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A scalable and facile synthetic process for N-(pyridin-4-yl)piperazine-1-carboxamide hydrochloride, a novel Rho kinase inhibitor with an unsymmetrical urea structure currently under investigation for the treatment of central nervous system disorders, was established. After optimisation of the reaction conditions, N-(pyridin-4-yl)piperazine-1-carboxamide hydrochloride was synthesised from 4-aminopyridine and N,N′-carbonyldiimidazole through acylation, deprotection and salt formation. This new procedure affords the product in 53% overall yield with high purity and it can be easily scaled up for production.

Rho kinase (ROCK) regulates the functions of several target proteins via its kinase activity. Therefore, ROCK activity inhibition may provide new possibilities of controlling the in vitro neuronal differentiation of embryonic stem (ES) cells. When we investigated the effects of the ROCK inhibitor Y-27632 on ES cell differentiation, we found that this inhibitor promoted the differentiation of these cells into neurons. Furthermore, we found that ROCK inhibition may promote the neuronal differentiation of ES cells by activating extracellular signal-regulated kinase (ERK) involved in the ERK signaling pathway. In this study, we investigated the effects of specific inhibitors of several cellular signaling components on the promotion of neuronal differentiation in ES cells to clarify the roles of cellular signaling pathways in the ROCK inhibitor-mediated cell differentiation process. Our results suggest that ERK may be activated via the Ras/Raf/MEK, the PI3K/PKC, or the Cdc42/Rac signaling pathways in the ROCK inhibitor-mediated promotion of neuronal differentiation in ES cells.

Background and Purpose Vasodilatation may contribute to the neuroprotective and vascular anti-remodelling effect of the tissue transglutaminase 2 (TG2) inhibitor cystamine. Here, we hypothesized that inhibition of TG2 followed by blockade of smooth muscle calcium entry and/or inhibition of Rho kinase underlies cystamine vasodilatation. Experimental Approach We used rat mesenteric small arteries and RT-PCR, immunoblotting, and measurements of isometric wall tension, intracellular Ca2+ ([Ca2+]i), K+ currents (patch clamp), and phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and myosin regulatory light chain, in our experiments. Key Results RT-PCR and immunoblotting revealed expression of TG2 in mesenteric small arteries. Cystamine concentration-dependently inhibited responses to phenylephrine, 5-HT and U46619 and for extracellular potassium. Selective inhibitors of TG2, LDN 27129 and T101, also inhibited phenylephrine contraction. An inhibitor of PLC suppressed cystamine relaxation. Cystamine relaxed and reduced [Ca2+]i in phenylephrine-contracted arteries. In potassium-contracted arteries, cystamine induced less relaxation without changing [Ca2+]i, and these relaxations were blocked by mitochondrial complex inhibitors. Blockers of Kv7 channels, XE991 and linopirdine, inhibited cystamine relaxation and increases in voltage-dependent smooth muscle currents. Cystamine and the Rho kinase inhibitor Y27632 reduced basal MYPT1-Thr855 phosphorylation, but only Y27632 reduced phenylephrine-induced increases in MYPT1-Thr855 and myosin regulatory light chain phosphorylation. Conclusions and Implications Cystamine induced vasodilatation by inhibition of receptor-coupled TG2, leading to opening of Kv channels and reduction of intracellular calcium, and by activation of a pathway sensitive to inhibitors of the mitochondrial complexes I and III. Both pathways may contribute to the antihypertensive and neuroprotective effect of cystamine.

Introduction It remains controversial whether statins have a beneficial effect on pulmonary arterial hypertension (PAH). This study is intended to evaluate whether statin, co-administered with Rho-kinase inhibitor, could enhance its efficacy. Although Rho-kinase inhibitors, including fasudil, have been reported to improve pulmonary hypertension in experimental and clinical studies, the combination of these agents has not been tested in the treatment of pulmonary hypertension (PH). Material and methods The effects of such a regimen on hemodynamics, right ventricle hypertrophy, and Rho-associated protein kinase (ROCK) activity in experimental monocrotaline (MCT)-induced pulmonary hypertension were examined. Fourteen days after monocrotaline injection (60 mg/kg), male rats were treated orally for another 14 days with fasudil (15 mg/kg per day), or with a combination of fasudil + rosuvastatin (10 mg/kg per day). Results The drug combination reversed the MCT-induced increase in right ventricle pressure (RVP) and reduced right ventricular hypertrophy (RV/LV + S ratio) more than Rho kinase inhibitor alone. The simultaneous administration of fasudil and rosuvastatin caused a further decrease of RhoA kinase activity in isolated lung tissues as compared to fasudil alone. Conclusions The results indicate that rosuvastatin intensifies the beneficial effects of Rho-kinase inhibitor on the Rho/Rho-kinase pathway and such a combination may represent an option for the treatment of pulmonary arterial hypertension.

This study evaluated the effects of intravitreal injection of fasudil (IVF), a Rho-kinase inhibitor, in cases of recent-onset nonarteritic anterior ischemic optic neuropathy (NAION). In this interventional case series, 13 eyes of 13 patients diagnosed with NAION within 14 days of onset were included. The affected eyes received a 0.025 mg/0.05 mL IVF. Functional and structural outcomes were assessed 1 and 3 months following treatment. Best corrected visual acuity (BCVA) was the main outcome measured, with mean deviation (MD) index of the VF test and peripapillary retinal nerve fiber layer thickness as secondary measures. There was a statistically significant improvement in the patients' BCVA 1 and 3 months following IVF; BCVA improved from 1.69 ± 0.55 logMAR at baseline to 0.98 ± 0.47 and 0.93 ± 0.51 logMAR at 1 and 3 months, respectively (P = .004). The change in BCVA was not significant between month 1 and month 3 (P = .22). Peripapillary retinal nerve fiber layer thickness decreased from 173.5 ± 29.28 µm in the baseline evaluation to 85.8 ± 8.8 µm at 1 month, and 62.9 ± 5.97 µm at 3 months (P = .003). MD values changed from 24.60 ± 3.80 to 21.0 ± 6.10 and 20.5 ± 6.50 at 1 and 3 months, respectively (P = .007 and .005, respectively). This pilot study suggests that IVF may be an effective treatment for patients with recent-onset NAION. Larger studies are required to establish the therapeutic role of fasudil for NAION.

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.

Bone mesenchymal stem cells (BMSCs) are a well-known donor graft source due to their potential for self-renewal and differentiation into multi-lineage cell types, including osteoblasts that are critical for fracture healing. Fasudil (FAS), a Rho kinase inhibitor, has been proven to induce the differentiation of bone marrow stem cells (BMSCs) into neuron-like cells. However, its role in the osteogenesis of BMSCs remain uncertain. Herein, we for the first time studied the effects of FAS on osteogenic differentiation in a mouse fracture model and further explored the involved mechanisms in mouse BMSCs. The results showed that FAS stimulated bone formation in the fracture mouse model. Additionally, at 30 μM, FAS significantly promotes alkaline phosphatase activity, mineralization, and the expression of osteogenic markers COL-1, RUNX2 and OCN in murine BMSCs. Blocking of P38 by SB202190 significantly reversed the effects of FAS, in vitro, suggesting that P38, but not ERK or JNK activation is required for FAS-induced osteogenesis. Collectively, our results indicate that FAS may be a promising agent for promoting fracture healing.

The contraction of gastrointestinal (GI) smooth muscles is regulated by both Ca(2+)-dependent and Ca(2+) sensitization mechanisms. Proline-rich tyrosine kinase 2 (Pyk2) is involved in the depolarization-induced contraction of vascular smooth muscle via a Ca(2+) sensitization pathway. However, the role of Pyk2 in GI smooth muscle contraction is unclear. The spontaneous contraction of colonic smooth muscle was measured by using isometric force transducers. Protein and phosphorylation levels were determined by using western blotting. Pyk2 protein was expressed in colonic tissue, and spontaneous colonic contractions were inhibited by PF-431396, a Pyk2 inhibitor, in the presence of tetrodotoxin (TTX). In cultured colonic smooth muscle cells (CSMCs), PF-431396 decreased the levels of myosin light chain (MLC20) phosphorylated at Ser19 and ROCK2 protein expression, but myosin light chain kinase (MLCK) expression was not altered. However, Y-27632, a Rho kinase inhibitor, increased phosphorylation of Pyk2 at Tyr402 and concomitantly decreased ROCK2 levels; the expression of MLCK in CSMCs did not change. The expression of P(Tyr402)-Pyk2 and ROCK2 was increased when CSMCs were treated with Ach. Pyk2 is involved in the process of colonic smooth muscle contraction through the RhoA/ROCK pathway. These pathways may provide very important targets for investigating GI motility disorders.

We investigated the effect of a new Rho kinase inhibitor, SB772077B (SB77) on aqueous outflow facility (OF) in human eyes using human organ-cultured anterior segment (HOCAS). IOP was monitored for 24 h post-treatment with either SB77 (0.1/10/50 µM) or vehicle after a stable baseline pressure. The hydrodynamic pattern of aqueous outflow was analysed by labelling outflow pathway with red fluorescent microspheres. The effect of SB77 on cell morphology, actin stress fibers, focal adhesions, ECM, status of RhoA activation and myosin light chain phosphorylation (p-MLC) were evaluated and compared with Y27632, by immunostaining using primary human trabecular meshwork (HTM) cells. Following 24 h treatment, SB77 increased OF by 16% at 0.1 µM (N = 6), 29% at 10 µM (N = 8; p = 0.018) and 39% at 50 µM (N = 8; p = 0.004) in human eyes. There was an overall increase in tracer quantity and in area along inner wall of Schlemm’s canal. Treatment with SB77 showed no evidence of cytotoxicity and caused a significant reduction in the expression of fibrotic markers compared to Y27632. The present findings indicate that SB77 treatment was effective in enhancing OF and reducing fibrotic markers in an ex vivo model. Thus SB77 may be a potential clinical candidate for the management of glaucoma.

To investigate the influence of the selective Rho-kinase (ROCK) inhibitor, fasudil, on the mRNA level of proinflammatory factors and the retinal vascular development in mice with oxygen-induced retinopathy (OIR). C57BL/6J mice underwent standard protocol for OIR induction from postnatal days 7 to 12. Subsequently, they received a daily intraperitoneal injection of fasudil or sodium chloride from P12 to P16. Analyses were performed using vascular staining on retinal flat mounts, RNA expression by qPCR, and immunohistochemistry on paraffin sections. On retinal flat mounts, the proportion of avascular area and tuft formation did not differ between the fasudil and NaCl group. Immunohistochemical staining revealed a less intense staining with inflammatory markers after fasudil. Nevertheless, there were no differences on RNA level between the two groups. In conclusion, our findings support that daily systemic application of fasudil does not decrease retinal neovascularization in rodents with oxygen-induced retinopathy. The results of our study together with the controversial results on the effects of different ROCK inhibitors from the literature makes it apparent that effects of ROCK inhibition are more complex, and further studies are necessary to analyze its potential therapeutic effects.

Fasudil, a Rho-kinase inhibitor, has shown outstanding therapeutic effects against cerebral vasospasm after subarachnoid hemorrhage (SAH) in humans. Studies show various biological effects of fasudil in the cardiovascular system. We conducted a preclinical systematic review to determine the efficacy and possible mechanisms of fasudil on animal models of myocardial ischemia/reperfusion (I/R) injury. Nineteen studies involving 400 animals were identified after searching 8 databases for articles published till June 2018. The methodological quality was assessed by the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) 10-item checklist. The data were analyzed using Rev-Man 5.3 software, and the score of study quality ranged from 3 to 6 points. Compared to the control group, fasudil treated animals showed reduced myocardial infarct size (P < 0.05), lower levels of cardiac enzymes (P < 0.05) and cardiac troponin T (P < 0.05), improved systolic and diastolic functions (P < 0.05), and increased degree of decline in the ST-segment (P < 0.05). The possible mechanisms of fasudil action against myocardial I/R injury are improvement in coronary vasodilation, inhibition of apoptosis and oxidative stress, relieving inflammation, and reduction in endoplasmic reticulum stress and metabolism. In conclusion, fasudil exerts a cardio-protective function through multiple signaling pathways in animal models of myocardial I/R injury.

Oleuropein and 3-hydroxytyrosol (3-HT) are natural polyphenols present in olive oil that are known to exhibit potent anti-oxidant activities and exert protective effects against a number of human diseases. In the liver, olive oil polyphenols have been demonstrated to prevent hepatocellular carcinoma (HCC) cell growth. However, little is known about their effects against HCC cell migration. Therefore, the present study investigated whether or not oleuropein and 3-HT were involved in the suppression of transforming growth factor-α (TGF-α)-induced migration of human HCC cells using human HCC-derived HuH7 cells. The TGF-α-induced migration of HuH7 cells was significantly and dose-dependently suppressed by oleuropein and 3-HT. This study group demonstrated previously that the TGF-α-induced activation of AKT and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) were involved in HuH7 cell migration. In addition to these protein kinases, the present study examined the involvement of TGF-α-induced activation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and Rho kinase in HuH7 cell migration. TGF-α-induced HuH7 cell migration was decreased by SB203580, a p38 MAPK inhibitor, and Y27632, a Rho kinase inhibitor. However, PD98059, an inhibitor of the upstream kinase activating ERK, did not suppress the TGF-α-induced migration of HuH7 cells. Although AKT, SAPK/JNK, p38 MAPK and Rho kinase pathways were suggested to be involved in the TGF-α-induced migration of HuH7 cells, 10-30 µM 3-HT did not exhibit any suppressive effect on the TGF-α-stimulated activities of these kinases. The results of the present study suggest that olive oil polyphenols suppressed the TGF-α-induced migration of HCC cells.

To evaluate the effect of topically administrated ripasudil, a rho kinase inhibitor, on blood flow in the optic nerve head (ONH) of normal rats. Ripasudil (0.4%) or placebo was administered in the right eye of normal Brown Norway rats in a double-blind manner. Laser speckle flowgraphy was measured in the ONH of the right eye 20 or 40 min after a single instillation and before and after 7 or 14 days of twice daily instillation. Mean blur rate was evaluated in the total area (MA), the vessel region (MV), and the tissue region (MT). Intraocular pressure (IOP), blood pressure, ocular perfusion pressure (OPP), and heart rate were also recorded at each time point. After a single instillation, MV was significantly larger at 40 min than 20 min in the ripasudil group (P = 0.044) and was significantly lower in the placebo group (P = 0.023). MA and MV 40 min after instillation were significantly larger in the ripasudil group than in the placebo group (P = 0.022 and P = 0.006, respectively). After continuous instillation, MA and MV in the ripasudil group significantly increased from baseline after 7 and 14 days of treatment (both P

AimsTo investigate morphological changes in type VI collagen in the human trabecular meshwork associated with the rho kinase inhibitor ripasudil.MethodsThis cross-sectional study evaluated the effects of ripasudil eye drop administration (RA) or no ripasudil eye drop administration (NRA) in patients with primary open-angle glaucoma (POAG; age range 60–80 years) who underwent conventional outflow reconstruction between December 2015 and September 2016 at Tokai University Hachioji Hospital. The juxtacanalicular tissue was removed and imaged using transmission electron microscopy. Type VI collagen comprises cross-banded aggregates with transverse bands 30 nm apart repeating every 105 nm. The transverse bands are called the outer rod-like region (ORR) and the intervals are called the inner rod-like region (IRR). The waveform intensity in the type VI collagen was analysed in electron micrographs using Fourier transformation to detect the IRR and ORR borders.ResultsTen eyes of 10 patients were included (n=5/group). The baseline characteristics did not differ significantly between groups. ORR width was significantly smaller in the RA group (37.85±3.43 nm) than in the NRA group (50.62±5.23 nm, pConclusionsRipasudil administration in patients with POAG induced morphological changes in type VI collagen. Patients with POAG administered RA had a significantly smaller ORR width and a significantly greater IRR width than patients with POAG not administered RA.

Purpose: This study investigated the hypotensive effect of RKI-1447, a Rho kinase inhibitor, in a porcine ex vivo pigmentary glaucoma model. Methods: Twenty-eight porcine anterior chambers were perfused with medium supplemented with 1.67 × 107 pigment particles/mL for 48 hours before treatment with RKI-1447 (n = 16) or vehicle control (n = 12). Intraocular pressure (IOP) was recorded and outflow facility was calculated. Primary trabecular meshwork cells were exposed to RKI-1447 or vehicle control; effects on the cytoskeleton, motility, and phagocytosis were evaluated. Result: Compared to baseline, the perfusion of pigment caused a significant increase in IOP in the RKI-1447 group (P = 0.003) at 48 hours. Subsequent treatment with RKI-1447 significantly reduced IOP from 20.14 ± 2.59 mmHg to 13.38 ± 0.91 mmHg (P = 0.02). Pigment perfusion reduced the outflow facility from 0.27 ± 0.03 at baseline to 0.18 ± 0.02 at 48 hours (P < 0.001). This was partially reversed with RKI-1447. RKI-1447 caused no apparent histological changes in the micro- or macroscopic TM appearance. RKI-1447-treated primary TM cells showed significant disruption of the actin cytoskeleton both in the presence and absence of pigment (P < 0.001) but no effect on TM migration was observed. Pigment-treated TM cells exhibited a reduction in TM phagocytosis, which RKI-1447 reversed. Conclusion: RKI-1447 significantly reduces IOP by disrupting TM stress fibers and increasing TM phagocytosis. These features may make it useful for the treatment of secondary glaucomas with an increased phagocytic load.

Fasudil, a Rho kinase (ROCK) inhibitor, effectively inhibits disease severity in a mouse model of Alzheimer's disease (AD). However, given its significant limitations, including a relatively narrow safety window and poor oral bioavailability, Fasudil is not suitable for long-term use. Thus, screening for ROCK inhibitor(s) that are more efficient, safer, can be used orally and suitable for long-term use in the treatment of neurodegenerative disorders is required. The main purpose of the present study is to explore whether FSD-C10, a novel ROCK inhibitor, has therapeutic potential in amyloid precursor protein/presenilin-1 transgenic (APP/PS1 Tg) mice, and to determine possible mechanisms of its action. The results showed that FSD-C10 effectively improved learning and memory impairment, accompanied by reduced expression of amyloid-β 1-42 (Aβ1-42), Tau protein phosphorylation (P-tau) and β-site APP-cleaving enzyme in the hippocampus and cortex area of brain. In addition, FSD-C10 administration boosted the expression of synapse-associated proteins, such as postynaptic density protein 95, synaptophsin, α-amino 3-hydroxy-5-methyl-4-isoxa-zolep-propionate receptor and neurotrophic factors, e,g., brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. Taken together, our results demonstrate that FSD-C10 has therapeutic potential in the AD mouse model, possibly through inhibiting the formation of Aβ1-42 and P-tau, and promoting the generation of synapse-associated proteins and neurotrophic factors.

Background: Interleukin-17 (IL-17) and Rho-kinase (ROCK) play an important role in regulating the expression of inflammatory mediators, immune cell recruitment, hyper-responsiveness, tissue remodeling, and oxidative stress. Modulation of IL-17 and ROCK proteins may represent a promising approach for the treatment of this disease.Objective: To study the effects of an anti-IL17 neutralizing antibody and ROCK inhibitor treatments, separately and in combination, in a murine model of chronic allergy-induced lung inflammation.Methods: Sixty-four BALBc mice, were divided into eight groups (n = 8): SAL (saline-instilled); OVA (exposed-ovalbumin); SAL-RHOi (saline and ROCK inhibitor), OVA-RHOi (exposed-ovalbumin and ROCK inhibitor); SAL-anti-IL17 (saline and anti-IL17); OVA-anti-IL17 (exposed-ovalbumin and anti-IL17); SAL-RHOi-anti-IL17 (saline, ROCK inhibitor and anti-IL17); and OVA-RHOi-anti-IL17 (exposed-ovalbumin, anti-IL17, and ROCK inhibitor). A 28-day protocol of albumin treatment was used for sensitization and induction of pulmonary inflammation. The anti-IL17A neutralizing antibody (7.5 μg per treatment) was administered by intraperitoneal injection and ROCK inhibitor (Y-27632) intranasally (10 mg/kg), 1 h prior to each ovalbumin challenge (days 22, 24, 26, and 28).Results: Treatment with the anti-IL17 neutralizing antibody and ROCK inhibitor attenuated the percentage of maximal increase of respiratory system resistance and respiratory system elastance after challenge with methacholine and the inflammatory response markers evaluated (CD4+, CD8+, ROCK1, ROCK2, IL-4, IL-5, IL-6, IL-10 IL-13, IL-17, TNF-α, TGF-β, NF-κB, dendritic cells, iNOS, MMP-9, MMP-12, TIMP-1, FOXP3, isoprostane, biglycan, decorin, fibronectin, collagen fibers content and gene expression of IL-17, VAChT, and arginase) compared to the OVA group (p < 0.05). Treatment with anti-IL17 and the ROCK inhibitor together resulted in potentiation in decreasing the percentage of resistance increase after challenge with methacholine, decreased the number of IL-5 positive cells in the airway, and reduced, IL-5, TGF-β, FOXP3, ROCK1 and ROCK2 positive cells in the alveolar septa compared to the OVA-RHOi and OVA-anti-IL17 groups (p < 0.05).Conclusion: Anti-IL17 treatment alone or in conjunction with the ROCK inhibitor, modulates airway responsiveness, inflammation, tissue remodeling, and oxidative stress in mice with chronic allergic lung inflammation.

Objective This study aims to study the effect of Rho kinase inhibitor fasudil on the expression endothelin-1 (ET-1) and nitric oxide (NO) in rats with hypoxic pulmonary hypertension (HPH). Methods Twenty-four male SD rats were randomly divided into three groups: control group, model group (HPH group) and HPH+fasudil group. The rat HPH model was established by intermittent hypoxia (IH) at atmospheric pressure. Mean pulmonary artery pressure (mPAP), right ventricular hypertrophy index (RVHI), ET-1 and NO levels, and pulmonary vascular structural changes were observed in all groups. Results MPAP, RVHI and ET-1 levels were significantly higher in HPH group than in control group, while NO was significantly lower than in control group. In addition, mPAP, RVHI and ET-1 were significantly lower in the HPH+fasudil group than in the HPH group. In the HPH group, ET-1 level was significantly and positively correlated with mPAP and RVHI, NO was negatively correlated with mPAP and RVHI levels, and ET-1 level was significantly and negatively correlated with NO level. In the HPH group, pulmonary arteriolar walls were generally thickened, and lumen stenosis was obvious; while after fasudil treatment, pulmonary arteriolar wall thickening and stenosis degree were significantly reduced. Conclusion Fasudil can significantly reduce ET-l level and increase NO level in HPH rats, suppressing the development of pulmonary arterial hypertension.

Background: Glaucoma is a progressive optic neuropathy causing visual impairment and Retinal Ganglionic Cells (RGCs) death gradually posing a need for neuroprotective strategies to minimize the loss of RGCs and visual field. It is recognized as a multifactorial disease, Intraocular Pressure (IOP) being the foremost risk factor. ROCK inhibitors have been probed for various possible indications, such as myocardial ischemia, hypertension, kidney diseases. Their role in neuroprotection and neuronal regeneration has been suggested to be of value in the treatment of neurological diseases, like spinal-cord injury, Alzheimer’s disease and multiple sclerosis but recently Rho-associated Kinase inhibitors have been recognized as potential antiglaucoma agents. Evidence Synthesis: Rho-Kinase is a serine/threonine kinase with a kinase domain which is constitutively active and is involved in the regulation of smooth muscle contraction and stress fibre formation. Two isoforms of Rho-Kinase, ROCK-I (ROCK β) and ROCK-II (ROCK α) have been identified. ROCK II plays a pathophysiological role in glaucoma and hence the inhibitors of ROCK may be beneficial to ameliorate the vision loss. These inhibitors decrease the intraocular pressure in the glaucomatous eye by increasing the aqueous humour outflow through the trabecular meshwork pathway. They also act as anti-scarring agents and hence prevent post-operative scarring after the glaucoma filtration surgery. Their major role involves axon regeneration by increasing the optic nerve blood flow which may be useful in treating the damaged optic neurons. These drugs act directly on the neurons in the central visual pathway, interrupting the RGC apoptosis and therefore serve as a novel pharmacological approach for glaucoma neuroprotection. Conclusion: Based on the results of high-throughput screening, several Rho kinase inhibitors have been designed and developed comprising of diverse scaffolds exhibiting Rho kinase inhibitory activity from micromolar to subnanomolar ranges. This diversity in the scaffolds with inhibitory potential against the kinase and their SAR development will be intricated in the present review. Ripasudil is the only Rho kinase inhibitor marketed to date for the treatment of glaucoma. Another ROCK inhibitor AR-13324 has recently passed the clinical trials whereas AMA0076, K115, PG324, Y39983 and RKI-983 are still under trials. In view of this, a detailed and updated account of ROCK II inhibitors as the next generation therapeutic agents for glaucoma will be discussed in this review.

Click here to listen to the PodcastBackground/aimsTo compare the efficacy of combined intravitreal injection of bevacizumab and a Rho-kinase inhibitor, fasudil (intravitreal bevacizumab (IVB)/intravitreal fasudil (IVF)), with IVB alone for centre-involving diabetic macular oedema (DME).MethodsIn this prospective randomised clinical trial, 44 eyes with centre-involving DME were randomised into two groups. The combined group received three consecutive injections of IVB (1.25 mg) and IVF (50 µM/L) monthly, while the monotherapy group received only one IVB (1.25 mg) injection per month for 3 months. Changes in best-corrected visual acuity (BCVA) and central macular thickness (CMT) were compared between the two groups at months 3 and 6. The primary outcome measure was the mean change in BCVA at month 6.ResultsMean BCVA was significantly improved in both groups at month 3 (PConclusionAdjunctive intravitreal injection of a Rho-kinase inhibitor may enhance and prolong the therapeutic effects of anti-vascular endothelial growth factor drugs for centre- involving DME.

Objective: The Rho GTPase/Rho kinase pathway is an important target in glaucoma treatment. This study investigated the hypotensive effect of RKI-1447, a Rho kinase inhibitor developed for cancer treatment, in a porcine ex vivo pigmentary glaucoma model. Materials and Methods: Twenty-eight fresh porcine anterior chambers were perfused with pigment medium (1.67 × 107 pigment particles/mL) for 48 hours before being subjected to the RKI-1447 (n = 16) or the vehicle control (n = 12). Another twelve eyes with normal medium perfusion served as the control. The intraocular pressure (IOP) was recorded at two-minute intervals and the outflow facility was calculated. To investigate the intracellular mechanism of the IOP reduction, primary trabecular meshwork cells were exposed to RKI-1447 or the vehicle control and then analyzed for changes in cytoskeleton, motility, and phagocytosis. Results: Compared to the baseline, the perfusion of pigment caused a significant increase in IOP in the RKI-1447 group (P = 0.003) at 48 hours. Subsequent treatment with RKI-1447 significantly reduced IOP from 20.14 ± 2.59 mmHg to 13.38 ± 0.91 mmHg (P = 0.02). Pigment perfusion reduced the outflow facility from 0.27 ± 0.03 at baseline to 0.18 ± 0.02 at 48 hours (P < 0.001). This was partially reversed with RKI-1447. RKI-1447 exhibited no apparent changes in the micro- or macroscopic appearance, including histology. Primary TM cells exposed to RKI-1447 showed a significant disruption of the actin cytoskeleton both in the presence and absence of pigment exposure (P < 0.001) but no effect on TM migration was observed. Pigment-treated TM cells exhibited a reduction in TM phagocytosis, which RKI reversed. Conclusions: RKI-1447 is a novel ROCK inhibitor that significantly reduces IOP by disrupting TM stress fibers and increasing TM phagocytosis. These features may make it especially useful for the treatment of secondary glaucomas with an increased phagocytosis load but also for other open angle glaucomas.

Cell migration is an essential part of many (patho)physiological processes in the body, including keratinocyte re-epithelialization of healing wounds. Recent interest in the mechanobiology of tissues suggests that physical forces and mechanical cues from the wound bed (in addition to biochemical signals) may also play an important role in the healing process. Previously, we explored this possibility and found that polyacrylamide (PA) gel stiffness affected primary human keratinocyte behavior and that mechanical deformations in soft (~1.2 kPA) PA gels produced by neighboring cells appeared to influence the process ofde novoepithelial sheet formation. In order to clearly demonstrate that keratinocytes do respond to such deformations, we conducted a series of experiments where we observed the response of single keratinocytes to a prescribed local substrate deformation that mimicked a neighboring cell or evolving multicellular aggregate via a servo-controlled microneedle. We also examined the effect of adding either Y27632, a rho kinase inhibitor, or blebbistatin, a non-muscle myosin II inhibitor, on the response of the cells to PA gel deformations. The results of this study indicate that keratinocytes do sense and respond to mechanical signals comparable to those that originate from substrate displacements imposed by neighboring cells, a finding that could have important implications for the process of keratinocyte re-epithelialization that takes place during normal and pathologic wound healing. Furthermore, the Rho/ROCK pathway and the engagement of NM II are both essential to the observed process of substrate deformation-directed keratinocyte migration.

Aim To examine putative interaction between adrenergic and muscarinic contractile activation in the human urinary outflow tract. Methods Tissue from the trigone and prostatic urethra was obtained from 12 cystectomy and 16 prostatectomy specimen. Contractions were elicited by exposure to exogenous agonists before and after inhibition of Rho kinase and protein kinase c (PKC). Immunofluorescence and Western-blot studies were performed using antibodies to muscarinic M3-receptors (M3-R) and alpha1A-adrenoreceptors (alpha1A-AR). The study is registered with ClinicalTrials.gov, number NCT01227447. Results There was strong co-localization of M3-R and alpha1A-AR on trigonal and urethral myocytes. Western blot analysis revealed a significantly higher expression of alpha1A-AR in the superficial than in the deep trigone. Phenylephrine (PE, 1 μm) augmented contractions induced by carbachol (CA, 3 μm) to more than threefold control in the male superficial trigone, and to about sevenfold control in the proximal urethra. No such potentiation could be detected in female bladder outlet. Both PKC inhibitor GF 109203X and Rho kinase inhibitor Y-27632 reduced responses to 1 μM PE as well as to 3 μM CA significantly. However, the synergistic effect of the combined intervention remained proportionally unaffected. Conclusions Muscarinic and adrenergic receptor activation exerts a strong synergistic effect in the male human bladder trigone and proximal urethra.

金沢大学医薬保健研究域医学系, Class II phosphoinositide 3-kinases (PI3Ks), PI3K-C2α and PI3K-C2β, are highly homologous and distinct from class I and class III PI3Ks in catalytic products and domain structures. In contrast to class I and class III PI3Ks, physiological roles of PI3K-C2α and PI3K-C2β are not fully understood. Because we previously demonstrated that PI3K-C2α is involved in vascular smooth muscle contraction, we studied the phenotypes of smooth muscle-specific knockout (KO) mice of PI3K-C2α and PI3K-C2β. The pup numbers born from single PI3K-C2α-KO and single PI3K-C2β-KO mothers were similar to those of control mothers, but those from double KO (DKO) mothers were smaller compared with control mice. However, the number of intrauterine fetuses in pregnant DKO mothers was similar to that in control mice. Both spontaneous and oxytocin-induced contraction of isolated uterine smooth muscle (USM) strips was diminished in DKO mice but not in either of the single KO mice, compared with control mice. Furthermore, contraction of USM of DKO mice was less sensitive to a Rho kinase inhibitor. Mechanistically, the extent of oxytocin-induced myosin light chain phosphorylation was greatly reduced in USM from DKO mice compared with control mice. The oxytocin-induced rise in the intracellular Ca2+ concentration in USM was similar in DKO and control mice. However, Rho activation in the intracellular compartment was substantially attenuated in DKO mice compared with control mice, as evaluated by fluorescence resonance energy transfer imaging technique. These data indicate that both PI3K-C2α and PI3K-C2β are required for normal USM contraction and parturition mainly through their involvement in Rho activation., Embargo Period 12 months

Recently drug-induced senescence has gained momentum as a new approach in cancer therapy. It is accepted that senescent cells display typical phenotypic features including flattened, enlarged, and multinucleated cell morphology. However, it is not well elucidated how these morphological alterations occur. The current study evaluates the possible role of Rho/Rho kinase pathway in cardiac glycoside-induced senescent cell morphology in HeLa cells. Our results indicate that the administration of cardiac glycosides, ouabain, digoxin, bufalin, to HeLa cells induced cellular senescence leading to an increase in the volume, area and maximum thickness of the cells. Although preincubation of specific Rho kinase inhibitor Y-27632 did not inhibit the occurrence of cardiac glycoside-induced senescence in cells, it reduced the cell area and cell volume. Inhibition of Rho by CT04 produced similar results as seen for the preincubation of Y-27632. In addition, inhibition of Rock caused a decrease in increased actin stress fibers in senescent cells induced by ouabain. Additionally, preincubation of Y-27632 decreased the ouabain-induced the phosphorylation of MYPT and cofilin. In conclusion, Rock inhibition-mediated alteration of senescent cell morphology may be associated with the decreased actin stress fibers formation. Since it is known that secretory activity is accompanied by the changes of cell morphology, these morphological alterations observed by the inhibition of Rho/Rho kinase pathway may also lead to important secretory functions of senescent cells.

Background: Rho-kinase (ROCK) activation in hepatic stellate cells (HSC) is a key mechanism promoting liver fibrosis and portal hypertension (PTH). Specific delivery of ROCK-inhibitor Y-27632 (Y27) to HSC targeting mannose-6-phosphate-receptors reduces portal pressure and fibrogenesis. In decompensated cirrhosis, presence of ascites is associated with reduced renal perfusion. Since in cirrhosis, platelet-derived growth factor receptor beta (PDGFRβ) is upregulated in the liver as well as the kidney, this study coupled Y27 to human serum albumin (HSA) substituted with PDGFRβ-recognizing peptides (pPB), and investigated its effect on PTH in cirrhotic rats. Methods: In vitro collagen contraction assays tested biological activity on LX2 cells. Hemodynamics were analyzed in BDL and CCl4 cirrhotic rats 3h, 6h and 24h after i.v. administration of Y27pPBHSA (0.5/1mg/kg b.w). Phosphorylation of moesin and myosin light chain (MLC) assessed ROCK activity in liver, femoral muscle, mesenteric artery, kidney and heart. Findings: Three Y27 molecules were coupled to pPBHSA as confirmed by HPLC/MS, which was sufficient to relax LX2 cells. In vivo, Y27pPBHSA-treated rats exhibited lower portal pressure, hepatic vascular resistance without effect on systemic vascular resistance, but a tendency towards lower cardiac output compared to non-treated cirrhotic rats. Y27pPBHSA reduced intrahepatic resistance by reduction of phosphorylation of moesin and MLC in Y27pPBHSA-treated cirrhotic rats. Y27pPBHSA was found in the liver of rats up to 6 hours after its injection, in the HSC demonstrated by double-immunostainings. Interestingly, Y27pPBHSA increased renal arterial flow over time combined with an antifibrotic effect as shown by decreased renal acta2 and col1a1 mRNA expression. Interpretation: Targeting the ROCK inhibitor Y27 to PDGFRI² decreases portal pressure with potential beneficial effects in the kidney. This unique approach should be tested in human cirrhosis. Funding: J. Trebicka is supported by grants from the Deutsche Forschungsgemeinschaft Grant SFB TRR57 P18, European Commission DirectorateGeneral for Research and Innovation Grant 668031, and Cellex Foundation. Declaration of Interest: The authors have no conflict of interest. Ethical Approval: All experiments were performed in accordance with the German Animal Protection Law and the Guidelines of the animal care facility (Haus fur experimentelle Therapie, University Portal and renal effects of targeted Rho-kinase inhibitor Clinics Bonn, Germany), and approved by the North Rhine-Westphalia State Agency for Nature, Environment, and Consumer Protection (LANUV, file reference LANUV NRW, 8402.04.2014.A137).

Stem cells play a crucial role in maintaining and repairing tissues during homeostasis and following injury. The efficient procurement of high quantity and quality of skin stem cells is important for both laboratory studies and clinical applications. Here, we describe a one-step isolation procedure to efficiently obtain both epidermal and dermal cell population from human skin specimen, based on the different influence of the Rho kinase inhibitor Y27632 on the growth of epidermal and dermal cells during the initial culture. Compared with the conventional methods, our protocol shows that it is simpler and less time consuming and can efficiently obtain the high quality of skin stem cells and can maintain the stem cell features after culture expansion.

Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense may be hindered by invasive isolation methods and/or improper culture conditions to support stem cell maintenance and other potential mechanisms contributing to long-term subcultivation in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases.

Background/Aims: Intravascular bubbles can exert pleiotropic detrimental effects, partly by inducing endothelial microparticles (EMPs) production, which play critical roles in cell communication and vascular inflammation cascades. However, the underlying mechanisms remain unclear. This study aimed to delineate the possible mechanisms involving bubble-induced EMPs formation. Methods: Human umbilical vein endothelial cells (HUVECs) were contacted by bubbles and EMPs level in supernatant were quantified by flow cytometry. Cytoplasmic calcium (Ca2+) was measured by the Ca2+ binding dyes Fluo-3 AM and flippase activity was assessed by translocation rate of fluorescent phosphatidylserine (PS) analogue NBD-PS. Protein levels of phospho-myosin light chain (MLC, a Rho kinase substrate) and phospho-extracellular signal-regulated kinase 1 or 2 (ERK1/2) were determined by western blotting. The score of actin colocalization was assessed by phalloidin-FITC using an immunofluorescent microscopy. Results: EMPs level markedly increased after bubble stimulus. Cytoplasmic calcium (Ca2+) significantly elevated (P< 0.05), flippase activity decreased (P< 0.05), protein levels of phospho- MLC and phospho- ERK1/2 significantly increased (P< 0.05, P < 0.05), and the score of actin colocalization markedly reduced (P< 0.05) in bubble-stimulated HUVECs. All the above changes except the increase in phospho-ERK1/2 can be reversed by Ca2+ channel blocker LaCl3 (P< 0.05). Additionally, MLC phosphorylation was significantly inhibited and actin colocalization markedly increased by Rho kinase inhibitor pretreatment and more importantly, bubble-induced EMPs markedly decreased. Conclusions: These results demonstrate that bubble stimulates EMPs formation by cytoplasmic Ca2+ elevation and subsequently activating Rho kinase pathway and cytoskeleton reorganization. Simultaneously, cytoplasmic Ca2+ inhibits the flippase activity and subsequently increases phosphatidylserine exposure, which also contributes to EMPs formation.

The generation of tissue-engineered epithelial models is often hampered by the limited proliferative capacity of primary epithelial cells. This study aimed to isolate normal tonsillar keratinocytes (NTK) from human tonsils, increase the lifespan of these cells using the Rho kinase inhibitor Y-27632 and to develop tissue-engineered equivalents of healthy and infected tonsil epithelium. The proliferation rate of isolated NTK and expression of c-MYC and p16INK4A were measured in the absence or presence of the inhibitor. Y-27632-treated NTK were used to generate tissue-engineered tonsil epithelium equivalents using de-epithelialized dermis that were then incubated with Streptococcus pyogenes to model bacterial tonsillitis, and the expression of pro-inflammatory cytokines was measured by cytokine array and ELISA. NTK cultured in the absence of Y-27632 rapidly senesced whereas cells cultured in the presence of this inhibitor proliferated for over 30 population doublings without changing their phenotype. Y-27632-treated NTK produced a multi-layered differentiated epithelium that histologically resembled normal tonsillar surface epithelium and responded to S. pyogenes infection by increased expression of pro-inflammatory cytokines including CXCL5 and IL-6. NTK can be isolated and successfully cultured in vitro with Y-27632 leading to a markedly prolonged lifespan without any deleterious consequences to the cell morphology. This functional tissue-engineered equivalent of tonsil epithelium will provide a valuable tool for studying tonsil biology and host-pathogen interactions in a more physiologically relevant manner.

Mesenchymal stem cells (MSCs) are a powerful source for cell therapy in degenerative diseases. The migration ability of MSCs is an important factor that enhances the therapeutic effect of the cells when they are transplanted into target tissues or organs. Hypoxia and the endothelial barrier, which are representative migration microenvironmental factors, are known to be regulated by the integrin-mediated pathway in several cancers. However, their regulatory mechanisms in MSCs remain unclear. Here, the objectives of the study were to compare the expression of markers related to integrin-mediated signaling in placenta-derived MSCs (PDMSCs) dependent on hypoxia and co-cultured with human umbilical vein endothelial cells (HUVECs) and to evaluate their correlations between migration ability and microenvironmetal factors including hypoxia and endothelial cells. The migration abilities of PDMSCs exposed to hypoxic conditions were significantly increased compared with normal fibroblasts (WI-38) and control (P

Purpose: To investigate the effect of the Rho-kinase inhibitor, Y27632, on pig corneal endothelial cell (pCEC) culture, and on inflammation and immune regulation of the responses of human cells to pCECs.Methods: pCECs were cultured with/without Y27632 to assess cell proliferation and in vitro wound healing assay. The level of MCP-1 and VEGF in pCECs stimulated with human TNF-α were measured. Proliferation of human PBMCs stimulated with pCECs, and cytokine production in human T cells, and monocyte migration after stimulation were investigated.Results: Y27632 promoted pCEC proliferation, prevented pCEC death, and enhanced in vitro wound healing. After stimulation, there were significantly lower levels of MCP-1 and VEGF measured in pCECs cultured with Y27632, and significantly reduced human PBMC proliferation, cytokine production, and monocyte migration.Conclusions: The application of the Rho-kinase inhibitor will be beneficial when culturing pCECs, and may provide a novel therapy to reduce inflammat...

This work aims to explore the efficacy of Rho kinase inhibitor Fasudil on cognitive impairment induced by chronic cerebral hypoperfusion in rats. A total of 32 male adult Sprague Dawley (SD) rats were randomly divided into three groups: treatment group, control group and sham-operated group for severe carotid artery stenosis model. After two weeks, 8.35 mg/kg Fasudil and physiological saline were intraperitoneally applied twice per day in treatment group and control group, respectively. Morris water maze test was performed in each group to detect the changes of cognitive function and observe the hippocampal pathomorphology in rats after eight weeks. The average escape latency distinctly shortened (P 0.05). Fasudil effectively improved hippocampal pathomorphology. Rho kinase inhibitor obviously ameliorated cognitive impairment induced by chronic cerebral hypoperfusion in rats.

Despite numerous reported attempts, successful isolation of genuine embryonic stem cells of cattle has been rare. Previous studies have shown that Thiazovivin, a Rho-associated kinase inhibitor, improves the survival and self-renewal of human embryonic stem cells. The present study demonstrates the effect of Thiazovivin on the derivation of embryo-derived stem-like cells. Attachment rates of blastocyst and embryonic cell clumps onto feeder cells in the Thiazovivin treatment group were greater than those of the control group. The pluripotency markers of the OCT4 and NANOG genes, and the adhesion molecule E-cadherin were increased by Thiazovivin treatment. This study suggests that Thiazovivin treatment improves the maintenance of stemness in a putative stem-like cell populations of cattle by promoting the expression of pluripotency marker genes, as well as enhancing the expression of the E-cadherin gene, resulting in an increase in cell adhesion.

Vascular remodeling in response to hemodynamic alterations is a physiological process that requires coordinated signaling between endothelial, inflammatory and vascular smooth muscle cells (VSMCs). Extensive experimental and clinical studies have indicated the critical role of the Ras homolog gene family, member A/Rho‑associated kinase (ROCK) signaling pathway in the pathogenesis of cardiovascular disease, where ROCK activation has been demonstrated to promote inflammation and remodeling through inducing the expression of proinflammatory cytokines and adhesion molecules in endothelial cells and VSMCs. However, the role of ROCK in flow‑induced vascular remodeling has not been fully defined. The current study aimed to investigate the effect of the ROCK signaling pathway in flow‑induced vascular remodeling by comparing the responses to partial carotid artery ligation in mice treated with fasudil (a ROCK inhibitor) and untreated mice. Intima‑media thickness and neointima formation were evaluated by morphology. VSMC proliferation and inflammation of the vessel wall were assessed by immunohistochemistry. In addition, the expression levels of ROCK and the downstream effectors of ROCK, myosin light chain (MLC) and phosphorylated‑MLC (p‑MLC), were quantified by western blot analysis. Following a reduction in blood flow, ROCK1 and p‑MLC expression increased in the untreated left common carotid arteries (LCA). Fasudil‑treated mice developed a significantly smaller intima‑media thickness compared with the untreated mice. Quantitative immunohistochemistry of the fasudil‑treated LCA indicated that there was a reduction in proliferation when compared with untreated vessels. There were fewer CD45+ cells observed in the fasudil‑treated LCA compared with the untreated LCA. In conclusion, the expression of ROCK was enhanced in flow‑induced carotid artery remodeling and ROCK inhibition as a result of fasudil treatment may attenuate flow‑induced carotid artery remodeling.

BACKGROUND Rho kinase (ROCK) pathway is associated with various cellular functions, such as smooth muscle contraction, inflammatory response, and cell proliferation. The spontaneously hypertensive rat (SHR) is commonly used genetically hypertensive rat model which develops hyperplastic morphological abnormalities in the ventral prostate. We investigated whether administration of hydroxyfasudil, a ROCK inhibitor, could reduce the levels of growth factors, inflammatory markers, and morphological abnormalities in the ventral prostate of the SHR. METHODS Twelve-week-old SHRs were treated with hydroxyfasudil (1 mg/kg/day, i.p.) or vehicle once daily for another 6 weeks. Wistar Kyoto (WKY) rats treated with vehicle were used as normotensive controls. At 18 weeks of age, blood pressure and heart rate were measured by the tail cuff method. Then the rats were sacrificed, and the ventral prostates were removed. The levels of ROCK activity, growth factors (TGF-β1 and bFGF), a smooth muscle differentiation marker (α-SMA) and an inflammatory cytokine (IL-6) in the ventral prostate were measured by ELISA and western blot. A histological evaluation in each group was also performed. RESULTS There were significant increases in blood pressure, prostate weight, prostate body weight ratio, and tissue levels of ROCK activity, TGF-β1, bFGF, α-SMA, and IL-6 in the SHR compared to the WKY rat. Histological examination of the ventral prostate showed morphological abnormalities such as a higher degree of proliferation in the glandular epithelial and stromal area in the SHR compared to the WKY rat. Treatment with hydroxyfasudil reduced the elevated ROCK activity, TGF-β1, bFGF, α-SMA, and IL-6 found in the ventral prostate of the SHR. Moreover, treatment with hydroxyfasudil decreased the morphological abnormalies in the SHR ventral prostate. CONCLUSIONS Treatment with hydroxyfasudil decreased the growth factors, an inflammatory cytokine, and morphological abnormalies in the SHR ventral prostate. These results suggest that chronic treatment with hydroxyfasudil may inhibit the progression of prostatic hyperplasia in the SHR. Prostate 75:1774–1782, 2015. © 2015 Wiley Periodicals, Inc.

The idea of direct differentiation of somatic cells into other differentiated cell types has attracted a great interest recently. Rho-kinase inhibitor Y-27632 (ROCKi) is a potential drug molecule, which has been reported to support the gene expressions typical for the chondrocytes, thus restricting their phenotypic conversion to fibroblastic cells upon the cellular expansion. In this study, we have investigated the short-term biological responses of ROCKi to human primary foreskin fibroblasts. The fibroblast cells were exposed to 1 and 10 μM ROCKi treatments. A proteomics analysis revealed expression changes of 56 proteins, and a further protein pathway analysis suggested their association with the cell morphology, the organization, and the increased cellular movement and the proliferation. These functional responses were confirmed by a Cell-IQ time-lapse imaging analysis. Rho-kinase inhibitor treatment increased the cellular proliferation up to twofold during the first 12 h, and a wound model based migration assay showed 50% faster filling of the mechanically generated wound area. Additionally, significantly less vinculin-associated focal adhesions were present in the ROCKi-treated cells. Despite the marked changes in the cell behavior, ROCKi was not able to induce the expression of the chondrocyte-specific genes, such as procollagen α1 (II) and aggrecan.

With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferation of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell–cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen transmission that might arise from animal-derived materials.

MVs are released in response to several stress agents, in an attempt to prevent continued cellular damage. After an initial stimulus of prostate cancer cells with sublytic C5b-9 and activation of MV release through PKC, cells take at least 20 min to fully recover their ability to microvesiculate. This release of MVs through activation of sublytic C5b-9 was inhibited by the PKC inhibitor bisindoylmaleimide I but not the Rho kinase inhibitor, Y27632. After stimulus there is a rise of 79 nMs(-1) over 11 s, reaching a peak [Ca(2+)]i of 920 nM. The concentration of cytosolic calcium then falls steadily at 2.4 nMs(-1) over 109 s reaching baseline levels (50-100 nM) within 10-15 min. In PC3 cells the rate of release of MVs from stimulated cells also reaches a minimum within 10-15 min. Using fura-2 AM-loaded cells, upon stimulation, cells were found to release MVs with a concentration of intravesicular calcium estimated at ∼ 430 nM.

Preliminary proteomics studies between tonic vs. phasic smooth muscles identified three distinct protein spots identified to be those of transgelin (SM22). The latter was found to be distinctly downregulated in the internal anal sphincter (IAS) vs. rectal smooth muscle (RSM) SMC. The major focus of the present studies was to examine the differential molecular control mechanisms by SM22 in the functionality of truly tonic smooth muscle of the IAS vs. the adjoining phasic smooth muscle of the RSM. We monitored SMC lengths before and after incubation with pFLAG-SM22 (for SM22 overexpression), and SM22 small-interfering RNA. pFLAG-SM22 caused concentration-dependent and significantly greater relaxation in the IAS vs. the RSM SMCs. Conversely, temporary silencing of SM22 caused contraction in both types of the SMCs. Further studies revealed a significant reverse relationship between the levels of SM22 phosphorylation and the amount of SM22-actin binding in the IAS and RSM SMC. Data showed higher phospho-SM22 levels and decreased SM22-actin binding in the IAS, and reverse to be the case in the RSM SMCs. Experiments determining the mechanism for SM22 phosphorylation in these smooth muscles revealed that Y-27632 (Rho kinase inhibitor) but not Gö-6850 (protein kinase C inhibitor) caused concentration-dependent decreased phosphorylation of SM22. We speculate that SM22 plays an important role in the regulation of basal tone via Rho kinase-induced phosphorylation of SM22.

Aim Hydrogen sulphide (H2S) exhibits a dual modulation of isolated artery tension. This study investigated the vasoconstrictive effect of sulphur sodium hydride (NaHS), a donor of gaseous H2S, on rat coronary artery. Methods The contractile response of isolated arteries was recorded using a wire myograph. Fluo-3/AM was used to load vascular smooth muscle, and intracellular calcium was determined using confocal laser microscopy. The protein expression of Rho kinase was examined using Western blot. Results NaHS induced concentration-dependent contractions of rat coronary artery, and the contraction reached approx. 65% of 60 mm KCl-induced contraction. The NaHS-induced contraction was elevated following the removal of endothelium or the use of the nitric oxide synthase inhibitor L-NAME. The cyclooxygenase inhibitor indomethacin reduced NaHS-induced contraction. The Rho kinase inhibitor Y-27632 significantly attenuated NaHS-induced vasoconstriction. Furthermore, NaHS elevated the protein expression of Rho kinase. NaHS-induced contraction was completely abolished in a Ca2+-free solution and suppressed by the Ca2+ influx blocker nifedipine (100 nm). NaHS also significantly increased the change rate of Ca2+ fluorescence intensity. However, treatment with a Cl−/HCO3− exchanger blocker, K+ channel blockers, the mitogen-activated protein kinase inhibitor U-0126 or cyclic adenosine monophosphate did not affect contraction. Species-dependent differences in NaHS-induced vasoconstriction were observed because these effects were only modest in dog coronary artery and absent in rabbit coronary artery. Conclusions NaHS induces the contraction of rat coronary artery, which is dependent on the activation of Ca2+ influx. Rho kinase likely participates in the vasoconstriction.

Ocular hypertension arising from increased resistance to aqueous humor (AH) outflow through the trabecular meshwork is a primary risk factor for open-angle glaucoma, a leading cause of blindness. Ongoing efforts have found little about the molecular and cellular bases of increased resistance to AH outflow through the trabecular meshwork in ocular hypertension patients. To test the hypothesis that dysregulated Rho GTPase signaling and a resulting fibrotic activity within the trabecular meshwork may result in ocular hypertension, we investigated the effects of expressing a constitutively active RhoA GTPase (RhoAV14) in the AH outflow pathway in Sprague-Dawley rats by using lentiviral vector-based gene delivery. Rats expressing RhoAV14 in the iridocorneal angle exhibited a significantly elevated intraocular pressure. Elevated intraocular pressure in the RhoAV14-expressing rats was associated with fibrotic trabecular meshwork and increased levels of F-actin, phosphorylated myosin light chain, α-smooth muscle actin, collagen-1A, and total collagen in the trabecular AH outflow pathway. Most of these changes were ameliorated by topical application of Rho kinase inhibitor. Human autopsy eyes from patients with glaucoma exhibited significant increases in levels of collagen-1A and total collagen in the trabecular AH outflow pathway. Collectively, these observations indicate that increased fibrogenic activity because of dysregulated RhoA GTPase activity in the trabecular AH outflow pathway increases intraocular pressure in a Rho kinase-dependent manner.

Background Activation of the Rho–Rho-kinase pathway has been shown to cause vasoconstriction in renal afferent arterioles. Vascular dysfunction plays a pivotal role in triggering reperfusion injury after kidney transplantation. Therefore, the effect of a Rho-kinase inhibitor, added to the preservation solution, on renal function after 18 h of storage at 4 °C was evaluated. Methods Porcine kidneys were preserved with cold HTK-solution. During preservation, in the study group, HTK was supplemented with the Rho-kinase inhibitor HA1077, whereas the control group received no further treatment ( n = 6, respectively). Kidney function after 18 h of storage at 4 °C was evaluated by 90 min of isolated reperfusion in vitro . Results Rho-kinase inhibition (RKI) was associated with significantly higher renal perfusate flow compared to the control group. Endothelial function, as measured by perfusate levels of nitric oxide and gene expression of eNOS, was significantly increased in the study group. In our model, RKI also significantly improved glomerular function (clearance of creatinine) as well as tubular cell integrity as reflected by reduced fractional sodium excretion and release of fatty acid binding protein, a specific tubular cell marker. Conclusion Our results indicate that blocking the Rho-kinase pathway during cold preservation may lead to a better graft function upon reperfusion.

Myogenic responses by resistance vessels are a key component of autoregulation in brain, thus playing a crucial role in regulating cerebral blood flow and protecting the blood–brain barrier against potentially detrimental elevations in blood pressure. Although cerebrovascular disease is often accompanied by alterations in myogenic responses, mechanisms that control these changes are poorly understood. Peroxisome proliferator–activated receptor γ has emerged as a regulator of vascular tone. We hypothesized that interference with peroxisome proliferator–activated receptor γ in smooth muscle would augment myogenic responses in cerebral arteries. We studied transgenic mice expressing a dominant-negative mutation in peroxisome proliferator–activated receptor γ selectively in smooth muscle (S-P467L) and nontransgenic littermates. Myogenic tone in middle cerebral arteries from S-P467L was elevated 3-fold when compared with nontransgenic littermates. Rho kinase is thought to play a major role in cerebrovascular disease. The Rho kinase inhibitor, Y-27632, abolished augmented myogenic tone in middle cerebral arteries from S-P467L mice. CN-03, which modifies RhoA making it constitutively active, elevated myogenic tone to ≈60% in both strains, via a Y-27632–dependent mechanism. Large conductance Ca 2+ -activated K + channels (BK Ca ) modulate myogenic tone. Inhibitors of BK Ca caused greater constriction in middle cerebral arteries from nontransgenic littermates when compared with S-P467L. Expression of RhoA or Rho kinase-I/II protein was similar in cerebral arteries from S-P467L mice. Overall, the data suggest that peroxisome proliferator–activated receptor γ in smooth muscle normally inhibits Rho kinase and promotes BK Ca function, thus influencing myogenic tone in resistance arteries in brain. These findings have implications for mechanisms that underlie large- and small-vessel disease in brain, as well as regulation of cerebral blood flow.

In endothelial cells, the RhoGTPase Rac1 stabilizes VE-cadherin trans-dimers in mature adherens junctions by counteracting actomyosin tension., The role of the RhoGTPase Rac1 in stabilizing mature endothelial adherens junctions (AJs) is not well understood. In this paper, using a photoactivatable probe to control Rac1 activity at AJs, we addressed the relationship between Rac1 and the dynamics of vascular endothelial cadherin (VE-cadherin). We demonstrated that Rac1 activation reduced the rate of VE-cadherin dissociation, leading to increased density of VE-cadherin at AJs. This response was coupled to a reduction in actomyosin-dependent tension across VE-cadherin adhesion sites. We observed that inhibiting myosin II directly or through photo-release of the caged Rho kinase inhibitor also reduced the rate of VE-cadherin dissociation. Thus, Rac1 functions by stabilizing VE-cadherin trans-dimers in mature AJs by counteracting the actomyosin tension. The results suggest a new model of VE-cadherin adhesive interaction mediated by Rac1-induced reduction of mechanical tension at AJs, resulting in the stabilization of VE-cadherin adhesions.

Lipid emulsions are widely used for the treatment of systemic toxicity that arises from local anesthetics. The goal of this in vitro study was to examine the cellular mechanism associated with the lipid emulsion-mediated attenuation of vasodilation induced by a toxic dose of bupivacaine in isolated endothelium-denuded rat aorta. The effects of lipid emulsion on vasodilation induced by bupivacaine, mepivacaine, and verapamil were assessed in isolated aorta precontracted with phenylephrine, the Rho kinase stimulant NaF, and the protein kinase C activator phorbol 12,13-dibutyrate (PDBu). The effects of Rho kinase inhibitor Y-27632 on contraction induced by phenylephrine or NaF were assessed. The effects of bupivacaine on intracellular calcium concentrations ([Ca(2+)]i) and tension induced by NaF were simultaneously measured. The effects of bupivacaine alone and lipid emulsion plus bupivacaine on myosin phosphatase target subunit 1 (MYPT1) phosphorylation induced by NaF were examined in rat aortic vascular smooth muscle cells. In precontracted aorta, the lipid emulsion attenuated bupivacaine-induced vasodilation but had no effect on mepivacaine-induced vasodilation. Y-27632 attenuated contraction induced by either phenylephrine or NaF. The lipid emulsion attenuated verapamil-induced vasodilation. Compared with phenylephrine-induced precontracted aorta, bupivacaine-induced vasodilation was slightly attenuated in NaF-induced precontracted aorta. The magnitude of the bupivacaine-induced vasodilation was higher than that of a bupivacaine-induced decrease in [Ca(2+)]i. Bupivacaine attenuated NaF-induced MYPT1 phosphorylation, whereas lipid emulsion pretreatment attenuated the bupivacaine-induced inhibition of MYPT1 phosphorylation induced by NaF. Taken together, these results suggest that lipid emulsions attenuate bupivacaine-induced vasodilation via the attenuation of inhibition of MYPT1 phosphorylation evoked by NaF.