine formation. Experiments using integrated bioinformatics analysis and coimmunoprecipitation established that ROCK1 inhibited pThr 172 -AMPKα by binding to receptor-interacting serine/threonine kinase 4 (RIPK4). These results suggest that fasudil accelerated wound repair and improved angiogenesis at least partially through the ROCK1/RIPK4/AMPK pathway. Fasudil may be a potential treatment for refractory wounds in diabetic patients., (© 2024. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

GSK-3β, IKK-β, and ROCK-1 kinases are implicated in the pathomechanism of Alzheimer's disease due to their involvement in the misfolding and accumulation of amyloid β (Aβ) and tau proteins, as well as inflammatory processes. Among these kinases, GSK-3β plays the most crucial role. In this study, we present compound 62 , a novel, remarkably potent, competitive GSK-3β inhibitor (IC 50 = 8 nM, K i = 2 nM) that also exhibits additional ROCK-1 inhibitory activity (IC 50 = 2.3 µM) and demonstrates anti-inflammatory and neuroprotective properties. Compound 62 effectively suppresses the production of nitric oxide (NO) and pro-inflammatory cytokines in the lipopolysaccharide-induced model of inflammation in the microglial BV-2 cell line. Furthermore, it shows neuroprotective effects in an okadaic-acid-induced tau hyperphosphorylation cell model of neurodegeneration. The compound also demonstrates the potential for further development, characterized by its chemical and metabolic stability in mouse microsomes and fair solubility.

Epithelial tissue forms and maintains a critical barrier function in the body. A novel culture design aimed at promoting uniform maturation of epithelial cells using liquid materials is described. Culturing Madin-Darby canine kidney (MDCK) cells at the liquid-liquid interface yielded reduced migration and stimulated active cell growth. Similar to solid-liquid interfaces, cells cultured on a fibronectin-coated liquid-liquid interface exhibited active migration and growth, ultimately reaching a confluent state. These cells exhibited reduced stress fiber formation and adopted a cobblestone-like shape, which led to their even distribution in the culture vessel. To inhibit stress fiber formation and apoptosis, the exposure of cells on liquid-liquid interfaces to Y27632, a specific inhibitor of the Rho-associated protein kinase (ROCK), facilitated tight junction formation (frequency of ZO-2-positive cells, F Z  = 0.73). In Y27632-exposed cells on the liquid-liquid interface, the value obtained by subtracting the standard deviation of the ratio of nucleus densities in each region that compartmentalized a culture vessel from 1, denoted as H LN , was 0.93 ± 0.01, indicated even cell distribution in the culture vessel at t = 72 h. The behavior of epithelial cells on liquid-liquid interfaces contributes to the promotion of their uniform maturation., (© 2024. The Author(s).)

Skin damage requires efficient immune cell responses to restore organ function. Epidermal-resident immune cells known as Langerhans cells use dendritic protrusions to surveil the skin microenvironment, which contains keratinocytes and peripheral axons. The mechanisms governing Langerhans cell dendrite dynamics and responses to tissue damage are poorly understood. Using skin explants from adult zebrafish, we show that Langerhans cells maintain normal surveillance following axonal degeneration and use their dendrites to engulf small axonal debris. By contrast, a ramified-to-rounded shape transition accommodates the engulfment of larger keratinocyte debris. We find that Langerhans cell dendrites are populated with actin and sensitive to a broad-spectrum actin inhibitor. We show that Rho-associated kinase (ROCK) inhibition leads to elongated dendrites, perturbed clearance of large debris, and reduced Langerhans cell migration to epidermal wounds. Our work describes the dynamics of Langerhans cells and involvement of the ROCK pathway in immune cell responses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Glaucoma is a chronic neurodegenerative disease that poses a significant threat of irreversible blindness worldwide. Current treatments for glaucoma focus on reducing intraocular pressure (IOP), which is the only modifiable risk factor. Traditional anti-glaucomatous agents, including carbonic anhydrase inhibitors, beta-blockers, alpha-2 agonists, and prostaglandin analogs, work by either improving uveoscleral outflow or reducing aqueous humor production. Rho kinase (ROCK) inhibitors represent a novel class of anti-glaucomatous drugs that have emerged from bench to bedside in the past decade, offering multifunctional characteristics. Unlike conventional medications, ROCK inhibitors directly target the trabecular meshwork outflow pathway. This review aims to discuss the mechanism of ROCK inhibitors in reducing IOP, providing neuroprotection, and preventing fibrosis. We also highlight recent studies and clinical trials evaluating the efficacy and safety of ROCK inhibitors, compare them with other clinical anti-glaucomatous medications, and outline future prospects for ROCK inhibitors in glaucoma treatment.

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

Penetrating keratoplasty remains the most common treatment to restore vision for corneal diseases. Immune rejection after corneal transplantation is one of the major causes of graft failure. In recent years, Rho-associated protein kinase (ROCK) inhibitors have been found to be associated with the activation of the STATs pathway and are widely studied in autoimmune diseases. Therefore, it may be possible that the ROCK inhibitors also participate in the local and systemic immune regulation in corneal transplantation through activation of the STATs pathway and affect the CD4 + T cell differentiation. This study aimed to explore the role of ROCK-STATs pathway in the occurrence of immune rejection in corneal transplantation by applying Y27632, a ROCK inhibitor, to the recipient mice and peripheral CD4 + T cells. We found that Y27632 significantly up-regulated the phosphorylation level of STAT5 in both spleen and lymph nodes, down-regulated the phosphorylation level of STAT3 in the CD4 + T cells in the spleen. It also increased the proportion of CD4 + CD25 + Foxp3 + Helios + Tregs while decreased CD4 + IL17A + -Th17 cells. Moreover, Y27632 also reduced the proportion of dendritic cells in both spleen and lymph nodes, as well as the expression level of CD86 on their surfaces in the spleen, while the proportion of macrophages was not affected. The expression levels of ROCK1, ROCK2, CD11c and IL-17A mRNA were also found to be low in the graft tissue while the expression of Helios was upregulated. Rho-kinase inhibitor can modulate the balance of Tregs/Th17 by regulating the phosphorylation levels of both STAT3 and STAT5, thereby inhibiting the occurrence of immune rejection in allogeneic corneal transplantation., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na + /K + -ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All authors have read the journal's policy on conflicts of interest and authorship agreement., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Pancreatic cancer is characterized by a densely fibrotic stroma. The fibrotic stroma hinders the intratumoral penetration of nanomedicine and diminishes therapeutic efficacy. Fibrosis is characterized by an abnormal organization of extracellular matrix (ECM) components, namely the abnormal deposition and/or orientation of collagen and fibronectin. Abnormal ECM organization is chiefly driven by pathological signaling in pancreatic stellate cells (PSCs), the main cell type involved in fibrogenesis. However, whether targeting signaling pathways involved in abnormal ECM organization improves the intratumoral penetration of nanomedicines is unknown. Here, we show that targeting transforming growth factor-β (TGFβ)/Rho-associated kinase (ROCK) 1/2 signaling in PSCs normalizes ECM organization and concomitantly improves macromolecular permeability of the fibrotic stroma. Using a 3-dimensional cell culture model of the fibrotic pancreatic cancer microenvironment, we found that pharmacological inhibition of TGFβ or ROCK1/2 improves the permeation of various macromolecules. By using an isoform-specific pharmacological inhibitor and siRNAs, we show that targeting ROCK2, but not ROCK1, alone is sufficient to normalize ECM organization and improve macromolecular permeability. Moreover, we found that ROCK2 inhibition/knockdown attenuates Yes-associated protein (YAP) nuclear localization in fibroblasts co-cultured with pancreatic cancer cells in 3D. Finally, pharmacological inhibition or siRNA-mediated knockdown of YAP normalized ECM organization and improved macromolecular permeability. Our results together suggest that the TGFβ/ROCK2/YAP signaling axis may be therapeutically targeted to normalize ECM organization and improve macromolecular permeability to augment therapeutic efficacy of nanomedicines in pancreatic cancer., Competing Interests: Declaration of competing interest Takuya Miyazaki is the CEO and Horacio Cabral is the co-founder and the head of drug discovery of Red Arrow Therapeutics, Inc. All other authors declare no conflict of interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Glaucoma is a leading cause of irreversible blindness which preferentially affects older individuals. No medications or therapies which are currently in our arsenal actually treat glaucoma itself. We know that intraocular pressure (IOP) is currently the only modifiable risk factor for glaucoma. The primary treatments for glaucoma include medications, laser therapies, and surgical therapies. The Rho kinase inhibitors are the newest class of medications currently on the market and in development for topical IOP-lowering therapy. Studies have shown their ability to lower eye pressure individually and in combination with other medications. Their ability to potentially provide neuroprotective effects for disease modification also gives this class exciting potential for glaucoma treatment., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Rho-associated kinases (ROCKs) are crucial during the adipocyte differentiation process. KD025 (Belumosudil) is a newly developed inhibitor that selectively targets ROCK2. It has exhibited consistent efficacy in impeding adipogenesis across a spectrum of in vitro models of adipogenic differentiation. Given the novelty of this treatment, a comprehensive systematic review has not been conducted yet. This systematic review aims to fill this knowledge void by providing readers with an extensive examination of the rationale behind KD025 and its impacts on adipogenesis. Preclinical evidence was gathered owing to the absence of clinical trials. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed, and the study's quality was assessed using the Joanna Briggs Institute (JBI) Checklist Critical Appraisal Tool for Systematic Reviews. In various in vitro models, such as 3T3-L1 cells, human orbital fibroblasts, and human adipose-derived stem cells, KD025 demonstrated potent anti-adipogenic actions. At a molecular level, KD025 had significant effects, including decreasing fibronectin (Fn) expression, inhibiting ROCK2 and CK2 activity, suppressing lipid droplet formation, and reducing the expression of proadipogenic genes peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα). Additionally, KD025 resulted in the suppression of fatty acid-binding protein 4 (FABP4 or AP2) expression, a decrease in sterol regulatory element binding protein 1c (SREBP-1c) and Glut-4 expression. Emphasis must be placed on the fact that while KD025 shows potential in preclinical studies and experimental models, extensive research is crucial to assess its efficacy, safety, and potential therapeutic applications thoroughly and directly in human subjects., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Autoimmune myocarditis is the limited or diffuse inflammation of the myocardium due to dysfunctional cellular and humoral immunity mechanisms. We constructed mouse models of experimental autoimmune myocarditis (EAM) using peptide MyHC-α614-629. On the day after secondary immunization, the mice were intraperitoneally injected with Rho kinase (ROCK) inhibitor Y-27632. On day 21, the cardiac tissues were harvested and weighed. The hearts of EAM mice were significantly enlarged and whitened. Furthermore, body weight (BW) slowly increased during the treatment period, the heart weight (HW) and the ratio of HW/eventual BW were increased, and inflammatory infiltration and fibrosis were aggravated in the myocardial tissue. Y-27632 treatment improved the aforementioned phenotypic and pathological features of EAM mice. Mechanistic analysis revealed a significant increase in Notch1, Hes1, Jag2, Dil1, Toll-like receptor (Tlr) 2, and interleukin (IL)-1β expression in the myocardial tissue of EAM mice. Notably, IL-1β expression was correlated with that of Notch1 and Tlr2. Following Y-27632 treatment, the expression of key target genes of the Notch signaling pathway (Notch1, Hes1, Dil1, and Jag2) and Tlr2 were obviously decreased. Y-27632 treatment also decreased the number of monocytes in the spleen of EAM mice. Thus, ROCK inhibitor Y-27632 exerted a protective effect in EAM mice by downregulating IL-1β expression. This study aimed to provide a reference point for the future treatment of myocarditis in clinical settings., (© 2024. The Author(s).)

Rho kinase inhibitor fasudil exerts therapeutic effects against vasospasms. In this study, we aimed to compare its suppressive effects on serotonin (5-HT)- and noradrenaline (NAd)-induced contractions of human endothelium-denuded internal thoracic arteries (ITAs) and saphenous veins (SVs). NAd and 5-HT induced concentration-dependent contractions in both ITAs and SVs. However, fasudil (3 µmol/L) pretreatment decreased these constrictor-induced contractions in both ITAs and SVs. Fasudil exerted similar inhibitory effects on 5-HT and NAd in ITAs. However, in SVs, fasudil exerted stronger inhibitory effects on NAd-induced contractions than on 5-HT-induced contractions. Therefore, inhibitory effects of fasudil on 5-HT-induced contractions were stronger in ITAs than in SVs. Overall, these results suggest that Rho kinases exert different effects on the two vasoconstrictors in SVs, but not in ITAs, thus explaining their different graft patencies.

Background: Allergic asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness, inflammation and remodeling. ROCK inhibitors have now been shown to have the potential to alleviate these symptoms, although the specific effects of a new ROCK inhibitor, GSK429286 A, remain underexplored., Objective: The aim of this study was to evaluate the therapeutic effects of a novel ROCK inhibitor, GSK429286 A, which exhibits a high affinity for both ROCK1 and ROCK2 isoforms, on allergic asthma in a guinea pig model, focusing on its effects on airway hyperresponsiveness, inflammation, and remodeling., Methods: To induce allergic asthma, guinea pigs were sensitized with ovalbumin for 28 days, and in the middle of sensitization they were treated with different doses of the RoCK inhibitor, GSK429286 A. The study evaluated the effect of the administered doses on the reduction of airway hyperresponsiveness, by measuring specific airway resistance (sRaw), and the number of coughs after citric acid inhalation. We also monitored the anti-inflammatory effect by measuring levels of inflammatory cytokines, IL-2, IL-4, IL-5, IL-13, and remodeling markers, such as collagen deposition, and goblet cell hyperplasia. In addition, we monitored the possible anti-remodeling effect of GSK429286 A by histopathological examination., Results: The ROCK inhibitor, GSK429286 A, showed an effect on suppressing airway hyperresponsiveness by reducing sRaw and the number of coughs in treated guinea pigs compared to controls. Our investigated drug suppressed the release of key mediators of inflammation, including IL-2, IL-4, and IL-5, thus demonstrating the effect of this ROCK inhibitor on the suppression of inflammation in the airways. Finally, GSK429286 A reduced markers of airway remodeling such as collagen deposition and goblet cell hyperplasia., Conclusion: GSK429286 A, an inhibitor of the ROCK pathway, exhibits significant anti-inflammatory and antiremodeling effects in a guinea pig model of allergic asthma. Indeed, we demonstrate its effect on suppressing airway hyperreactivity and reducing cough frequency. These findings suggest that GSK429286 A may be a promising therapeutic agent for allergic asthma, although further studies are needed to investigate its long-term efficacy, underlying mechanisms, and optimal dosing strategy., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

We previously reported that the sustained component of contraction induced by depolarizing stimulation by high K + concentration in rat caudal arterial smooth muscle involves a Ca 2+ -induced Ca 2+ sensitization mechanism whereby Ca 2+ entry through voltage-gated Ca 2+ channels activates proline-rich tyrosine kinase 2 (Pyk2), leading to activation of RhoA/Rho-associated kinase (ROCK). In the present study, we investigated a potential role for Pyk2-mediated RhoA/ROCK activation in contraction mediated by elevation of cytosolic free Ca 2+ concentration ([Ca 2+ ] i ) induced by a Ca 2+ ionophore, ionomycin, rather than by depolarizing stimulation. Ionomycin (60 µM) induced slow and sustained contraction of rat caudal arterial smooth muscle due to influx of Ca 2+ . Pre-treatment with a myosin light chain kinase (MLCK) inhibitor, ML-9 (30 µM), inhibited both the early phase (4 min) and the sustained phase (30 min) of ionomycin-induced contraction. On the other hand, a ROCK inhibitor, HA-1077 (3 µM), and Pyk2 inhibitors, sodium salicylate (10 mM) and PF-431396 (3 µM), suppressed only the sustained phase of ionomycin-induced contraction. A calmodulin (CaM) inhibitor, W-7 (150 µM), but not W-5 (150 µM), suppressed the early phase of contraction. Early or sustained increase of ionomycin-induced 20 kDa light chain of myosin (LC 20 ) phosphorylation was inhibited by each inhibitor in a manner similar to the attenuation of contraction. These results indicate that the early phase of ionomycin-induced contraction is mediated by MLCK activation by [Ca 2+ ] i elevation, whereas the sustained phase of ionomycin-induced contraction involves RhoA/ROCK activation and inhibition of myosin light chain phosphatase (MLCP) through CaM-independent Pyk2 activation by [Ca 2+ ] i elevation.

Background: The pathological manifestations of Alzheimer's disease (AD) include not only brain amyloid β protein (Aβ) containing neuritic plaques and hyperphosphorylated tau (p-- tau) containing neurofibrillary tangles but also microgliosis, astrocytosis, and neurodegeneration mediated by metabolic dysregulation and neuroinflammation., Methods: While antibody-based therapies targeting Aβ have shown clinical promise, effective therapies targeting metabolism, neuroinflammation, and p-tau are still an urgent need. Based on the observation that Ras homolog (Rho)-associated kinases (ROCK) activities are elevated in AD, ROCK inhibitors have been explored as therapies in AD models. This study determines the effects of fasudil, a ROCK inhibitor, on neuroinflammation and metabolic regulation in the P301S tau transgenic mouse line PS19 that models neurodegenerative tauopathy and AD. Using daily intraperitoneal (i.p.) delivery of fasudil in PS19 mice, we observed a significant hippocampal-specific decrease of the levels of phosphorylated tau (pTau Ser202/Thr205), a decrease of GFAP+ cells and glycolytic enzyme Pkm1 in broad regions of the brain, and a decrease in mitochondrial complex IV subunit I in the striatum and thalamic regions., Results: Although no overt detrimental phenotype was observed, mice dosed with 100 mg/kg/day for 2 weeks exhibited significantly decreased mitochondrial outer membrane and electron transport chain (ETC) protein abundance, as well as ETC activities., Conclusion: Our results provide insights into dose-dependent neuroinflammatory and metabolic responses to fasudil and support further refinement of ROCK inhibitors for the treatment of AD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: The traditional Chinese medicine formula, Yu's Enema Formula (YEF), has demonstrated potential in the treatment of Ulcerative Colitis (UC)., Objective: This study aimed to unveil the anti-UC mechanisms of YEF., Methods: Utilizing public databases, we obtained YEF and UC-related targets. GO and KEGG analyses were conducted via clusterProfiler and Reactome. The STRING database facilitated the construction of the PPI network, and hub targets were selected using cytoHubba. We used R software for differential expression and correlation analyses, and molecular docking was performed with PyMOL and AutoDock. HPLC analysis identified the compounds in YEF. For in vivo validation, a UC rat model was employed., Results and Discussion: 495 YEF-UC overlapping targets were identified. GO and KEGG analyses indicated enrichment in exogenous stimuli response, peptide response, positive MAPK cascade regulation, interleukin- related signaling, and the TLR4 cascade. Hub targets included CTNNB1, JUN, MAPK1, MAPK3, SRC, STAT3, TLR4, TP53, and RELA, which were often interconnected. Molecular docking revealed quercetin's strong binding affinity with CTNNB1, MAPK1, MAPK3, SRC, STAT3, TLR4, and TP53, consistent with HPLC analysis. In vivo experiments suggested that YEF has the potential to alleviate UC symptoms and protect the intestinal mucosal barrier by inhibiting the RhoA/ROCK pathway., Conclusion: YEF may safeguard the intestinal mucosal barrier in UC by targeting CTNNB1, MAPK1, MAPK3, SRC, STAT3, TLR4, and TP53, while blocking the RhoA/ROCK pathway., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Suboptimal response to conventional treatments in refractory diabetic macular edema (rDME) encourages efforts to identify new therapeutic options., Purpose: To evaluate the effect of three monthly intravitreal injections of a Rho-associated protein kinase (ROCK) inhibitor (Fasudil, Asahi Kasei Pharma Corporation, Tokyo, Japan) in eyes with rDME., Methods: Ten eyes of 10 patients with DME unresponsive to at least six previous intravitreal bevacizumab (IVB) injections were recruited and underwent 3 consecutive monthly intravitreal injection of 0.025mg/0.05mL Fasudil. Best-corrected visual acuity (BCVA) and central macular thickness (CMT) were evaluated as functional and anatomical response indicators, respectively., Results: The mean age was 60.1±5.1 years (range, 53-68). Five cases responded to treatment, two with both anatomical and functional responses (reduction of CMT from 521 to 395 and from 390 to 301 microns and improvement of BCVA from 0.3 to 0.1 LogMAR and 0.6 to 0.4 LogMAR, respectively) and three with only functional improvement (0.7 to 0.4; 0.7 to 0.4; and 0.3 to 0.1 LogMAR). Of note, cases with no significant change in CMT showed morphologic improvement of the retinal microstructure to some extent. No adverse event was observed during the study period., Conclusion: Monotherapy with intravitreal injection of ROCK inhibitors appears to have moderate visual benefits in eyes with DME refractory to IVB. Such effects may be functionally significant without obvious anatomical improvement., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Excessive fluoride intake poses health risks to humans and animals. Many studies have indicated that fluoride exposure can damage the cytoskeleton and synapses, which has negative effects on the intellectual development of humans and animals. Our previous study suggested that the RhoA/ROCK signalling pathway is activated by NaF exposure in HT-22 cells and plays a vital role in cytoskeletal assembly and synaptogenesis. However, the mechanism underlying RhoA/ROCK-mediated cytoskeletal injury induced by fluoride remains unclear. In this study, Neuro-2A cells and ICR mice were used to investigate the effects of RhoA/ROCK activation inhibition on NaF-induced synaptic dysfunction and cognitive impairment. We detected the expression of GAP, RhoA, ROCK1/2, and (p)-MLC in vivo and in vitro model. The results showed that NaF exposure activated the RhoA/ROCK/MLC signalling pathway. We measured the effects of RhoA/ROCK inhibition on synaptic injury and intellectual impairment induced by NaF exposure. In vitro, Y-27632 suppressed activated RhoA/ROCK, attenuated morphological and ultrastructural damage, and decreased the survival rate and synapse-functional protein expression caused by NaF. In vivo, the results showed that the RhoA/ROCK/MLC pathway was inhibited by fasudil and improved pathological damage in the hippocampus, cognitive impairment, and decreased expression of neurofunctional proteins induced by NaF. Overall, these results suggest that fasudil and Y-27632 can reverse neurotoxicity caused by fluoride exposure. Furthermore, inhibition of RhoA/ROCK may be a future treatment for CNS injury, and more detailed studies on other neurodegenerative disease models are required to confirm its effectiveness., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Schizophrenia (SCZ) is a severe psychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive deficits. Current antipsychotic treatment in SCZ improves positive symptoms but has major side effects and little impact on negative symptoms and cognitive impairment. The pathoetiology of SCZ remains unclear, but is known to involve small GTPase signaling. Rho kinase, an effector of small GTPase Rho, is highly expressed in the brain and plays a major role in neurite elongation and neuronal architecture. This study used a touchscreen-based visual discrimination (VD) task to investigate the effects of Rho kinase inhibitors on cognitive impairment in a methamphetamine (METH)-treated male mouse model of SCZ. Systemic injection of the Rho kinase inhibitor fasudil dose-dependently ameliorated METH-induced VD impairment. Fasudil also significantly suppressed the increase in the number of c-Fos-positive cells in the infralimbic medial prefrontal cortex (infralimbic mPFC) and dorsomedial striatum (DMS) following METH treatment. Bilateral microinjections of Y-27632, another Rho kinase inhibitor, into the infralimbic mPFC or DMS significantly ameliorated METH-induced VD impairment. Two proteins downstream of Rho kinase, myosin phosphatase-targeting subunit 1 (MYPT1; Thr696) and myosin light chain kinase 2 (MLC2; Thr18/Ser19), exhibited increased phosphorylation in the infralimbic mPFC and DMS, respectively, after METH treatment, and fasudil inhibited these increases. Oral administration of haloperidol and fasudil ameliorated METH-induced VD impairment, while clozapine had little effect. Oral administration of haloperidol and clozapine suppressed METH-induced hyperactivity, but fasudil had no effect. These results suggest that METH activates Rho kinase in the infralimbic mPFC and DMS, which leads to cognitive impairment in male mice. Rho kinase inhibitors ameliorate METH-induced cognitive impairment, perhaps via the cortico-striatal circuit., Competing Interests: Declaration of Competing Interest This study was funded in part by Sumitomo Pharma Co., Ltd., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)