Application Of Y-27632 Research Articles

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.

ROCK inhibitor increases proacinar cells in adult salivary gland organoids.

Salisphere-derived adult epithelial cells have been used to improve saliva production of irradiated mouse salivary glands. Importantly, optimization of the cellular composition of salispheres could improve their regenerative capabilities. The Rho Kinase (ROCK) inhibitor, Y27632, has been used to increase the proliferation and reduce apoptosis of progenitor cells grown in vitro. In this study, we investigated whether Y27632 could be used to improve expansion of adult submandibular salivary epithelial progenitor cells or to affect their differentiation potential in different media contexts. Application of Y27632 in medium used previously to grow salispheres promoted expansion of Kit+ and Mist1+ cells, while in simple serum-containing medium Y27632 increased the number of cells that expressed the K5 basal progenitor marker. Salispheres derived from Mist1CreERT2; R26TdTomato mice grown in salisphere media with Y27632 included Mist1-derived cells. When these salispheres were incorporated into 3D organoids, inclusion of Y27632 in the salisphere stage increased the contribution of Mist1-derived cells expressing the proacinar/acinar marker, Aquaporin 5 (AQP5), in response to FGF2-dependent mesenchymal signals. Optimization of the cellular composition of salispheres and organoids can be used to improve the application of adult salivary progenitor cells in regenerative medicine strategies.

The effects of Y-27632 on pial microvessels during global brain ischemia and reperfusion in rabbits

BackgroundGlobal brain ischemia-reperfusion during propofol anesthesia provokes persistent cerebral pial constriction. Constriction is likely mediated by Rho-kinase. Cerebral vasoconstriction possibly exacerbates ischemic brain injury. Because Y-27632 is a potent Rho-kinase inhibitor, it should be necessary to evaluate its effects on cerebral pial vessels during ischemia—reperfusion period. We therefore tested the hypotheses that Y-27632 dilates cerebral pial arterioles after the ischemia-reperfusion injury, and evaluated the time-course of cerebral pial arteriolar status after the ischemia-reperfusion.MethodsJapanese white rabbits were anesthetized with propofol, and a closed cranial window inserted over the left hemisphere. Global brain ischemia was produced by clamping the brachiocephalic, left common carotid, and left subclavian arteries for 15 min. Rabbits were assigned to cranial window perfusion with: (1) artificial cerebrospinal fluid (Control group, n = 7); (2) topical infusion of Y-27632 10−6 mol · L−1 for 30 min before the initiation of global brain ischemia (Pre group, n = 7); (3) topical infusion of Y-27632 10−6 mol · L−1 starting 30 min before ischemia and continuing throughout the study period (Continuous group, n = 7); and, (4) topical infusion of Y-27632 10−6 mol · L−1 starting 10 min after the ischemia and continuing until the end of the study (Post group, n = 7). Cerebral pial arterial and venule diameters were recorded 30 min before ischemia, just before arterial clamping, 10 min after clamping, and 5, 10, 20, 40, 60, 80, 100, and 120 min after unclamping.ResultsMean arterial blood pressure and blood glucose concentration increased significantly after global brain ischemia except in the Continuous group. In the Pre and Continuous groups, topical application of Y-27632 produced dilation of large (mean 18–19%) and small (mean; 25–29%) pial arteries, without apparent effect on venules. Compared with the Control and Pre groups, arterioles were significantly dilated during the reperfusion period in the Continuous and Post groups (mean at 120 min: 5–8% in large arterioles and 11–12% in small arterioles).ConclusionsY-27632 dilated cerebral pial arterioles during reperfusion. Y-27632 may enhance recovery from ischemia by preventing arteriolar vasoconstriction during reperfusion.

ROCK inhibitor primes human induced pluripotent stem cells to selectively differentiate towards mesendodermal lineage via epithelial-mesenchymal transition-like modulation

Robust control of human induced pluripotent stem cell (hIPSC) differentiation is essential to realize its patient-tailored therapeutic potential. Here, we demonstrate a novel application of Y-27632, a small molecule Rho-associated protein kinase (ROCK) inhibitor, to significantly influence the differentiation of hIPSCs in a lineage-specific manner. The application of Y-27632 to hIPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration and time-dependent manner. Such changes in cell and colony morphology were associated with decreased expression of E-cadherin, a cell-cell junctional protein, proportional to the increased exposure to Y-27632. Interestingly, gene and protein expression of pluripotency markers such as NANOG and OCT4 were not downregulated by an exposure to Y-27632 up to 36h. Simultaneously, epithelial-to-mesenchymal (EMT) transition markers were upregulated with an exposure to Y-27632. These EMT-like changes in the cells with longer exposure to Y-27632 resulted in a significant increase in the subsequent differentiation efficiency towards mesendodermal lineage. In contrast, an inhibitory effect was observed when cells were subjected to ectodermal differentiation after prolonged exposure to Y-27632. Collectively, these results present a novel method for priming hIPSCs to modulate their differentiation potential with a simple application of Y-27632.

Sphingosine 1-Phosphate Causes Airway Hyper-Reactivity by Rho-Mediated Myosin Phosphatase Inactivation

In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyper-reactivity in bronchial asthma. The effects of S1P on the response to methacholine was examined in the fura-2-loaded strips of guinea pig tracheal smooth muscle using simultaneous recording of the isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F(340)/F(380)). A 15-min pretreatment with S1P (>100 nM) markedly enhanced methacholine-induced contraction without elevating F(340)/F(380). This effect of S1P was suppressed in the presence of Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide], a selective inhibitor of Rho-kinase, in a concentration-dependent manner. Moreover, pretreatment with pertussis toxin caused an inhibition in S1P-induced hyper-reactivity to methacholine in a time- and concentration-dependent manner. In contrast, although S1P-induced Ca(2+) mobilization was attenuated by SKF96365 and verapamil, the subsequent response to methacholine was unaffected. A 15-min pretreatment with lower concentrations of S1P (<100 nM), which is clinically attainable, did not increase methacholine-induced contraction. However, when the incubation was lengthened to 6 h, S1P (<100 nM) enhanced the subsequent response to methacholine. Next, application of S1P to cultured human bronchial smooth muscle cells increased the proportion of active RhoA (GTP-RhoA) and phosphorylation of myosin phosphatase target subunit 1 (MYPT1). This phosphorylation of MYPT1 was significantly inhibited by application of Y-27632 and by pretreatment with pertussis toxin. Our findings demonstrate that exposure of airway smooth muscle to S1P results in airway hyper-reactivity mediated by Ca(2+) sensitization via inactivation of myosin phosphatase, which links G(i) and RhoA/Rho-kinase processes.

Rho-Kinase Inhibition Enhances Axonal Plasticity and Attenuates Cold Hyperalgesia after Dorsal Rhizotomy

Dorsal rhizotomy results in primary deafferentation of the dorsal horn with concomitant sprouting of spared intraspinal monoaminergic axons. Because descending monoaminergic systems are thought to mitigate nociceptive transmission from the periphery and because dorsal rhizotomy can result in neuropathic pain, we sought to determine whether the rhizotomy-induced sprouting response could be further augmented. Because myelin-derived molecules mask endogenous plasticity of CNS axons and because myelin-inhibitory signaling occurs through the Rho-GTPase pathway, we inhibited Rho-pathway signaling after cervical dorsal rhizotomy in rats. An increase in the density of serotonergic- and tyrosine hydroxylase-positive fibers was seen in the dorsal horn 1 week after septuple rhizotomy, and axon density continued to increase for at least 1 month. One week after septuple rhizotomy, administration of intrathecal Y-27632, an antagonist of Rho-kinase (ROCK), increased the density of both fiber types over vehicle-treated controls. To examine behavioral effects of both cervical rhizotomy and ROCK inhibition, we examined responses to evoked pain: mechanical and thermal allodynia and cold hyperalgesia in the forepaw were examined after single, double, and quadruple rhizotomies of dorsal roots of the brachial plexus. The most notable behavioral outcome was the development of cold hyperalgesia in the affected forepaw after rhizotomies of the C7 and C8 dorsal roots. Application of Y-27632 both attenuated cold hyperalgesia and induced monoaminergic plasticity after C7/8 rhizotomy. Thus, inhibition of Rho-pathway signaling both promoted the sprouting of intact supraspinal monoaminergic fibers and alleviated pain after dorsal rhizotomy.

Inhibition of sustained hypoxic vasoconstriction by Y-27632 in isolated intrapulmonary arteries and perfused lung of the rat.

We have examined the effects of Y-27632, a specific inhibitor of Rho-activated kinases (ROCK I and ROCK II) upon sustained hypoxic pulmonary vasoconstriction (HPV) in both rat isolated small intrapulmonary arteries (IPA) and perfused rat lungs in situ. Y-27632 (100 nM - 3 microM) was found to cause a concentration-dependent inhibition of acute sustained HPV in rat IPA. Application of Y-27632 (10-600 nM) in perfused rat lungs caused no change in basal perfusion pressure, but was found to inhibit HPV in a concentration-dependent manner, resulting in complete ablation of the pressor response to hypoxia at a concentration of 600 nM. Furthermore, addition of Y-27632 at any point during hypoxia caused a reversal of HPV in perfused rat lungs. These results suggest that activation of Rho-associated kinase may be a pivotal step in the generation of sustained HPV.