Rho-associated Coiled-coil Containing Protein Kinase Inhibitor Research Articles

Space microgravity improves proliferation of human iPSC-derived cardiomyocytes.

SummaryIn microgravity, cells undergo profound changes in their properties. However, how human cardiac progenitors respond to space microgravity is unknown. In this study, we evaluated the effect of space microgravity on differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiac progenitors compared with 1G cultures on the International Space Station (ISS). Cryopreserved 3D cardiac progenitors were cultured for 3 weeks on the ISS. Compared with 1G cultures, the microgravity cultures had 3-fold larger sphere sizes, 20-fold higher counts of nuclei, and increased expression of proliferation markers. Highly enriched cardiomyocytes generated in space microgravity showed improved Ca2+ handling and increased expression of contraction-associated genes. Short-term exposure (3 days) of cardiac progenitors to space microgravity upregulated genes involved in cell proliferation, survival, cardiac differentiation, and contraction, consistent with improved microgravity cultures at the late stage. These results indicate that space microgravity increased proliferation of hiPSC-cardiomyocytes, which had appropriate structure and function.

Renal ROCK Activation and Its Pharmacological Inhibition in Patients With Diabetes

Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine/threonine kinase with essential roles in cytoskeletal functions. Substantial evidence implicates ROCK as a critical regulator in the inception and progression of diabetic nephropathy through a mechanism involving mesangial fibrosis, podocyte apoptosis, and endothelial inflammation. Despite these experimental observations, human data is lacking. Here we show that the phosphorylated form of myosin phosphatase targeting subunit 1 (MYPT1), a ROCK substrate, was increased in both the glomerular and tubulointerstitial areas in patients with histologically confirmed diabetic nephropathy. We also conducted a retrospective pilot analysis of data from patients with diabetes to assess the renoprotective effects of fasudil, an ATP-competitive ROCK inhibitor licensed in Japan for the prevention of vasospasm following subarachnoid hemorrhage. Fifteen subjects (male, n = 8; female, n = 7; age 65.7 ± 14.7 years; body height, 161.1 ± 12.6 cm; body weight, 57.6 ± 13.7 kg; body mass index, 22.4 ± 3.7 kg/m2) were enrolled to evaluate blood pressure and the renal outcome after fasudil treatment. Of note, proteinuria was significantly reduced at the end of the fasudil treatment without affecting the blood pressure or estimated glomerular filtration rate. Taken together, these findings suggest that the administration of fasudil could be associated with a better renal outcome by inhibiting the ROCK activity in patients with diabetes.

Y-27632, a ROCK inhibitor, improved laser-induced shock wave (LISW)-induced cochlear synaptopathy in mice

Recently, a pathological condition called cochlear synaptopathy has been clarified, and as a disorder of the auditory nerve synapses that occurs prior to failure of hair cells, it has been recognized as a major cause of sensorineural hearing loss. However, cochlear synaptopathy is untreatable. Inhibition of rho-associated coiled-coil containing protein kinase (ROCK), a serine-threonine protein kinase, has been reported to have neuroprotective and regenerative effects on synaptic pathways in the nervous system, including those in the inner ear. We previously demonstrated the regenerative effect of the ROCK inhibitor, Y-27632, on an excitotoxic cochlear nerve damage model in vitro. In this study, we aimed to validate the effect of ROCK inhibition on mice with cochlear synaptopathy induced by laser-induced shock wave (LISW) in vivo. After the elevation of ROCK1/2 expression in the damaged cochlea was confirmed, we administered Y-27632 locally via the middle ear. The amplitude of wave I in the auditory brainstem response and the number of synapses in the Y-27632-treated cochlea increased significantly. These results clearly demonstrate that ROCK inhibition has a promising clinical application in the treatment of cochlear synaptopathy, which is the major pathology of sensorineural hearing loss.

ROCK inhibitors enhance the production of large lipid-enriched 3D organoids of 3T3-L1 cells

Since the recent discovery of prostaglandin-associated peri-orbitopathy, a great deal of interest has developed concerning the side effects of anti-glaucoma medications toward periocular fatty tissue, especially their adipogenesis. Two- or three-dimension (2D or 3D) cultures of the 3T3-L1 cells were employed to elucidate the effects of the Rho-associated coiled-coil containing protein kinase inhibitor (ROCK-i) the anti-glaucoma drug, Ripasudil, and other ROCK-i, such as Y27632 on adipogenesis. Ultrastructure by electron microscopy and physical stiffness measurements by a micro-squeezer demonstrated the 3D organoids had essentially matured during the 7-day culture. The effects of ROCK-i on 3D organoid sizes, lipid staining, the mRNA expression of adipogenesis related genes, Pparγ, Cebpa and Leptin, and extracellular matrix (ECM) including collagen (COL) 1, 4 and 6, and fibronectin, and physical stiffness were then conducted. Upon adipogenesis, the sizes, lipid staining and mRNA expressions of adipogenesis related genes, Col 4 and Col 6 were dramatically increased, and were further enhanced by ROCK-i. Micro-squeezer analysis demonstrated that adipogenesis resulted in a marked less stiffed 3D organoid and this was further enhanced by ROCK-i. Our present study indicates that ROCK-i significantly enhanced the production of large lipid-enriched 3T3-L1 3D organoids.

Prostaglandin F2α and EP2 agonists, and a ROCK inhibitor modulate the formation of 3D organoids of Grave's orbitopathy related human orbital fibroblasts

3D organoid cultures were used to elucidate the periocular effects of several anti-glaucoma drugs including a prostaglandin F2α analogue (bimatoprost acid; BIM-A), EP2 agonist (omidenepag; OMD) or a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor (ripasudil; Rip) on Grave's orbitopathy (GO) related orbital fatty tissue. 3D organoids were prepared from GO related human orbital fibroblasts (GHOFs) obtained from patients with GO. The effects of either 100 nM BIM-A, 100 nM OMD or 10 μM Rip on the 3D GHOFs organoids were examined with respect to organoid size, physical properties by a micro-squeezer, and the mRNA expression of extracellular matrix (ECM) proteins including collagen (COL) 1, COL 4, COL 6, and fibronectin (FN), ECM regulatory genes including lysyl oxidase (LOX), Connective Tissue Growth Factor (CTGF) and inflammatory cytokines including interleukin-1β (IL1β) and interleukin-6 (IL6). The size of the 3D GHOFs organoids decreased substantially in the presence of BIM-A, but also increased substantially in the presence of the others (OMD or Rip). The physical stiffness of the 3D GHOFs organoids was significantly decreased by Rip. BIM-A caused significantly the down-regulation of three ECM genes, Col 1, Col 6 and Fn, and two ECM regulatory genes and the up-regulation of IL6. In the presence of OMD, two ECM genes, Col 1 and Fn, and LOX were significantly down-regulated but IL1β and IL6 were significantly up-regulated. In the case of Rip, Col 1, FN and CTGF were significant down-regulated. Our present findings indicate that anti-glaucoma drugs modulate the structures and physical properties 3D GHOFs organoids in different manners by modifying the gene expressions of ECM, ECM regulatory factors and inflammatory cytokines. The results indicate that the benefits and demerits of anti-glaucoma medications need to be scrutinized carefully, in cases of patients with GO.

A visible light-controllable Rho kinase inhibitor based on a photochromic phenylazothiazole.

Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine kinase whose inhibitors are useful for the regulation of the actomyosin system. Here, we developed a photoswitchable ROCK inhibitor based on a phenylazothiazole scaffold. The reversible trans-cis isomerization by visible light stimuli enabled us to manipulate ROCK activities in vitro and in cells.

Regenerative Effect of a ROCK Inhibitor, Y-27632, on Excitotoxic Trauma in an Organotypic Culture of the Cochlea.

In the past, most inner ear diseases were thought to start with the impairment of the sensory epithelium of the cochlea before subsequently progressing to secondary neural degeneration. However, recent studies show that loss of primary synapses accompanied by excitotoxic degeneration of peripheral axons is likely to be the underlying pathology in sensorineural hearing loss. Rho-associated coiled-coil containing protein kinase (ROCK) inhibition has been reported to have neuroprotective and regenerative effects on synaptic pathways. Therefore, we analyzed the effect of ROCK inhibition using Y-27632 in a model of peripheral axonal damage in the spiral ganglion neurons created using the glutamate agonists, N-methyl-D-aspartate (NMDA) and kainic acid, to induce excitotoxic trauma in the explanted cochlea. The number of axons projecting to hair cells in the cochlea treated with Y-27632 was significantly greater than those in the cochlea treated only with NMDA + kainic acid. Furthermore, there was a significant increase in synapses between the spiral ganglion and the inner hair cells in the cochlea treated with Y-27632. The findings of this study suggest that ROCK inhibition could be a potential strategy for the regeneration of peripheral axons in the spiral ganglion and synapse formation in the inner hair cells of a cochlea that has sustained excitotoxic injury, which is one of the primary etiologies of inner ear disease.

Efficient primary culture model of patient‑derived tumor cells from colorectal cancer using a Rho‑associated protein kinase inhibitor and feeder cells.

Invitro culture of patient‑derived tumor cells offers many advantages in the development of novel therapies for colorectal cancer. Although various culture systems have been developed, the long‑term expansion of patient‑derived tumor cells remains challenging. The present results suggested that tumor cells isolated from colorectal cancer patient‑derived xenografts can be efficiently immortalized in conditioned medium from irradiated feeder cells containing Y‑27632, a rho‑associated coiled‑coil containing protein kinase (ROCK) inhibitor. Patient‑derived tumor cells proliferated rapidly, reaching 90‑95%confluence in ~6days. Short tandem repeat analysis suggested that these tumor tissues and cultured cells presented 13identical short tandem repeat loci, including Amelogenin, PentaE, PentaD, D2S1338 and D19S433. Their epithelial phenotype was confirmed by staining for epithelial cell adhesion molecule and cytokeratin20, whereas vimentin was used as a mesenchymal marker. When cells were transferred to 3Dcultures, they continued to proliferate, forming well‑defined tumor spheroids. Expression levels of human telomerase reverse transcriptase and C‑Myc mRNA were increased in cultured cells. Finally, immortalized cells were used for the screening of 65anticancer drugs approved by the Food and Drug Administration, allowing the identification of gene‑drug associations. In the present study, primary culture models of colorectal cancer were efficiently established using a ROCK inhibitor and feeder cells, and this approach could be used for personalized treatment strategies for patients with colorectal cancer.

ROCK inhibitor Y‑27632 protects rats against cerebral ischemia/reperfusion‑induced behavioral deficits and hippocampal damage.

Cerebral ischemic injury is a major cause of death and long‑term disability worldwide that leads to neurological and behavioral deficits, and for which successful treatments are still lacking. Ras homolog family member A (RhoA) and Rho‑associated coiled‑coil containing protein kinase (ROCK) are associated with the growth of neurons and the movement of neuronal growth cones. RhoA/ROCK inhibitors have been demonstrated to promote the recovery of motor function following nerve injury, but the underlying mechanism requires further investigation. The present study aimed to investigate the effects of the ROCK inhibitor Y‑27632 on middle cerebral artery occlusion (MCAO)‑induced cerebral ischemic injury. Rats were randomly assigned to the Control, Y‑27632, MCAO+Vehicle or MCAO+Y‑27632 group. Firstly, infarct volume, cognitive ability and cerebral injury were assessed. Secondly, indicators of cerebral inflammation, oxidative stress and apoptosis were evaluated. Finally, the expression of recombinant glial fibrillary acidic protein (GFAP) and allograft inflammatory factor1(AIF1) in the brain were measured to assess the activation of astrocytes and microglia, respectively. The results showed that Y‑27632 effectively increased the survival rate and behavioral performance of rats, and attenuated the cerebral injury, oxidative stress and cerebral inflammation levels following MCAO. The disturbance in hippocampal neurons caused by MCAO was also alleviated following treatment with Y‑27632. Neuronal apoptosis was also decreased following Y‑27632 treatment, as demonstrated by the TUNEL assay and the expression levels of Caspases‑3, 8 and 9 and Bax/Bcl‑2 ratio. The levels of GFAP and AIF1 were increased by MCAO and further promoted by Y‑27632, indicating the activation of astrocytes and microglia. In conclusion, the present study offered evidence of a protective effect of Y‑27632 administration on cerebral ischemia/reperfusion induced behavioral and hippocampal damage by activating astrocytes and microglia.

ROCK Inhibition Induces Terminal Adipocyte Differentiation and Suppresses Tumorigenesis in Chemoresistant Osteosarcoma Cells.

Tumors comprise heterogeneous cell types including cancer stem cells (CSC), progenitor cells, and differentiated cells. Chemoresistance is a potential cause of relapse and a key characteristic of CSC, but the development of novel therapeutic approaches for targeting these cells has been limited. We previously established osteosarcoma-initiating (OSi) cells by introducing the gene for c-Myc into bone marrow stromal cells of Ink4a/Arf knockout mice. These OSi cells are composed of two distinct clones: highly tumorigenic cells (AX cells), similar to bipotent committed osteochondral progenitor cells, and tripotent cells of low tumorigenicity (AO cells), similar to mesenchymal stem cells. Here we show that depolymerization of the actin cytoskeleton induces terminal adipocyte differentiation and suppresses tumorigenesis in chemoresistant OSi cells. In contrast to AX cells, AO cells were highly resistant to conventional chemotherapeutic agents such as doxorubicin and were thus identified as chemoresistant cells. Inhibition of Rho-associated coiled-coil containing protein kinase (ROCK) elicited terminal adipocyte differentiation in chemoresistant AO cells through negative regulation of the transcriptional coactivator megakaryoblastic leukemia 1 associated with actin depolymerization. The clinically administered ROCK inhibitor fasudil significantly suppressed growth in vitro and tumorigenicity in vivo of chemoresistant AO cells as well as of OSi cells. Our findings thus suggest a new therapeutic strategy based on the induction of trans-terminal differentiation via modulation of actin cytoskeleton dynamics for therapy-resistant osteosarcoma stem cells. SIGNIFICANCE: These findings suggest that induction of trans-terminal differentiation through regulation of actin dynamics is a potential novel therapeutic approach for targeting chemoresistant stem-like tumor cells.

Targeting ROCK/LIMK/cofilin signaling pathway in cancer.

Rho-associated coiled-coil-containing protein kinase (ROCK)/Lin11, Isl-1 and Mec-3 kinase (LIMK)/cofilin-signaling cascades are stimulated by receptor tyrosine kinases, G protein-coupled receptors, integrins and its ligands, growth factors, hormones, fibronectin, collagen, and laminin. Activated signaling cascades can cause transit from normal cells to cancer cells by modulating actin/filament dynamics. In various cancers including breast, prostate, and colorectal cancers, high expression or activity of each cascade protein is significantly associated with poor survival rate of patients as well as aggressive metastasis. Silencing ROCK, LIMK, or cofilin can abrogate their activities and inhibit cancer cell growth, invasion, and metastasis. Therefore ROCK/LIMK/cofilin signaling proteins might be good candidates to develop cancer prevention strategies or therapeutics. Currently, netarsudil, a ROCK inhibitor, is only used in clinical patients for glaucoma or ocular hypertension, but not for cancer. In this review, we will discuss comprehensive ROCK/LIMK/cofilin signaling pathway in cancers and its inhibitors for developing cancer therapy.

ROCK Inhibitor Y27632 Induced Morphological Shift and Enhanced Neurite Outgrowth-Promoting Property of Olfactory Ensheathing Cells via YAP-Dependent Up-Regulation of L1-CAM.

Olfactory ensheathing cells (OECs) are heterogeneous in morphology, antigenic profiles and functions, and these OEC subpopulations have shown different outcomes following OEC transplantation for central nervous system (CNS) injuries. Morphologically, OECs are divided into two subpopulations, process-bearing (Schwann cells-like) and flattened (astrocytes-like) OECs, which could switch between each other and are affected by extracellular and intracellular factors. However, neither the relationship between the morphology and function of OECs nor their molecular mechanisms have been clarified. In the present study, we first investigated morphological and functional differences of OECs under different cytokine exposure conditions. It demonstrated that OECs mainly displayed a process-bearing shape under pro-inflammatory conditions (lipopolysaccharide, LPS), while they displayed a flattened shape under anti-inflammatory conditions [interleukin-4 (IL-4) and transforming growth factor-β1 (TGF-β1)]. The morphological changes were partially reversible and the Rho-associated coiled-coil-containing protein kinase (ROCK)/F-actin pathway was involved. Functionally, process-bearing OECs under pro-inflammatory conditions showed increased cellular metabolic activity and a higher migratory rate when compared with flattened OECs under anti-inflammatory conditions and significantly promoted neurite outgrowth and extension. Remarkably, the morphological shift towards process-bearing OECs induced by ROCK inhibitor Y27632 enhanced the neurite outgrowth-promoting property of OECs. Furthermore, as the downstream of the ROCK pathway, transcriptional co-activator Yes-associated protein (YAP) mediated morphological shift and enhanced the neurite outgrowth-promoting property of OECs through upregulating the expression of the neural adhesion molecule L1-CAM. Our data provided evidence that OECs with specific shapes correspond to specific functional phenotypes and opened new insights into the potential combination of OECs and small-molecule ROCK inhibitors for the regeneration of CNS injuries.

RHOA activity in expanding blastocysts is essential to regulate HIPPO-YAP signaling and to maintain the trophectoderm-specific gene expression program in a ROCK/actin filament-independent manner.

What molecular signals are required to maintain the functional trophectoderm (TE) during blastocyst expansion of the late stage of preimplantation development? The activity of ras homology family member A (RHOA) GTPases is necessary to retain the expanded blastocyst cavity and also to sustain the gene expression program specific to TE. At the early stages of preimplantation development, the precursor of the TE lineage is generated through the molecular signals that integrate RHOA, RHO-associated coiled-coil containing protein kinase (ROCK), the apicobasal cell polarity, and the HIPPO-Yes-associated protein (YAP) signaling pathway. By contrast, molecular mechanisms regulating the maintenance of the TE characteristics at the later stage, which is crucial for blastocyst hatching and implantation, are scarcely understood. Expanding mouse blastocysts, obtained from crosses of the F1 (C57BL6 × DBA/2) strain, were exposed to chemical agents that interfere with RHOA, ROCK, or the actin cytoskeleton for up to 8 h, and effects on the blastocyst cavity, HIPPO-YAP signaling, and cell lineage-specific gene expression profiles were examined. Mouse embryos at the embryonic stage E3.5 (expanding blastocysts) and E4.5 (fully expanded blastocysts) were treated with RHOA inhibitor (C3 exoenzyme), ROCK inhibitor (Y27632), or actin filament disruptors (cytochalasin B and latrunculin A). The integrity of the blastocyst cavity was evaluated based on the gross morphology. Effects on HIPPO-YAP signaling were assessed based on the presence of nuclearized YAP protein by immunofluorescence staining and the expression of YAP/TEA domain family member (TEAD) target genes by quantitative RT-PCR (qRT-PCR). The impact of these disruptors on cell lineages was evaluated based on expression of the TE-specific and inner cell mass-specific marker genes by qRT-PCR. The integrity of the apicobasal cell polarity was assessed by localization of protein kinase C zeta (PRKCZ; apical) and scribbled planar cell polarity (SCRIB; basal) proteins by immunofluorescence staining. For comparisons, cultured cell lines, NIH/3T3 (mouse fibroblast) and P19C5 (mouse embryonal carcinoma), were also treated with RHOA inhibitor, ROCK inhibitor, and actin filament disruptors for up to 8 h, and effects on HIPPO-YAP signaling were assessed based on expression of YAP/TEAD target genes by qRT-PCR. Each experiment was repeated using three independent batches of embryos (n = 40-80 per batch) or cell collections. Statistical analyses of data were performed, using one-way ANOVA and two-sample t-test. Inhibition of RHOA deflated the cavity, diminished nuclear YAP (P < 0.01), and down-regulated the YAP/TEAD target and TE-specific marker genes in both E3.5 and E4.5 blastocysts (P < 0.05), indicating that the maintenance of the key TE characteristics is dependent on RHOA activity. However, inhibition of ROCK or disruption of actin filament only deflated the blastocyst cavity, but did not alter HIPPO-YAP signaling or lineage-specific gene expressions, suggesting that the action of RHOA to sustain the TE-specific gene expression program is not mediated by ROCK or the actomyosin cytoskeleton. By contrast, ROCK inhibitor and actin filament disruptors diminished YAP/TEAD target gene expressions in cultured cells to a greater extent than RHOA inhibitor, implicating that the regulation of HIPPO-YAP signaling in expanding blastocysts is distinctly different from that in the cell lines. Furthermore, the apicobasal cell polarity proteins in the expanding blastocyst were mislocalized by ROCK inhibition but not by RHOA inhibition, indicating that cell polarity is not linked to regulation of HIPPO-YAP signaling. Taken together, our study suggests that RHOA activity is essential to maintain the TE lineage in the expanding blastocyst and it regulates HIPPO-YAP signaling and the lineage-specific gene expression program through mechanisms that are independent of ROCK or actomyosin cytoskeleton. Not applicable. This study was conducted using one species, the mouse. Direct translation of the experiments and findings to human fertility preservation and ART requires further investigations. The elucidation of the mechanisms of TE formation is highly pertinent to fertility preservation in women. Our findings may raise awareness among providers of ART that the TE is sensitive to disturbance even in the late stage of blastocyst expansion and that rational approaches should be devised to avoid conditions that may impair the TE and its function. This study was funded by grants from the Ingeborg v.F. McKee Fund of the Hawaii Community Foundation (16ADVC-78882 to V.B.A.), and the National Institutes of Health (P20 GM103457 and R03 HD088839 to V.B.A.). The authors have no conflict of interest to declare.

Effects of ripasudil, a ROCK inhibitor, on retinal edema and nonperfusion area in a retinal vein occlusion murine model

Rho-associated coiled-coil containing protein kinase (ROCK) inhibitors are used to treat glaucoma patients and have protective effects on ischemic states. However, it is poorly understood how the ROCK pathway affects the pathological signs of retinal vein occlusion (RVO). In this study, we evaluated the effects of ripasudil, a ROCK inhibitor, on a murine RVO model. In vivo, RVO was induced by retinal vein laser irradiation in mice, and evaluated with ripasudil. In vitro, the effects of ripasudil were examined on tight junction protein integrity in human retinal microvascular endothelial cells (HRMECs). Moreover, we investigated the expression level of the phosphorylated myosin phosphatase target protein (MYPT)-1 after administration of ripasudil. Ripasudil significantly prevented deterioration, such as retinal edema, reduced the size of the nonperfusion area, and improved retinal blood flow. Ripasudil treatment inhibited disintegration of ZO-1 in HRMECs. Administration of ripasudil suppressed retinal phosphorylation of MYPT-1 in a murine RVO model. These findings indicate that ripasudil might be as a possible therapeutic agent for RVO.

Morphomechanical and structural changes induced by ROCK inhibitor in breast cancer cells

The EMT phenomenon is based on tumour progression. The cells lose their physiologic phenotype and assumed a mesenchymal phenotype characterized by an increased migratory capacity, invasiveness and high resistance to apoptosis. In this process, RHO family regulates the activation or suppression of ROCK (Rho-associated coiled-coil containing protein kinase) which in turn regulates the cytoskeleton dynamics. However, while the biochemical mechanisms are widely investigated, a comprehensive and careful estimation of biomechanical changes has not been extensively addressed. In this work, we used a strong ROCK inhibitor, Y-27632, to evaluate the effects of inhibition on living breast cancer epithelial cells by a biomechanical approach. Atomic Force Microscopy (AFM) was used to estimate changes of cellular elasticity, quantified by Young's modulus parameter. The morphometric alterations were analyzed by AFM topographies and Confocal Laser Scanning Microscopy (CLSM). Our study revealed a significant modification in the Young's modulus after treatment, especially as regards cytoskeletal region. Our evidences suggest that the use of Y-27632 enhanced the cell rigidity, preventing cell migration and arrested the metastasization process representing a potential powerful factor for cancer treatment.

The ROCK inhibitor, thiazovivin, inhibits human corneal endothelial‑to‑mesenchymal transition/epithelial‑to‑mesenchymal transition and increases ionic transporter expression.

Corneal diseases exhibit a high prevalence and are prone to cause blindness; furthermore, maintaining the morphology and ionic transporter expression in corneal endothelial cells (CECs) is crucial for treatment of these diseases. This study aimed to investigate the effects of the novel Rho associated coiled-coil containing protein kinase (ROCK) inhibitor, thiazovivin (2,4-disubstituted thiazole, TZV), on human corneal endothelial-to-mesenchymal transition/epithelial-to-mesenchymal transition (EndMT/EMT), cell morphology, junction proteins and ionic transporter expression in human CECs (HCECs) in vitro and then to clarify the mechanisms of action of TZV. In the present study, primary HCECs were cultured in vitro and passaged. The expression levels of adhesion proteins (E-cadherin and N-cadherin), the EndMT/EMT marker, α smooth muscle actin (α-SMA), the tight junction protein, Zonula occludens-1 (ZO-1), and the ionic transporter, Na+/K+-ATPase, were detected by immunofluorescence. The proliferative ability of the HCECs was determined by CCK-8 assay. The mRNA expression of the EndMT/EMT-inducing gene, Snail, was examined by RT-PCR. The protein expression levels of ROCK1/2 were evaluated by western blot analysis. The HCECs were cultured with TZV at various concentrations (2, 4, or 6 µM) for different periods of time (24 or 48 h). We found that the the cell states of the HCECs co-cultured with 4 µM TZV for 48 h reached the optimum, and corneal EndMT/EMT was inhibited, as evidenced by the significantly upregulated expression of ZO-1 and E-cadherin, and the markedly downregulated expression of N-cadherin and α-SMA. Furthermore, the cells exhibited a normal, tightly connected hexagonal or pentagonal morphology. Additionally, the protein expression of ROCK1/2 and the mRNA expression of Snail were significantly decreased. However, there was no significant difference between the TZV-treated and the control groups as regards HCEC proliferative ability. These findings suggested that the ROCK inhibitor, TZV (4 µM), was effective in improving the morphology, cell junctions and ionic transporter expression of HCECs by inhibiting EndMT/EMT, but had no effect on HCEC proliferation.

Vascular Normalization by ROCK Inhibitor: Therapeutic Potential of Ripasudil (K-115) Eye Drop in Retinal Angiogenesis and Hypoxia.

In this study, we investigated the therapeutic potential of a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor ripasudil (K-115) eye drop on retinal neovascularization and hypoxia. In vitro, human retinal microvascular endothelial cells (HRMECs) were pretreated with ripasudil and then stimulated with VEGF. ROCK activity was evaluated by phosphorylation of myosin phosphatase target protein (MYPT)-1. Endothelial migration and cell viability were assessed by cell migration and MTT assay, respectively. The concentration of ripasudil in the retina was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vivo, normal saline, 0.4%, or 0.8% ripasudil were administered three times a day to mice with oxygen-induced retinopathy (OIR). The areas of neovascularization and avascular retina were also quantified with retinal flat-mounts at postnatal day (P) 15, P17, or P21. The retinal hypoxic area was evaluated using hypoxia-sensitive drug pimonidazole by immunohistochemistry at P17. The vascular normalization was also evaluated by immunohistochemistry at P17. Ripasudil but not fasudil significantly reduced VEGF-induced MYPT-1 phosphorylation in HRMECs at 30 μmol/L. Ripasudil significantly inhibited VEGF-induced HRMECs migration and proliferation. The concentration of ripasudil in the retina was 3.8 to 10.4 μmol/L and 6.8 to 14.8 μmol/L after 0.4% and 0.8% ripasudil treatment, respectively. In the 0.4% and 0.8% ripasudil treated OIR mice, the areas of neovascularization as well as avascular area in the retina was significantly reduced compared with those of saline-treated mice at P17 and P21. Pimonidazole staining revealed that treatment with 0.4% and 0.8% ripasudil significantly inhibited the increase in the hypoxic area compared with saline. 0.8% ripasudil could cause intraretinal vascular sprouting and increase retinal vascular perfusion. Novel ROCK inhibitor ripasudil eye drop has therapeutic potential in the treatment of retinal hypoxic neovascular diseases via antiangiogenic effects as well as vascular normalization.

Effects of K-115 (Ripasudil), a novel ROCK inhibitor, on trabecular meshwork and Schlemm's canal endothelial cells.

Ripasudil hydrochloride hydrate (K-115), a specific Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, was the first ophthalmic solution developed for the treatment of glaucoma and ocular hypertension in Japan. Topical administration of K-115 decreased intraocular pressure (IOP) and increased outflow facility in rabbits. This study evaluated the effect of K-115 on monkey trabecular meshwork (TM) cells and Schlemm’s canal endothelial (SCE) cells. K-115 induced retraction and rounding of cell bodies as well as disruption of actin bundles in TM cells. In SCE-cell monolayer permeability studies, K-115 significantly decreased transendothelial electrical resistance (TEER) and increased the transendothelial flux of FITC-dextran. Further, K-115 disrupted cellular localization of ZO-1 expression in SCE-cell monolayers. These results indicate that K-115 decreases IOP by increasing outflow facility in association with the modulation of TM cell behavior and SCE cell permeability in association with disruption of tight junction.

Mechanisms of favorable effects of Rho kinase inhibition on myocardial remodeling and systolic function after experimental myocardial infarction in the rat

The objective of this study was to determine the molecular mechanisms by which cardiac Rho-associated coiled-coil containing protein kinase (ROCK) activation after myocardial infarction (MI) does intervene in cardiac systolic function decline and remodeling. Simultaneous measurement of different cardiac ROCK target proteins levels, in vivo left ventricular (LV) systolic function, myocardial fibrosis and hypertrophy in rats with MI under ROCK inhibition with fasudil. Seven days after MI, the ventricular mass increased significantly by 5.6% in the MI group and was reduced with fasudil. LV systolic dysfunction improved significantly with fasudil whereas cardiac ROCK activation was reduced to sham levels. The ROCK inhibitor also reduced increased cardiac levels of both ROCK1 and ROCK2 isoforms, cardiomyocyte ROCK2 fluorescence levels and β-myosin heavy chain (MHC) levels in addition to myocardial collagen volume fraction decline. Compared with sham rats, troponin phosphorylation levels after MI were similar and ROCK inhibition reduced them. MI significantly increased phosphorylation levels of extracellular-signal-regulated kinase (ERK) 42 and ERK 44 by twofold and 63%, respectively, whereas in the fasudil-treated MI group these levels were similar to those in the sham group. MI significantly increased phosphorylated levels of the transcription factor GATA-4 and the ROCK inhibitor normalized them. LV systolic dysfunction after MI was strongly associated with cardiac ROCK activation and subsequent phosphorylation of ROCK target proteins that promote ventricular remodeling such as β-MHC and the ERK/GATA-4 pathway. ROCK inhibition with fasudil significantly improved systolic function, diminished myocardial fibrosis and normalized β-MHC and ERK/GATA-4 phosphorylation levels.

ROCK inhibitor Y-27632 inhibits the growth, migration, and invasion of Tca8113 and CAL-27 cells in tongue squamous cell carcinoma.

The objective of this study is to determine the effects of Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor Y-27632 on the growth, invasion, and migration of Tca8113 and CAL-27 cells in tongue squamous cell carcinoma (TSCC). The methods of the study are as follows: After being routinely cultured for 24h, Tca8113 and CAL-27 cells were treated with Y-27632 solution. The morphological change of Y-27632-treated cells was observed under an optical microscope and an inverted microscope; MTT assay was performed to measure the optical density (OD) of cells and calculate cell growth inhibition rate; the change of apoptosis was detected by AnnexinV-FITC/PI assay; cell invasion and migration were measured by Transwell assay. The results were as follows: (1) With increasing concentration of Y-27632, cell morphology changed and cell apoptosis appeared; (2) MTT assay showed that inhibition effect of Y-27632 on Tca8113 and CAL-27 cells was enhanced with increasing concentrations and time (all P < 0.01); (3) Apoptosis showed that, compared with controls, the number of apoptosis cells in experimental groups was significantly increased (all P < 0.01). Apoptosis rate was elevated with increasing concentrations of Y-27632; (4) Transwell assay showed, after a treatment with Y-27632, the number of migrated and invaded Tca8113 and CAL-27 cells in each group was statistically different (all P < 0.01); compared with controls, the number of migrated cell in groups treated with Y-27632 was decreased and less Tca8113 and CAL-27 cells in experimental groups passed through polycarbonate membrane (all P < 0.05). The study concludes that Y-27632 can inhibit the growth, invasion, and migration of Tca8113 and CAL-27 cells, suggesting that Y-27632 may be therapeutically useful in TSCC.