Y-27632 acts beyond ROCK inhibition to maintain epidermal stem-like cells in culture.

Conditional reprogramming is a cell culture technique that effectively immortalizes epithelial cells with normal genotypes by renewing epidermal stem cells. Y-27632, a compound that promotes conditional reprogramming through an unknown mechanism, was developed to inhibit the two Rho-associated kinase (ROCK) isoforms. We used human foreskin keratinocytes (HFKs) to study the role of Y-27632 in conditional reprogramming and learn how ROCKs control epidermal stem cell renewal. In conditional reprogramming, Y-27632 increased HFK adherence to culture dishes, progression through S, G2 and M phases of the cell cycle, and epidermal stem cell marker levels. Although this correlated with ROCK inhibition by Y-27632, we generated CRISPR-Cas9-mediated HFK ROCK knockouts to test the direct role of ROCK inhibition. Knockout of single ROCK isoforms was insufficient to disrupt ROCK activity or promote HFK propagation without Y-27632. Although ROCK activity was reduced, HFKs with double knockout of ROCK1 and ROCK2 still required Y-27632 to propagate. Y-27632 was the most effective among the ROCK inhibitors we tested at promoting HFK proliferation and epidermal stem cell marker expression. Thus, the ability of Y-27632 to promote an epidermal stem cell state in conditional reprogramming not only depends upon ROCK inhibition but also acts via as-yet-unidentified mechanisms. Epidermal stem cell renewal might in part be regulated by ROCKs, but also involves additional pathways.

Rho-kinase inhibitor alleviates CD4+T cell mediated corneal graft rejection by modulating its STAT3 and STAT5 activation.

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.

Tunneling nanotubes-based intercellular mitochondrial trafficking as a novel therapeutic target in dry eye.

Cell-to-cell mitochondria transfer via tunneling nanotubes (TNTs) has recently been revealed as a spontaneous way to protect damaged cells. Previously, we have reported mesenchymal stem cells (MSCs) can rescue retinal ganglion cell and corneal epithelium through intercellular mitochondrial trafficking. Mitochondrial damage and oxidative stress in corneal epithelial cells are vital in dry eye disease (DED). However, whether intercellular mitochondrial transfer is involved in the pathological and repair process of DED is currently unknown. Therefore, in this study, we designed a coculture system to evaluate the role of intercellular mitochondrial transfer between human corneal epithelial cells (CEC) in DED. In addition, we successfully discovered the ROCK inhibitor, Y-27632 as an intensifier to improve the efficiency of intercellular mitochondrial transport. As expected, the enhanced mitochondrial transfer promotes the regeneration of CECs. Moreover, through further exploration of mechanisms, it was demonstrated that F-actin-mediated cell morphological changes and cytoskeletal remodeling may be potential mechanisms for Y-27632 to induce mitochondrial metastasis. In conclusion, we established a new method for cell repair in DED that healthy CEC offered mitochondria to damaged CEC, providing a new insight into the cellular mechanism of corneal epithelium homeostatic regenerative therapeutics in DED.

Persistent Escherichia coli infection in renal tubular cells enhances calcium oxalate crystal-cell adhesion by inducing ezrin translocation to apical membranes via Rho/ROCK pathway.

Recent evidence has suggested that recurrent urinary tract infection (UTI) can cause not only infection stones but also metabolic stones (e.g., those containing calcium oxalate monohydrate or COM). However, precise mechanisms underlying UTI-induced metabolic stones remained unknown. In this study, Escherichia coli, the most common bacterium found in recurrent UTI was used to establish the in vitro model for persistent infection of renal epithelial cells. The promoting effects of persistent E. coli infection on kidney stone formation were validated by COM crystal-cell adhesion assay, followed by immunofluorescence study for changes in surface expression of the known COM crystal receptors. Among the five receptors examined, only ezrin had significantly increased level on the surface of persistently infected cells without change in its total level. Such translocation of ezrin to apical membranes was confirmed by Western blotting of apical membrane and cytosolic fractions and confocal microscopic examination. Additionally, persistent infection increased phosphorylation (Thr567) of ezrin. However, all of these changes induced by persistent E. coli infection were significantly inhibited by small-interfering RNA (siRNA) specific for ezrin or a Rho-associated kinase (ROCK)-specific inhibitor (Y-27632). In summary, this study provides a piece of evidence demonstrating that persistent infection by E. coli, one of the non-urease-producing bacteria, may contribute to COM metabolic stone formation by translocation of ezrin to apical membranes, thereby promoting COM crystal-cell adhesion. Such ezrin translocation was mediated via Rho/ROCK signaling pathway. These findings may, at least in part, explain the pathogenic mechanisms underlying recurrent UTI-induced metabolic kidney stone disease.

TNF-α Plus IL-1ß Induces Opposite Regulation of Cx43 Hemichannels and Gap Junctions in Mesangial Cells through a RhoA/ROCK-Dependent Pathway.

Connexin 43 (Cx43) is expressed in kidney tissue where it forms hemichannels and gap junction channels. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remains unknown. Here, analysis of ethidium uptake and thiobarbituric acid reactive species revealed that treatment with TNF-α plus IL-1ß increases Cx43 hemichannel activity and oxidative stress in MES-13 cells (a cell line derived from mesangial cells), and in primary mesangial cells. The latter was also accompanied by a reduction in gap junctional communication, whereas Western blotting assays showed a progressive increase in phosphorylated MYPT (a target of RhoA/ROCK) and Cx43 upon TNF-α/IL-1ß treatment. Additionally, inhibition of RhoA/ROCK strongly antagonized the TNF-α/IL-1ß-induced activation of Cx43 hemichannels and reduction in gap junctional coupling. We propose that activation of Cx43 hemichannels and inhibition of cell-cell coupling during pro-inflammatory conditions could contribute to oxidative stress and damage of mesangial cells via the RhoA/ROCK pathway.

Combined therapy (Rho-A-kinase inhibitor and chitosan/collagen porous scaffold) provides a supportive environment for endogenous regenerative processes after spinal cord trauma.

Due to the complexity of pathological processes in spinal cord injury (SCI), it is increasingly recognized that combined strategies are more effective than single treatments. The aim of the present study was to enhance neural tissue regeneration and axon regrowth using Rho-A-kinase inhibitor (Y-27632) in a rat SCI model (Th9 compression) and to bridge the lesion with a chitosan/collagen porous scaffold (ChC-PS) applied two weeks after SCI. In addition, to see the synergic effect of Y-27632 and ChC-PS, we combined these single therapeutic strategies to enhance the regenerative capacity of injured spinal cord tissue. The animals survived eight weeks. Application of Y-27632 modulated the inhibitory milieu by specifically targeting gray and white matter integrity, glial fibrillary acidic protein (GFAP)-immunoreactivity, and the outgrowth of neurofilaments and growth-associated protein-43 (GAP-43) immunoreactive axons across the lesion sites, leading to significant positive functional outcome from day 20 to 56. Compared to single treatments, combined Y-27632/ChC-PS therapy was more effective in neurofilaments and GAP-43 expression and GFAP immunoreactivity in the perilesional area of dorsal, lateral and ventral columns, and in enhancing the gray and white matter integrity throughout the cranio-caudal extent. The findings indicate that combined therapy provides a supportive environment for endogenous regenerative processes.

Comparative culture of human corneal endothelial cells following treatment with human platelet lysate/fibrin hydrogel versus Y-27632 ROCK inhibitor: in vitro and ex vivo study.

PURPOSE: The advancement of tissue engineering and cell therapy research has resulted in innovative therapeutic options for patients with corneal endothelial diseases. The aim of this study was to compare the potential effect of using human platelet lysate (HPL)/Fibrin hydrogel versus using a Y-27632 ROCK inhibitor, on the culture of human corneal endothelial cells (HCECs) under in vitro and ex vivo conditions. METHODS: HCECs were isolated from human donors and treated separately with HPL/Fibrin hydrogel, a Y-27632 ROCK inhibitor, and fetal bovine serum (FBS). MTT viability assay and cell counting were performed on the treated cells. Subsequently, we prepared ex vivo models of human corneal endothelial dysfunction and incubated them with DiI-labeled-HCECs. Specular and fluorescence microscopy were then performed on each of the ex vivo models. RESULTS: In comparison, similar viability results were achieved in the cells treated with HPL/Fibrin hydrogel versus those treated with the Y-27632 ROCK inhibitor, but both treatments showed higher viability than the control group (FBS). More importantly, based on the specular and fluorescence microscopic results, the HPL/Fibrin hydrogel and the Y-27632 ROCK inhibitor treatments showed similar inducible effects on the attachment of the cells to the Descemet membranes of the ex vivo models. CONCLUSION: HPL/Fibrin hydrogel and Y-27632 ROCK inhibitor have similar inducible effects on the viability and attachment of the HCECs. A definite advantage of treating cells with HPL/Fibrin hydrogel is that it serves as a xeno-free and biocompatible material which can have autologous applications in future usage by clinics.

ROCK inhibitor attenuates carbon blacks-induced pulmonary fibrosis in mice via Rho/ROCK/NF-kappa B pathway.

Exposure to carbon blacks (CBs) has been associated with the progression of pulmonary fibrosis, whereas the mechanism is still not clear. We therefore aimed to investigate the effect of RhoA/ROCK pathway on pulmonary fibrosis caused by CBs exposure. Western blot analysis indicated that CBs could promote the activation of RhoA/ROCK pathway and phosphorylation of p65 and IκBα in mice lung. However, ROCK inhibitor Y-27632 could attenuate phosphorylation levels of p65 and IκBα and restore histopathological changes of the lung tissue. Then, we evaluated the effect of RhoA/ROCK pathway on pulmonary fibrosis by detecting the expression levels of α-SMA, vimentin, and Collagen type-I (Col-I), which could be partly inhibited by Y-27632. It was assumed that inhibition of ROCK could be a promising therapeutic candidate for CBs-induced pulmonary fibrosis, which possibly through the blockage of RhoA/ROCK/NF-κB pathway.

Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632.

Cultured keratinocytes are desirable models for biological and medical studies. However, primary keratinocytes are difficult to maintain, and there has been little research on lingual keratinocyte culture. Here, we investigated the effect of Y-27632, a Rho kinase (ROCK) inhibitor, on the immortalization and characterization of cultured rat lingual keratinocyte (RLKs). Three Y-27632-supplemented media were screened for the cultivation of RLKs isolated from Sprague-Dawley rats. Phalloidin staining and TUNEL assay were applied to visualize cytoskeleton dynamics and cell apoptosis following Y-27632 removal. Label-free proteomics, RT-PCR, calcium imaging, and cytogenetic studies were conducted to characterize the cultured cells. Results showed that RLKs could be conditionally immortalized in a high-calcium medium in the absence of feeder cells, although they did not exhibit normal karyotypes. The removal of Y-27632 from the culture medium led to reversible cytoskeletal reorganization and nuclear enlargement without triggering apoptosis, and a total of 239 differentially expressed proteins were identified by proteomic analysis. Notably, RLKs derived from the non-taste epithelium expressed some molecular markers characteristic of taste bud cells, yet calcium imaging revealed that they rarely responded to tastants. Collectively, we established a high-calcium and feeder-free culture method for the long-term maintenance of RLKs. Our results shed some new light on the immortalization and differentiation of lingual keratinocytes.

Synergistic effects of Rho kinase inhibitor Y-27632 and Noggin overexpression on the proliferation and neuron-like cell differentiation of stem cells derived from human exfoliated deciduous teeth.

Stem cells from human exfoliated deciduous teeth (SHEDs) are highly proliferative, clonogenic, and multipotent stem cells with a neural crest cell origin. This property could be a desirable option for potential therapeutic applications. In this study, we focus on the effects of Rho kinase inhibitors Y-27632 and Noggin on the proliferation of SHEDs and their differentiation into neuron-like cells. SHEDs were extracted from 10 samples of deciduous teeth obtained from healthy children aged from 5 to 10. The passaged SHEDs were transfected with Noggin, Y-27632, or their combination. By means of MTT and colony formation assays, the effects of Y-27632 and Noggin on cell viability and colony formation were detected. Cellular morphology and neurosphere formation were observed under a microscope. Y-27632 transfection in SHEDs showed enhanced cell viability, colony formation, and neurosphere formation indicating that Y-27632 could promote cell proliferation of SHEDs. Furthermore, we observed that the SHEDs treated with Noggin in combination with Y-27632 displayed typical neuron-like cell morphology and reticular processes. Noggin or Y-27632 alone or in combination induced obviously increased NSE, Nestin, and GFAP levels, which were highest in SHEDs treated with the combination of Noggin and Y-27632. These findings suggest that Y-27632 promotes the proliferation of SHEDs, and Y-27632 and Noggin in combination have a synergistic effect on promoting differentiation of SHEDs into neuron-like cells.

Enhanced Vascular Smooth Muscle Calcium Sensitivity and Loss of Endothelial Vasodilator Influence Contribute to Myogenic Tone Development in Rat Radial Uterine Arteries during Gestation.

Uterine artery myogenic tone (MT) develops during pregnancy in hemochorial placentates such as rats and humans. The physiological reason for its appearance is not clear, and we reasoned that it may be a late pregnancy (LP) event in preparation for controlling hemorrhage during parturition. We also hypothesized that gestational increases in RhoA-induced vascular smooth muscle (VSM) calcium sensitivity are contributory and occur under the tonic influence of nitric oxide (NO). Second-order pre-placental radial arteries from early-pregnant (day 12, n = 5), mid-pregnant (day 16, n = 5) and LP (day 20, n = 20) rats were used in combination with arteriography, VSM calcium measurements, pharmacological RHO/Rho-associated protein kinase (ROCK) and nitric oxide synthase (NOS) inhibition, and Western blotting. A subgroup of LP animals (LP + LN; n = 5) treated with L-NAME from gestational days 10 to 20 were used to determine the effects of NOS inhibition on MT and RhoA expression. MT was evident throughout pregnancy, but its expression in pressurized vessels was masked by endothelial NO-induced vasodilation during early gestation. RhoA protein expression was upregulated in LP and attenuated by in vivo NOS inhibition (as was MT). In vitro RHO/ROCK inhibition decreased MT in a concentration-dependent manner without reducing VSM calcium. In summary, pressure-dependent uterine artery tone increases with gestational age due to a combination of RhoA-mediated increases in VSM calcium sensitivity and a loss of endothelial NO influence.

Teratogenesis in the chick embryo following post-gastrulation exposure to Y-27632 -effect of Y-27632 on embryonic development.

The pyridine derivative Y-27632 inhibits Rho-associated coiled-coil-containing protein kinase (ROCK) signaling, which is involved in numerous developmental processes during embryogenesis, primarily by controlling actin-cytoskeleton assembly and cell contractility. Somite formation requires rearrangement of the cytoskeleton and assists in major morphological mechanisms, including ventral body wall formation. Administration of Y-27632 impairs cytoskeletal arrangements in post-gastrulation chick embryos leading to ventral body wall defects (VBWD) at later stages of development. The aim of this study was to investigate the effect of Y-27632 on somite development in post-gastrulation chick embryos during early embryogenesis. After 60 h incubation, embryos in shell-less culture were treated with Y-27632 or vehicle for controls. Following administration, abnormality rates were assessed. In treatment groups, embryos showed a kinked longitudinal body axis. Western blot confirmed impaired ROCK downstream signaling by decreased expression of phosphorylated cofilin-2. Histology, Lysotracker studies and RT-PCR demonstrated increased cell death in somites, the neural tube and the ectoderm. RT-PCR and Western blot of factors known to be involved during somitogenesis revealed reduced expression in the treatment group compared to controls. We hypothesize that administration of Y-27632 disrupts somite development causing axial kinking and embryo malformation, which may lead to VBWD.

Should we keep rocking? Portraits from targeting Rho kinases in cancer.

Cancer targeted therapy, either alone or in combination with conventional chemotherapy, could allow the survival of patients with neoplasms currently considered incurable. In recent years, the dysregulation of the Rho-associated coiled-coil kinases (ROCK1 and ROCK2) has been associated with increased metastasis and poorer patient survival in several tumor types, and due to their essential roles in regulating the cytoskeleton, have gained popularity and progressively been researched as targets for the development of novel anti-cancer drugs. Nevertheless, in a pediatric scenario, the influence of both isoforms on prognosis remains a controversial issue. In this review, we summarize the functions of ROCKs, compile their roles in human cancer and their value as prognostic factors in both, adult and pediatric cancer. Moreover, we provide the up-to-date advances on their pharmacological inhibition in pre-clinical models and clinical trials. Alternatively, we highlight and discuss detrimental effects of ROCK inhibition provoked not only by the action on off-targets, but most importantly, by pro-survival effects on cancer stem cells, dormant cells, and circulating tumor cells, along with cell-context or microenvironment-dependent contradictory responses. Together these drawbacks represent a risk for cancer cell dissemination and metastasis after anti-ROCK intervention, a caveat that should concern scientists and clinicians.

Acute lung injury: The therapeutic role of Rho kinase inhibitors.

Acute lung injury (ALI) is a pulmonary illness with high rates of mortality and morbidity. Rho GTPase and its downstream effector, Rho kinase (ROCK), have been demonstrated to be involved in cell adhesion, motility, and contraction which can play a role in ALI. The electronic databases of Google Scholar, Scopus, PubMed, and Web of Science were searched to obtain relevant studies regarding the role of the Rho/ROCK signaling pathway in the pathophysiology of ALI and the effects of specific Rho kinase inhibitors in prevention and treatment of ALI. Upregulation of the RhoA/ROCK signaling pathway causes an increase of inflammation, immune cell migration, apoptosis, coagulation, contraction, and cell adhesion in pulmonary endothelial cells. These effects are involved in endothelium barrier dysfunction and edema, hallmarks of ALI. These effects were significantly reversed by Rho kinase inhibitors. Rho kinase inhibition offers a promising approach in ALI [ARDS] treatment.

Staphylococcus aureus impairs sinonasal epithelial repair: Effects in patients with chronic rhinosinusitis with nasal polyps and control subjects.

BACKGROUND: The effect of Staphylococcus aureus on nasal epithelial repair has never been assessed in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). OBJECTIVE: This study aimed to determine whether (1) nasal epithelial cell cultures from patients with CRSwNP and control subjects repair differently; (2) S aureus exoproducts compromise nasal epithelial repair; (3) S aureus alters lamellipodial dynamics; and (4) deleterious effects could be counteracted by the Rho-associated coiled-coil kinase inhibitor Y-27632. METHODS: Primary nasal epithelial cells (pNECs) collected during surgeries were cultured and injured under 3 conditions: (1) basal conditions, (2) exposed to S aureus exoproducts, and (3) exposed to S aureus exoproducts and Y-27632. Epithelial repair, lamellipodial dynamics, and cytoskeletal organization were assessed. RESULTS: Under basal conditions, pNEC cultures from patients with CRSwNP presented significantly lower repair rates and reduced lamellipodial protrusion length and velocity than those from control subjects. S aureus exoproducts significantly decreased repair rates and protrusion dynamics in both control subjects and patients with CRSwNP; however, the effect of S aureus on cell protrusions was more sustained over time in patients with CRSwNP. Under basal conditions, immunofluorescence assays showed significantly reduced percentages of cells with lamellipodia at the wound edge in patients with CRSwNP compared with control subjects. S aureus altered cell polarity and decreased the percentage of cells with lamellipodia in both groups. Finally, Y-27632 prevented the deleterious effects of S aureus exoproducts on CRSwNP repair rates, as well as on lamellipodial dynamics and formation. CONCLUSIONS: S aureus exoproducts significantly alter epithelial repair and lamellipodial dynamics on pNECs, and this impairment was more pronounced in patients with CRSwNP. Importantly, Y-27632 restored epithelial repair and lamellipodial dynamics in the presence of S aureus exoproducts.

Y-27632 Induces Neurite Outgrowth by Activating the NOX1-Mediated AKT and PAK1 Phosphorylation Cascades in PC12 Cells.

Y-27632 is known as a selective Rho-associated coiled coil-forming kinase (ROCK) inhibitor. Y-27632 has been shown to induce neurite outgrowth in several neuronal cells. However, the precise molecular mechanisms linking neurite outgrowth to Y-27632 are not completely understood. In this study, we examined the ability of Y-27632 to induce neurite outgrowth in PC12 cells and evaluated the signaling cascade. The effect of Y-27632 on the neurite outgrowth was inhibited by reactive oxygen species (ROS) scavengers such as N-acetyl cysteine (NAC) and trolox. Furthermore, Y-27632-induced neurite outgrowth was not triggered by NADPH oxidase 1 (NOX1) knockdown or diphenyleneiodonium (DPI), a NOX inhibitor. Suppression of the Rho-family GTPase Rac1, which is under the negative control of ROCK, with expression of the dominant negative Rac1 mutant (Rac1N17) prevented Y-27632-induced neurite outgrowth. Moreover, the Rac1 inhibitor NSC23766 prevented Y-27632-induced AKT and p21-activated kinase 1 (PAK1) activation. AKT inhibition with MK2206 suppressed Y-27632-induced PAK1 phosphorylation and neurite outgrowth. In conclusion, our results suggest that Rac1/NOX1-dependent ROS generation and subsequent activation of the AKT/PAK1 cascade contribute to Y-27632-induced neurite outgrowth in PC12 cells.

Rho kinase inhibitors reduce voltage-dependent Ca2+ channel signaling in aortic and renal microvascular smooth muscle cells.

Voltage-dependent L-type Ca2+ channels (L-VDCCs) and the RhoA/Rho kinase pathway are two predominant intracellular signaling pathways that regulate renal microvascular reactivity. Traditionally, these two pathways have been thought to act independently; however, recent evidence suggests that these pathways could be convergent. We hypothesized that Rho kinase inhibitors can influence L-VDCC signaling. The effects of Rho kinase inhibitors Y-27632 or RKI-1447 on KCl-induced depolarization or the L-VDCC agonist Bay K8644 were assessed in afferent arterioles using an in vitro blood-perfused rat juxtamedullary nephron preparation. Superfusion of KCl (30-90 mM) led to concentration-dependent vasoconstriction of afferent arterioles. Administration of Y-27632 (1, 5, and 10 µM) or RKI-1447 (0.1, 1, and 10 µM) significantly increased the starting diameter by 16-65%. KCl-induced vasoconstriction was markedly attenuated with 5 and 10 µM Y-27632 and with 10 µM RKI-1447 (P < 0.05 vs. KCl alone). Y-27632 (5 µM) also significantly attenuated Bay K8644-induced vasoconstriction (P < 0.05). Changes in intracellular Ca2+ concentration ([Ca2+]i) were estimated by fura-2 fluorescence during KCl-induced depolarization in cultured A7r5 cells and in freshly isolated preglomerular microvascular smooth muscle cells. Administration of 90 mM KCl significantly increased fura-2 fluorescence in both cell types. KCl-mediated elevation of [Ca2+]i in A7r5 cells was suppressed by 1-10 µM Y-27632 (P < 0.05), but 10 µM Y-27632 was required to suppress Ca2+ responses in preglomerular microvascular smooth muscle cells. RKI-1447, however, significantly attenuated KCl-mediated elevation of [Ca2+]i. Y-27632 markedly inhibited Bay K8644-induced elevation of [Ca2+]i in both cell types. The results of the present study indicate that the Rho kinase inhibitors Y-27632 and RKI-1447 can partially inhibit L-VDCC function and participate in L-VDCC signaling.

The effect of rho kinase inhibition on morphological and electrophysiological maturity in iPSC-derived neurons.

Induced pluripotent stem cell (iPSC)-derived neurons permit the study of neurogenesis and neurological disease in a human setting. However, the electrophysiological properties of iPSC-derived neurons are consistent with those observed in immature cortical neurons, including a high membrane resistance depolarized resting membrane potential and immature firing properties, limiting their use in modeling neuronal activity in adult cells. Based on the proven association between inhibiting rho kinase (ROCK) and increased neurite complexity, we seek to determine if short-term ROCK inhibition during the first 1-2 weeks of differentiation would increase morphological complexity and electrophysiological maturity after several weeks of differentiation. While inhibiting ROCK resulted in increased neurite formation after 24 h, this effect did not persist at 3 and 6 weeks of age. Additionally, there was no effect of ROCK inhibition on electrophysiological properties at 2-3, 6, or 12 weeks of age, despite an increase in evoked and spontaneous firing and a more hyperpolarized resting membrane potential over time. These results indicate that while there is a clear effect of time on electrophysiological maturity, ROCK inhibition did not accelerate maturity.

Activation of SIRT1 ameliorates LPS-induced lung injury in mice via decreasing endothelial tight junction permeability.

The integrity of the endothelial barrier is a determinant of the prognosis of lipopolysaccharide (LPS)-induced acute lung injury (ALI). In this study, we investigated whether and how Sirtuin 1 (SIRT1) maintained the vascular integrity during ALI. An experimental model of ALI was established in mice through intratracheal administration of LPS (10 mg/kg). LPS stimulation significantly increased the pulmonary permeability and decreased the expression of SIRT1 and tight junction proteins (TJs), including occludin, claudin-5, tight junction protein 1 and tight junction protein 2. Morphological studies showed that LPS induced obvious lung injury with inflammatory cell infiltration in the interstitial and alveolar space, hemorrhage, edema, and the thickened alveolar wall compared to the control mice. Intratracheal administration of the selective SIRT1 activator SRT1720 (6.25 mg/kg) significantly attenuated LPS-induced lung injury, lung hyper-permeability and increased TJs expression, whereas intratracheal administration of the selective SIRT1 inhibitor EX527 (6.25 mg/kg) aggravated LPS-induced ALI. Similar protective effects of SIRT1 on pulmonary cellular permeability were observed in primary human pulmonary microvascular endothelial cells treated with LPS (2 mg/mL) in vitro. We further demonstrated that the RhoA/ROCK signaling pathway was activated in SIRT1 regulation of tight junction permeability. The RhoA/ROCK inhibitor Y-27632 (10 µM) increased the expression of TJs and reversed LPS- or EX527-induced hyper-permeability. In conclusion, SIRT1 ameliorates LPS-induced lung injury via decreasing endothelial tight junction permeability, possibly via RhoA/ROCK signaling pathway. This finding may contribute to the development of new therapeutic approaches for lung injury.

An in vitro study of scarring formation mediated by human Tenon fibroblasts: Effect of Y-27632, a Rho kinase inhibitor.

Scar formation is the most common cause for failure of glaucoma filtration surgery because of increased fibroblast proliferation and activation. We have now examined the effect of Y-27632, a Rho-associated protein kinase (ROCK) inhibitor, on postsurgical scarring formation in human Tenon fibroblasts (HTFs). Collagen gel contraction assay was used to compare contractility activity of Y-27632 with several antiglaucoma drugs. Immunofluorescence and western blotting were used to examine expression of scar formation-related factors. We found that Y-27632 inhibited collagen gel contraction, as well as α-smooth muscle actin and vimentin expression; these were promoted by treatment with latanoprost, timolol, or transforming growth factor (TGF)-ß. To investigate the effect of Y-27632 in postsurgical scarring, we mimicked TGF-ß secretion by stimulating HTFs with TGF-ß prior to Y-27632 treatment. HTFs cultured in the presence of TGF-ß significantly increased gel contraction. In contrast, when HTFs were treated with 10µM Y-27632, contraction was significantly inhibited. Furthermore, Y-27632 reduced TGF-ß-induced phosphorylation of mitogen-activated protein kinase signalling. These results suggest that ROCK inhibitors may inhibit fibrosis by inhibiting transdifferentiation of Tenon fibroblasts into myofibroblasts and by inhibiting TGF-ß signalling after surgery through mitogen-activated protein kinase pathway suppression. These results implicate that ROCK inhibitors may improve outcomes after filtering surgery with a potential antiscarring effect, while latanoprost and timolol may induce fibrosis. SIGNIFICANCE OF THE STUDY: Scar formation is the primary cause of failure after glaucoma filtration surgery. A ROCK inhibitor, Y-27632, has been introduced as a novel potential antiglaucoma treatment to reduce intraocular pressure. The aim of our study was to elucidate the effect of Y-27632 on scarring formation after glaucoma filtration surgery, in direct comparison with other antiglaucoma drugs. Our findings thus suggested that Y-27632 may inhibit fibrosis and improve outcome after glaucoma filtration surgery through inhibition of transdifferentiation of Tenon fibroblasts into myofibroblasts, and the TGF-ß and MAPK signalling after surgery, while latanoprost and timolol may induce fibrosis.