Rho Kinase Inhibitor Research Articles

Influence of the calcium voltage-gated channel auxiliary subunit (CACNA2D1) absence on intraocular pressure in mice

The etiology of elevated intraocular pressure (IOP), a major risk factor for glaucoma (optic nerve atrophy), is poorly understood despite continued efforts. Although the gene variant of CACNA2D1 (encoding α2δ1), a calcium voltage-gated channel auxiliary subunit, has been reported to be associated with primary open-angle glaucoma, and the pharmacological mitigation of α2δ1 activity by pregabalin lowers IOP, the cellular basis for α2δ1 role in the modulation of IOP remains unclear. Our recent findings reveled readily detectable levels of α2δ1 and its ligand thrombospondin in the cytoskeletome fraction of human trabecular meshwork (TM) cells. To understand the direct role of α2δ1 in the modulation of IOP, we evaluated α2δ1 null mice for changes in IOP and found a moderate (∼10%) but significant decrease in IOP compared to littermate wild type control mice. Additionally, to gain cellular insights into α2δ1 antagonist (pregabalin) induced IOP changes, we assessed pregabalin's effects on human TM cell actin cytoskeletal organization and cell adhesive interactions in comparison with a Rho kinase inhibitor (Y27632), a known ocular hypotensive agent. Unlike Y27632, pregabalin did not have overt effects on cell morphology, actin cytoskeletal organization, or cell adhesion in human TM cells. These results reveal a modest but significant decrease in IOP in α2δ1 deficient mice, and this response appears to be not associated with the contractile and cell adhesive characteristics of TM cells based on the findings of pregabalin effects on isolated TM cells. Therefore, the mechanism by which pregabalin lowers IOP remains elusive.

Chemical Insights into Topical Agents in Intraocular Pressure Management: From Glaucoma Etiopathology to Therapeutic Approaches.

Glaucoma encompasses a group of optic neuropathies characterized by complex and often elusive etiopathology, involvihttng neurodegeneration of the optic nerve in conjunction with abnormal intraocular pressure (IOP). Currently, there is no cure for glaucoma, and treatment strategies primarily aim to halt disease progression by managing IOP. This review delves into the etiopathology, diagnostic methods, and treatment approaches for glaucoma, with a special focus on IOP management. We discuss a range of active pharmaceutical ingredients used in glaucoma therapy, emphasizing their chemical structure, pharmacological action, therapeutic effectiveness, and safety/tolerability profiles. Notably, most of these therapeutic agents are administered as topical formulations, a critical aspect considering patient compliance and drug delivery efficiency. The classes of glaucoma therapeutics covered in this review include prostaglandin analogs, beta blockers, alpha agonists, carbonic anhydrase inhibitors, Rho kinase inhibitors, and miotic (cholinergic) agents. This comprehensive overview highlights the importance of topical administration in glaucoma treatment, offering insights into the current state and future directions of pharmacological management in glaucoma.

Protocol for a randomized, placebo-controlled, double-blind phase IIa study of the safety, tolerability, and symptomatic efficacy of the ROCK-inhibitor Fasudil in patients with Parkinson's disease (ROCK-PD).

The Rho-kinase (ROCK) inhibitor Fasudil has shown symptomatic and disease-modifying effects in Parkinson's disease (PD) models in vitro and in vivo. In Japan, Fasudil has been approved for the treatment of subarachnoid haemorrhage since 1995 and shows a favourable safety profile. To investigate the safety, tolerability, and symptomatic efficacy of ROCK-inhibitor Fasudil in comparison to placebo in a randomized, national, multicenter, double-blind phase IIa study in patients with PD. We plan to include 75 patients with at least 'probable' PD (MDS criteria), Hoehn and Yahr stages 1-3, and age 30-80 years in 13 German study sites. Patients must be non-fluctuating and their response to PD medication must have been stable for 6 weeks. Patients will be randomly allocated to treatment with the oral investigational medicinal product (IMP) containing either Fasudil in two dosages, or placebo, for a total of 22 days. As primary analysis, non-inferiority of low/high dose of Fasudil on the combined endpoint consisting of occurrence of intolerance and/or treatment-related serious adverse events (SAEs) over 22 days will be assessed in a sequential order, starting with the lower dose. Secondary endpoints will include tolerability alone over 22 days and occurrence of treatment-related SAEs (SARs) over 22 and 50 days and will be compared on group level. Additional secondary endpoints include efficacy on motor and non-motor symptoms, measured on established scales, and will be assessed at several timepoints. Biomaterial will be collected to determine pharmacokinetics of Fasudil and its active metabolite, and to evaluate biomarkers of neurodegeneration. After positive evaluation by the competent authority and the ethics committee, patient recruitment started in the 3rd quarter of 2023. ROCK-PD is registered with Eudra-CT (2021-003879-34) and clinicaltrials.gov (NCT05931575). Results of this trial can pave way for conducting extended-duration studies assessing both symptomatic efficacy and disease-modifying properties of Fasudil.

Rho-Kinase Inhibition of Active Force and Passive Tension in Airway Smooth Muscle: A Strategy for Treating Airway Hyperresponsiveness in Asthma.

Rho-kinase inhibitors have been identified as a class of potential drugs for treating asthma because of their ability to reduce airway inflammation and active force in airway smooth muscle (ASM). Past research has revealed that, besides the effect on the ASM's force generation, rho-kinase (ROCK) also regulates actin filament formation and filament network architecture and integrity, thus affecting ASM's cytoskeletal stiffness. The present review is not a comprehensive examination of the roles played by ROCK in regulating ASM function but is specifically focused on passive tension, which is partially determined by the cytoskeletal stiffness of ASM. Understanding the molecular basis for maintaining active force and passive tension in ASM by ROCK will allow us to determine the suitability of ROCK inhibitors and its downstream enzymes as a class of drugs in treating airway hyperresponsiveness seen in asthma. Because clinical trials using ROCK inhibitors in the treatment of asthma have yet to be conducted, the present review focuses on the in vitro effects of ROCK inhibitors on ASM's mechanical properties which include active force generation, relaxation, and passive stiffness. The review provides justification for future clinical trials in the treatment of asthma using ROCK inhibitors alone and in combination with other pharmacological and mechanical interventions.

Multiscale imaging of corneal endothelium damage and Rho-kinase inhibitor application in mouse models of acute ocular hypertension.

We developed a multiscale optical imaging workflow, integrating and correlating visible-light optical coherence tomography, confocal laser scanning microscopy, and single-molecule localization microscopy to investigate mouse cornea damage from the in-vivo tissue level to the nanoscopic single-molecule level. We used electron microscopy to validate the imaged nanoscopic structures. We imaged wild-type mice and mice with acute ocular hypertension and examined the effects of Rho-kinase inhibitor application. We defined four types of intercellular tight junction structures as healthy, compact, partially-distorted, and fully-distorted types by labeling the zonula occludens-1 protein in the corneal endothelial cell layer. We correlated the statistics of the four types of tight junction structures with cornea thickness and intraocular pressure. We found that the population of fully-distorted tight junctions correlated well with the level of corneal edema, and applying Rho-kinase inhibitor reduced the population of fully-distorted tight junctions under acute ocular hypertension. Together, these data point to the utility of multiscale optical imaging in revealing fundamental biology relevant to disease and therapeutics.

Exploring the Ocular Absorption Pathway of Fasudil Hydrochloride towards Developing a Nanoparticulate Formulation with Improved Performance.

Rho-kinase (ROCK) inhibitors represent a new category of anti-glaucoma medications. Among them, Fasudil hydrochloride, a selective ROCK inhibitor, has demonstrated promising outcomes in glaucoma treatment. It works by inhibiting the ROCK pathway, which plays a crucial role in regulating the trabecular meshwork and canal of Schlemm's aqueous humor outflow. This study aims to investigate the ocular absorption pathway of Fasudil hydrochloride and, subsequently, develop a nanoparticle-based delivery system for enhanced corneal absorption. Employing the ionic gelation method and statistical experimental design, the factors influencing chitosan nanoparticle (Cs NP) characteristics and performance were explored. Fasudil in vitro release and ex vivo permeation studies were performed, and Cs NP ocular tolerability and cytotoxicity on human lens epithelial cells were evaluated. Permeation studies on excised bovine eyes revealed significantly higher Fasudil permeation through the sclera compared to the cornea (370.0 μg/cm2 vs. 96.8 μg/cm2, respectively). The nanoparticle size (144.0 ± 15.6 nm to 835.9 ± 23.4 nm) and entrapment efficiency range achieved (17.2% to 41.4%) were predominantly influenced by chitosan quantity. Cs NPs showed a substantial improvement in the permeation of Fasudil via the cornea, along with slower release compared to the Fasudil aqueous solution. The results from the Hen's Egg Test Chorioallantoic Membrane (HET-CAM) and Bovine Corneal Opacity and Permeability (BCOP) tests indicated good conjunctival and corneal biocompatibility of the formulated chitosan nanoparticles, respectively. Lens epithelial cells displayed excellent tolerance to low concentrations of these nanoparticles (>94% cell viability). To the best of our knowledge, this is the first report on the ocular absorption pathway of topically applied Fasudil hydrochloride where the cornea has been identified as a potential barrier that could be overcome using Cs NPs.

Sustained intestinal epithelial monolayer wound closure after transient application of a FAK-activating small molecule.

M64HCl, which has drug-like properties, is a water-soluble Focal Adhesion Kinase (FAK) activator that promotes murine mucosal healing after ischemic or NSAID-induced injury. Since M64HCl has a short plasma half-life in vivo (less than two hours), it has been administered as a continuous infusion with osmotic minipumps in previous animal studies. However, the effects of more transient exposure to M64HCl on monolayer wound closure remained unclear. Herein, we compared the effects of shorter M64HCl treatment in vitro to continuous treatment for 24 hours on monolayer wound closure. We then investigated how long FAK activation and downstream ERK1/2 activation persist after two hours of M64HCl treatment in Caco-2 cells. M64HCl concentrations immediately after washing measured by mass spectrometry confirmed that M64HCl had been completely removed from the medium while intracellular concentrations had been reduced by 95%. Three-hour and four-hour M64HCl (100 nM) treatment promoted epithelial sheet migration over 24 hours similar to continuous 24-hour exposure. 100nM M64HCl did not increase cell number. Exposing cells twice with 2-hr exposures of M64HCl during a 24-hour period had a similar effect. Both FAK inhibitor PF-573228 (10 μM) and ERK kinase (MEK) inhibitor PD98059 (20 μM) reduced basal wound closure in the absence of M64HCl, and each completely prevented any stimulation of wound closure by M64HCl. Rho kinase inhibitor Y-27632 (20 μM) stimulated Caco-2 monolayer wound closure but no further increase was seen with M64HCl in the presence of Y-27632. M64HCl (100 nM) treatment for 3 hours stimulated Rho kinase activity. M64HCl decreased F-actin in Caco-2 cells. Furthermore, a two-hour treatment with M64HCl (100 nM) stimulated sustained FAK activation and ERK1/2 activation for up to 16 and hours 24 hours, respectively. These results suggest that transient M64HCl treatment promotes prolonged intestinal epithelial monolayer wound closure by stimulating sustained activation of the FAK/ERK1/2 pathway. Such molecules may be useful to promote gastrointestinal mucosal repair even with a relatively short half-life.

Advancements in antiglaucoma medications: A comprehensive review

Abstract Glaucoma is a common ocular condition that has permanent effects and is a serious threat to everyone’s vision health. The course of the disease is still significantly influenced by increased intraocular pressure (IOP), also known as eye pressure. This emphasizes the significance of effective use of medications to treat this illness. Significant progress has been made in the development of glaucoma medications over time. There are several different ways to lower IOP using these drugs. This in-depth analysis examines the evolving field of pharmacological glaucoma treatments, covering a range of drug classes including beta-blockers, prostaglandin analogs, alpha agonists, carbonic anhydrase inhibitors, and rho kinase inhibitors. The mode of action, clinical effectiveness, and safety issues of each drug category are studied.

Descemet Endothelial Thickness Comparison Trial 1 (DETECT 1): outcome masked, placebo-controlled trial comparing two types of corneal transplant surgeries and effect of rho kinase inhibitors on endothelial cell loss protocol

BackgroundIt remains uncertain which endothelial keratoplasty (EK) technique yields the best outcomes while maintaining safety, particularly in eyes with coexisting ocular conditions. Moreover, the impact of endothelial cell loss (ECL)...

Change in Intraocular Pressure after Selective Laser Trabeculoplasty in North Indian Primary Open Angle Glaucoma Patients: A Prospective Analytical Study

Introduction: Glaucoma is the second leading cause of blindness in the world. The only known modifiable risk factor for the onset and progression of glaucoma is Intraocular Pressure (IOP). There are a limited number of studies documenting the IOP-lowering effect of Selective Laser Trabeculoplasty (SLT) in Primary Open Angle Glaucoma (POAG) patients, especially within the North Indian population. Understanding the effect of SLT on IOP reduction in this population is crucial for the proper management of glaucoma patients. Aim: To study the IOP-lowering effect of SLT in North Indian POAG patients (who had not achieved target IOP on two or more antiglaucoma drugs) after a period of six months. Materials and Methods: A hospital-based, single-arm, prepost type of prospective analytical study was conducted at Lady Hardinge Medical College and associated hospitals in New Delhi, India, from January 2021 to June 2022. POAG patients aged 18 years or older, who had not achieved the target IOP while on two or more Antiglaucoma Medications (AGM), were given the option of treatment with SLT. A total of 33 eyes were considered for the study as per sample size calculations. PreSLT, IOP was recorded along with other baseline parameters. The SLT procedure was carried out according to the standard protocol. IOP was recorded during follow-ups at one week, one month, three months, and six months. The Friedman test was used to compare IOP values, with a significance level set at 0.05. Spearman correlation was employed to explore the correlation between baseline IOP and the change in IOP at six months. Results: A total of 33 eyes were included in the study. Of these, 28 (84.8%) eyes had advanced glaucoma, while 5 (15.2%) eyes had moderate glaucoma according to International Classification of Diseases, Tenth Revision (ICD-10) staging. Patients were on two or more commonly used AGMs, including prostaglandin analogues, beta blockers, alpha agonists, topical carbonic anhydrase inhibitors, and rho kinase inhibitors. The mean number of topical AGMs that patients were using was 3.03. The number and type of AGMs for each patient remained consistent pre and post-SLT throughout the study period. The mean IOP (baseline) was 16.39±2.73 mmHg, and after six months, the mean IOP was 13.86±1.99 mmHg. The mean IOP reduced by 2.53 mm Hg (15.44%). There was a strong negative correlation between baseline IOP and the change in IOP at six months, which was statistically significant (rho=-0.78, p-value<0.001). Conclusion: The present study indicates that SLT can be considered an adjunctive treatment for lowering IOP in North Indian POAG patients. A strong negative correlation was found between baseline IOP and IOP at six months post-SLT, suggesting that a higher reduction can be expected in patients with elevated baseline IOP.

Netarsudil Associated Limbal Follicles: A Case Report

Primary Open Angle Glaucoma (POAG) is one of the leading causes of irreversible blindness worldwide. Netarsudil, a Rho-kinase inhibitor and norepinephrine transporter, has recently been approved for reducing intraocular pressure (IOP), which is the single most clinically modifiable risk factor for this condition. Here, a case of a 53-year-old female with POAG who developed follicles along the corneal limbus following treatment with Netarsudil eye drops has been reported. The patient had been non-compliant with her POAG treatment for the past five years and presented with diminished vision (right eye greater than left) and raised IOP of 28 mmHg and 26 mmHg in the right and left eyes, respectively. The patient was started on Netarsudil (0.02%) eye drops once daily. After three weeks, the IOP had reduced to 19 mmHg and 18 mmHg in the right and left eyes, respectively; however, the patient still complained of itching and redness. A slit-lamp examination showed few follicular lesions on the limbus. Initial treatment for allergic conjunctivitis was ineffective, leading to a worsening of symptoms. Netarsudil was then discontinued, and dorzolamide was initiated instead, resulting in the resolution of symptoms within one week. The patient continued on dorzolamide without further issues. While Netarsudil is effective in reducing IOP, many of its potential adverse effects may still be unknown due to limited clinical experience. The present case report aims to contribute to the understanding of the safety profile of this drug. More such reports in the future will enhance this knowledge and help clinicians manage similar adverse situations.

Evaluating the efficacy of Rho kinase inhibitor eye drops in the management of corneal edema: A single-center retrospective cohort study.

This study aimed to evaluate the efficacy of ripasudil in managing various corneal edema conditions. This single-center retrospective analysis was conducted at Hadassah Medical Center and involved 16 patients with 17 eyes. Patients were selected based on diagnostic criteria, primarily corneal edema. The conditions were as follows, listed by frequency: postcataract surgery (31.25%), postpenetrating keratoplasty (25%), post-Descemet's membrane endothelial keratoplasty (18.75%), Fuchs' endothelial corneal dystrophy (12.5%), status post-Ahmed glaucoma valve (6.25%), and status posttrabeculectomy (6.25%). The treatment regimen involved topical administration of ripasudil hydrochloride hydrate (Glanatec® 0.4%), administered three times a day or tailored to condition severity. Efficacy was assessed using pre- and posttreatment measurements of best-corrected visual acuity (BCVA), central corneal thickness (CCT), and endothelial cell count (ECC), along with slit-lamp and optical coherence tomography examinations. The average duration of ripasudil treatment was approximately 4.9 ± 2.2 months. Significant improvements were observed in BCVA, changing from a pretreatment value of 1.106 ± 0.817 logMAR to a posttreatment value of 0.56 ± 0.57 logMAR (P = 0.0308). CCT also showed a significant reduction, from 619.50 ± 56.36 μm pretreatment to 572.5 ± 75.48 μm posttreatment (P = 0.0479). ECC showed a marginal but not statistically significant increase, from 849.00 ± 570.72 cells/mm² pretreatment to 874.75 ± 625.59 cells/mm² posttreatment (P = 0.9010). The study provides robust evidence supporting the use of ripasudil in managing corneal edema. Significant improvements in key ocular metrics such as BCVA and CCT were observed, enhancing the overall quality of life for patients suffering from various forms of corneal edema.

The Role of Rho Kinase Inhibitors in Corneal Diseases.

The cornea, as the outermost layer of the eye, plays a crucial role in vision by focusing light onto the retina. Various diseases and injuries can compromise its clarity, leading to impaired vision. This review aims to provide a thorough overview of the pharmacological properties, therapeutic potential and associated risks of Rho-associated protein kinase (ROCK) inhibitors in the management of corneal diseases. The article focuses on four key ROCK inhibitors: Y-27632, fasudil, ripasudil, and netarsudil, providing a comparative examination. Studies supporting the use of ROCK inhibitors highlight their efficacy across diverse corneal conditions. In Fuchs' endothelial corneal dystrophy, studies on the application of Y-27632, ripasudil, and netarsudil demonstrated noteworthy enhancements in corneal clarity, endothelial cell density, and visual acuity. In pseudophakic bullous keratopathy, the injection of Y-27632 together with cultured corneal endothelial cells into the anterior chamber lead to enhanced corneal endothelial cell density and improved visual acuity. Animal models simulating chemical injury to the cornea showed a reduction of neovascularization and epithelial defects after application of fasudil and in a case of iridocorneal endothelial syndrome netarsudil improved corneal edema. Addressing safety considerations, netarsudil and ripasudil, both clinically approved, exhibit adverse events such as conjunctival hyperemia, conjunctival hemorrhage, cornea verticillata, conjunctivitis, and blepharitis. Monitoring patients during treatment becomes crucial to balancing the potential therapeutic benefits with these associated risks. In conclusion, ROCK inhibitors, particularly netarsudil and ripasudil, offer promise in managing corneal diseases. The comparative analysis of their pharmacological properties and studies supporting their efficacy underscore their potential therapeutic significance. However, ongoing research is paramount to comprehensively understand their safety profiles and long-term outcomes in diverse corneal conditions, guiding their optimal application in clinical practice.

Preventing Fluid Retention After Subarachnoid Haemorrhage During Administration of Endothelin Receptor Antagonist.

Prevention of delayed cerebral infarction (DCI) due to cerebral vasospasm after subarachnoid haemorrhage (SAH) has been done with intravenous Rho kinase inhibitors (ROCKI), ozagrel sodium (TXA2I), selective ROCKI infusion (ROCKI i.a.), and cerebrospinal fluid (CSF) drainage. The endothelin receptor antagonist (ERA, clazosentan) became available in 2022 and is said to be highly recommended for DCI prevention, while fluid retention such as pleural effusion and pulmonary oedema accumulation is often experienced. We investigated the relationship between patient background, fluid retention, and ERA. Ten consecutive SAH patients treated with ERA from July to December 2022 were included. We examined the results of blood sampling on admission, echocardiography, chest computed tomography (CT), with postoperative DCI, and hydrocephalus requiring cerebrospinal fluid shunt (hydro), and symptomatic fluid retention requiring albumin and furosemide (third fluid space). Two males and eight females, mean age 63years, mean preoperative World Federation Neurosurgical Surgeons (WFNS) grade 3.5, mean creatinine 0.94, mean brain natriuretic peptide (NT-proBNP). In 1883, two patients with Takotsubo cardiomyopathy and four patients with neurogenic pulmonary oedema are present. All patients underwent coil embolisation, and postoperative CSF drainage, ROCKI, TXA2I systemic administration, and ROCKI i.a. There were one DCI, three hydro, and five third fluid cases. Concerning the third fluid, the only significant difference was found in the age. An improvement in fluid retention after ERA discontinuation in old patients was shown. Our experience suggests that age may be the most influential factor. Based on these results, we have also found that by avoiding the use of ERA in patients older than 80years, strictly limiting the infusion volume when using ERA, and actively using the drugs for heart failure early on, the frequency of suffering from third fluid space is reduced.

Safety and Efficacy of the Rho-Kinase Inhibitor (Ripasudil) in Bleb Needling after Trabeculectomy: A Prospective Multicenter Study.

Ripasudil, a rho-associated protein kinase inhibitor ophthalmic solution, shows a protective effect in preventing excessive scarring in vitro. This study aims to evaluate the safety and efficacy of ripasudil for glaucoma patients submitted to the needling procedure. In this prospective, multicenter, single-arm study, we included 20 eyes of 20 patients with glaucoma who underwent the needling procedure without antimetabolites. All patients administered ripasudil after needling for three months. The primary endpoint of this study was the safety of ripasudil in patients, and the secondary endpoint was the change in IOP at 12 weeks after the needling procedure. No serious complications were found in the patients. One eye experienced pruritus and conjunctival follicle, while another eye had conjunctival follicle. These complications were transient and resolved quickly after discontinuation of ripasudil. The mean preoperative IOP was 14.6 ± 4.6 mmHg, which decreased to 11.0 ± 4.7 mmHg (p = 0.0062) at 1 week postoperatively. The IOP reduction effect continued to 12 weeks (11.8 ± 3.1 mmHg; p = 0.0448). The administration of the ROCK inhibitor, ripasudil, after the needling procedure is safe and effective in maintaining IOP for 12 weeks.

The role of angiotensin II activation of yes-associated protein/PDZ-binding motif signaling in hypertensive cardiac and vascular remodeling

Vascular remodeling is the pathogenic basis of hypertension and end organ injury, and the proliferation of vascular smooth muscle cells (VSMCs) is central to vascular remodeling. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are key effectors of the Hippo pathway and crucial for controlling cell proliferation, apoptosis and differentiation. The present study investigated the role of YAP/TAZ in cardiac and vascular remodeling of angiotensin II-induced hypertension. Ang II induced YAP/TAZ activation in the heart and aorta, which was prevented by YAP/TAZ inhibitor verteporfin. Treatment with verteporfin significantly reduced Ang II-induced cardiac and vascular hypertrophy with a mild reduction in systolic blood pressure (SBP), verteporfin attenuated Ang II-induced cardiac and aortic fibrosis with the inhibition of transform growth factor (TGF)β/Smad2/3 fibrotic signaling and extracellular matrix collagen I deposition. Ang II induced Rho A, extracellular signal-regulated kinase 1/2 (ERK1/2) and YAP/TAZ activation in VSMCs, either Rho kinase inhibitor fasudil or ERK inhibitor PD98059 suppressed Ang II-induced YAP/TAZ activation, cell proliferation and fibrosis of VSMCs. Verteporfin also inhibited Ang II-induced VSMC proliferation and fibrotic TGFβ1/Smad2/3 pathway. These results demonstrate that Ang II activates YAP/TAZ via Rho kinase/ERK1/2 pathway in VSMCs, which may contribute to cardiac and vascular remodeling in hypertension. Our results suggest that YAP/TAZ plays a critical role in the pathogenesis of hypertension and end organ damage, and targeting the YAP/TAZ pathway may be a new strategy for the prevention and treatment of hypertension and cardiovascular diseases.

Inhibition of Rho kinase (ROCK) impairs cytoskeletal contractility in human Müller glial cells without effects on cell viability, migration, and extracellular matrix production

The epiretinal membrane is a fibrocontractile tissue that forms on the inner surface of the retina, causing visual impairment ranging from mild to severe, and even retinal detachment. Müller glial cells actively participate in the formation of this membrane. Current research is constantly seeking for new therapeutic approaches that aim to prevent or treat cellular dysfunctions involved in the progression of this common fibrosis condition. The Rho GTPases signaling pathway regulates several processes associated with the epiretinal membrane, such as cell proliferation, migration, and contraction. Rho kinase (ROCK), an effector of the RhoA GTPase, is an interesting potential therapeutic target. This study aimed to evaluate the effects of a ROCK inhibitor (Y27632) on human Müller cells viability, growth, cytoskeletal organization, expression of extracellular matrix components, myofibroblast differentiation, migration, and contractility. Müller cells of the MIO-M1 lineage were cultured and treated for different periods with the inhibitor. Viability was evaluated by MTT assay and trypan blue exclusion method, and growth was evaluated by growth curve and BrdU incorporation assay. The actin cytoskeleton was stained with fluorescent phalloidin, intermediate filaments and microtubules were analyzed with immunofluorescence for vimentin and α-tubulin. Gene and protein expression of collagens I and V, laminin and fibronectin were evaluated by rt-PCR and immunofluorescence. Chemotactic and spontaneous cell migration were studied by transwell assay and time-lapse observation of live cells, respectively. Cell contractility was assessed by collagen gel contraction assay. The results showed that ROCK inhibition by Y27632 did not affect cell viability, but decreased cell growth and proliferation after 72 h. There was a change in cell morphology and organization of F-actin, with a reduction in the cell body, disappearance of stress fibers and formation of long, branched cell extensions. Microtubules and vimentin filaments were also affected, possibly because of F-actin alterations. The inhibitor also reduced gene expression and immunoreactivity of smooth muscle α-actin, a marker of myofibroblasts. The expression of extracellular matrix components was not affected by the inhibitor. Chemotactic cell migration showed no significant changes, while cell contractility was substantially reduced. No spontaneous migration of MIO-M1 cells was observed. In conclusion, pharmacological inhibition of ROCK in Müller cells could be a potentially promising approach to treat epiretinal membranes by preventing cell proliferation, contractility and transdifferentiation, without affecting cell viability.

Radial artery spasms impair invasive cardiological procedures' performance: a pharmacological approach to prevention and treatment.

The radial artery (RA) access is currently the gold standard to perform cardiovascular interventions. One of the more common limitations is radial artery spasm which is an often complication interrupting the procedure. Common risk factors associated with spasm include female gender, periprocedural anxiety, multiple puncture attempts, distal radial access, diabetes, hypertension, and smoking. The mechanism of spasm is complex and includes calmodulin and rho-kinase pathways leading to the smooth muscle contraction. Proper hydration, anxiety management, and adequate local anesthesia should be applied to decrease the risk of spasms. Radial cocktail is often used to prevent spasm. Its composition differs between catheterization laboratories and the effect is attributed either to the verapamil or nitroglycerin, with contradictory results of different studies. Balbay maneuver is also an effective mean of prevention. Hydrophilic-coated devices can be used both to avoid spasms or reverse them. Radial angiography can be used to differentiate spasm from a tortuosity and choose proper method of management. Fasudil, a Rho-kinase inhibitor, has been reported as a pharmacological method to prevent spasm and reverse radial artery spasm.

Role of Rho Kinase in Regulating Arterial Stiffness in Anesthetized Rabbits.

The effects of Rho kinase inhibitors fasudil and ripasudil on arterial stiffness were assessed using anesthetized rabbits, where the aortic β and femoral β were measured as a stiffness parameter at each arterial region. Intravenous administration of fasudil and ripasudil dose-dependently decreased blood pressure and femoral vascular resistance and increased femoral arterial blood flow, which appeared according to their in vitro potencies for Rho kinase inhibition. Both drugs increased the aortic β but decreased the femoral β at hypotensive doses. These results suggest that the inhibition of Rho kinase contributes to reducing elastic recoil in the aorta and an increase in compliance in the femoral artery. In addition, the Rho kinase-associated Ca2+-independent mechanism of arterial vascular smooth muscle contraction may be essential in the regulation of femoral arterial stiffness.

Use of a Tissue-Targeting Recombinant Human Interleukin-22 Fusion Molecule (F-652) for the Treatment of Advanced Refractory Lower GI Acute Gvhd

Background: Steroid-refractory (SR) gastrointestinal acute GVHD (GI aGVHD) is a serious complication after allogeneic hematopoietic transplantation (allo-HCT), and subsequent lines of treatment following steroid failure are often ineffective. The JAK 1/2 inhibitor ruxolitinib is the only FDA-approved treatment for SR aGVHD, and patients for whom ruxolitinib is not effective or not tolerated have limited therapeutic options and high mortality. Interleukin-22 (IL-22) is a tissue-protective IL-10-family cytokine that has been shown to support intestinal mucosa after damage, directly signaling to the intestinal epithelium and promoting its survival, regeneration, and production of innate antimicrobial molecules. F-652, a recombinant human (rh)IL-22 fusion protein composed of an IL-22 dimer and IgG2 Fc fragment, has recently been studied in a phase 2 trial for patients with newly diagnosed lower GI aGVHD, demonstrating a promising day 28 treatment response rate and improved gut microbial health. Given this, we examined F-652 administration in 3 patients with severe SR GI aGVHD. Methods: Three patients with hematologic malignancies underwent MUD allo-HCT with tacrolimus and methotrexate GVHD prophylaxis ( Table). The patients were initially treated for GI aGVHD with methylprednisolone at a dose of 2 mg/kg/day without response. All patients presented with or progressed to stage 4 gut aGVHD and failed several lines of therapy, including ruxolitinib. All patients also had multiple episodes of bacteremia and at least some degree of GVHD-associated GI bleeding. F-652 treatment was initiated under single-patient-use authorization as the 5 th-7 th line of therapy and was administered at a dose of 45 mcg/kg IV weekly. In vitro cultures suggested that rho kinase inhibition did not interfere with trophic effects of IL-22 on intestinal epithelium, so patients were permitted to continue belumosidil upon F-652 initiation. While there was concern that JAK inhibition could compromise the activity of IL-22, continuation of ruxolitinib was considered on a case-by-case basis. C-reactive protein (CRP) is an acute phase reactant induced by IL-22, and circulating CRP levels served as a pharmacodynamic marker of in vivo biologic activity of F-652. Results: Consistent CRP elevations were observed 3 days after F-652 dosing in patients A and C, both of whom demonstrated a reduction in GVHD symptoms following F-652 treatment. These CRP elevations occurred despite concurrent use of belumosidil in Patient A and ruxolitinib in Patient C. CRP levels increased in Patient B after the first two doses, but not the third ( Figure). By day 28 following F-652 initiation, 2-of-3 patients (A and C) achieved a partial response, with decreased stool output and resolution of GI bleeding. Patient A had experienced 21 episodes of bacteremia prior to initiating rhIL-22 therapy but continued to demonstrate a partial response at day 56 and resolutiuon of bacteremias. Further improvement during the continued treatment (12 total doses) allowed for hospital discharge after 14 months of hospitalization. Patient B received 4 failed lines of therapy beyond ruxolitinib and was experiencing GI bleeding necessitating frequent transfusions prior to F-652 initiation. The symptoms persisted following F-652 treatment, and the patient passed at day 149 post-HCT. Patient C continues on active F-652 treatment and remains in a partial response as of this submission (> 50 days after treatment initiation). Overall, F-652 treatment has been well tolerated, and no significant adverse events attributable to the drug were observed. Conclusions: This is the first report of IL-22 therapy in SR aGVHD. Treatment was well tolerated, and 2-of-3 patients achieved a response following rhIL-22 administration after failing multiple previous therapies. Whereas most treatments for GVHD target immune cells and deepen immunosuppression, IL-22 is understood to act directly upon epithelial cells where it can support tissue recovery, improve barrier function, and promote innate antimicrobial immunity. CRP elevations post-treatment suggest that IL-22 administration may be able to exert these effects even with concurrent use of a JAK inhibitor. Our findings support further development of this approach and provide proof-of-concept for the use of tissue-supportive strategies to enhance the recovery of damaged gastrointestinal mucosa in advanced lower GI aGVHD.