Effect Of Rho-kinase Inhibitor Research Articles

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

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

Rho kinase inhibitors ameliorate cognitive impairment in a male mouse model of methamphetamine-induced schizophrenia

Schizophrenia (SCZ) is a severe psychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive deficits. Current antipsychotic treatment in SCZ improves positive symptoms but has major side effects and little impact on negative symptoms and cognitive impairment. The pathoetiology of SCZ remains unclear, but is known to involve small GTPase signaling. Rho kinase, an effector of small GTPase Rho, is highly expressed in the brain and plays a major role in neurite elongation and neuronal architecture. This study used a touchscreen-based visual discrimination (VD) task to investigate the effects of Rho kinase inhibitors on cognitive impairment in a methamphetamine (METH)–treated male mouse model of SCZ. Systemic injection of the Rho kinase inhibitor fasudil dose-dependently ameliorated METH-induced VD impairment. Fasudil also significantly suppressed the increase in the number of c-Fos–positive cells in the infralimbic medial prefrontal cortex (infralimbic mPFC) and dorsomedial striatum (DMS) following METH treatment. Bilateral microinjections of Y-27632, another Rho kinase inhibitor, into the infralimbic mPFC or DMS significantly ameliorated METH-induced VD impairment. Two proteins downstream of Rho kinase, myosin phosphatase-targeting subunit 1 (MYPT1; Thr696) and myosin light chain kinase 2 (MLC2; Thr18/Ser19), exhibited increased phosphorylation in the infralimbic mPFC and DMS, respectively, after METH treatment, and fasudil inhibited these increases. Oral administration of haloperidol and fasudil ameliorated METH-induced VD impairment, while clozapine had little effect. Oral administration of haloperidol and clozapine suppressed METH-induced hyperactivity, but fasudil had no effect. These results suggest that METH activates Rho kinase in the infralimbic mPFC and DMS, which leads to cognitive impairment in male mice. Rho kinase inhibitors ameliorate METH-induced cognitive impairment, perhaps via the cortico-striatal circuit.

Effect and mechanism of Rho kinase inhibitor on intestinal injury in septic rats

To explore the effect of Rho kinase inhibitor on intestinal injury in septic rats and its possible mechanism. Thirty-two male Sprague-Dawley (SD) rats were randomly divided into sham operation group (Sham group), Rho kinase inhibitor Y-27632 control group (Y+Sham group), sepsis model group [cecal ligation and puncture (CLP) group] and Y-27632 pretreatment group (Y+CLP group), with 8 rats in each group. Rat sepsis model was reproduced by CLP. The rats in the Sham group and Y+Sham group were only separated and moved the cecum without ligation and perforation. The rats in the Y+Sham group and Y+CLP group were pretreated with intraperitoneal injection of Y-27632 solution 5 mg/kg 15 minutes before operation; the rats in the Sham group and CLP group were intraperitoneally injected with the same amount of phosphate buffered saline (PBS). Twenty-four hours after operation, the heart blood was collected and the serum diamine oxidase (DAO) content was determined by enzyme-linked immunosorbent assay (ELISA). Then the small intestine tissue was collected, the pathological changes of the intestinal tissue were observed under the light microscope after hematoxylin-eosin (HE) staining, and Chiu's score was performed. The positive expressions of Rho-related coiled-coil kinase 1 (ROCK1) and nuclear factor-κB (NF-κB) in intestinal tissue were detected by immunohistochemistry. ELISA was used to detect the levels of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in intestinal tissue homogenate. The intestinal tissue structure of the Sham group and Y+Sham group was intact and the mucosa was arranged neatly. Compared with the Sham group, the intestinal mucosa of the CLP group was arranged disorderly, with a large number of inflammatory cells infiltration, and the Chiu's score was significantly increased (3.83±0.27 vs. 0.12±0.11, P < 0.05), indicating that those rats suffered from septic intestinal injury. Compared with the CLP group, the degree of necrosis of intestinal epithelial cells in the Y+CLP group was reduced, a small amount of inflammatory cells infiltration was seen, and the Chiu's score was significantly decreased (2.85±0.21 vs. 3.83±0.27, P < 0.05), indicating that Y-27632 pretreatment could alleviate intestinal injury in septic rats. Compared with the Sham group, the positive expressions of intestinal tissue ROCK1 and NF-κB, the contents of serum DAO and intestinal homogenate TNF-α in the CLP group were significantly increased [ROCK1 expression (A value): 0.19 (0.18, 0.22) vs. 0.10 (0.09, 0.11), NF-κB expression (A value): 0.40±0.02 vs. 0.15±0.01, DAO (ng/L): 287.81±23.31 vs. 144.92±17.72, TNF-α (ng/L): 101.08±5.62 vs. 74.81±5.56, all P < 0.05], the level of intestinal homogenate IL-10 was significantly decreased (μg/L: 55.16±5.20 vs. 95.95±7.53, P < 0.05). Compared with the CLP group, the positive expressions of intestinal tissue ROCK1, NF-κB, the contents of serum DAO and intestinal homogenate TNF-α in the Y+CLP group were significantly decreased [ROCK1 expression (A value): 0.15 (0.13, 0.18) vs. 0.19 (0.18, 0.22), NF-κB expression (A value): 0.28±0.01 vs. 0.40±0.02, DAO (ng/L): 243.34±19.76 vs. 287.81±23.31, TNF-α (ng/L): 90.41±8.79 vs. 101.08±5.62, all P < 0.05], while the level of intestinal homogenate IL-10 was significantly increased (μg/L: 66.15±5.74 vs. 55.16±5.20, P < 0.05), indicating that the protective effect of Y-27632 pretreatment on sepsis intestinal injury rats might be related to the regulation of RhoA/ROCK1/NF-κB signaling pathway. Rho kinase inhibitors can reduce intestinal injury in septic rats, and the mechanism may be related to inhibiting RhoA/ROCK1/NF-κB signaling pathway and reducing intestinal inflammation in septic rats.

Roles of RAGE/ROCK1 Pathway in HMGB1-Induced Early Changes in Barrier Permeability of Human Pulmonary Microvascular Endothelial Cell.

BackgroundHigh mobility group box 1 (HMGB1) causes microvascular endothelial cell barrier dysfunction during acute lung injury (ALI) in sepsis, but the mechanisms have not been well understood. We studied the roles of RAGE and Rho kinase 1 (ROCK1) in HMGB1-induced human pulmonary endothelial barrier disruption.MethodsIn the present study, the recombinant human high mobility group box 1 (rhHMGB1) was used to stimulate human pulmonary microvascular endothelial cells (HPMECs). The endothelial cell (EC) barrier permeability was examined by detecting FITC-dextran flux. CCK-8 assay was used to detect cell viability under rhHMGB1 treatments. The expression of related molecules involved in RhoA/ROCK1 pathway, phosphorylation of myosin light chain (MLC), F-actin, VE-cadherin and ZO-1 of different treated groups were measured by pull-down assay, western blot and immunofluorescence. Furthermore, we studied the effects of Rho kinase inhibitor (Y-27632), ROCK1/2 siRNA, RAGE-specific blocker (FPS-ZM1) and RAGE siRNA on endothelial barrier properties to elucidate the related mechanisms.ResultsIn the present study, we demonstrated that rhHMGB1 induced EC barrier hyperpermeability in a dose-dependent and time-dependent manner by measuring FITC-dextran flux, a reflection of the loss of EC barrier integrity. Moreover, rhHMGB1 induced a dose-dependent and time-dependent increases in paracellular gap formation accompanied by the development of stress fiber rearrangement and disruption of VE-cadherin and ZO-1, a phenotypic change related to increased endothelial contractility and endothelial barrier permeability. Using inhibitors and siRNAs directed against RAGE and ROCK1/2, we systematically determined that RAGE mediated the rhHMGB1-induced stress fiber reorganization via RhoA/ROCK1 signaling activation and the subsequent MLC phosphorylation in ECs.ConclusionHMGB1 is capable of disrupting the endothelial barrier integrity. This study demonstrates that HMGB1 activates RhoA/ROCK1 pathway via RAGE, which phosphorylates MLC inducing stress fiber formation at short time, and HMGB1/RAGE reduces AJ/TJ expression at long term independently of RhoA/ROCK1 signaling pathway.

The effect of Rho Kinase inhibition on corneal nerve regeneration in vitro and in vivo

PurposeImpairment of corneal nerves can lead to neurotrophic keratopathy accompanied with severe ocular surface damage, which due to limited treatment options, can result in severe visual deterioration. This study evaluates a possible new treatment by enhancing the corneal nerve regeneration using a Rho Kinase inhibitor (Y27632). ROCK is known to play an important role in regulating cell morphology, adhesion and motility but little is known about its role in corneal nerve regeneration. MethodsEffects of ROCK inhibition on murine peripheral nerves was assessed in single cell- and wound healing assays as well as a 3D in vitro model. Furthermore, Sholl analysis evaluating neuronal branching and life-death assays evaluating toxicity of the inhibitor were performed. An in vivo mouse model was established, with monitoring weekly corneal nerve regrowth using confocal microscopy. Additionally, corneal nerve fiber length was evaluated by immunofluorescence staining. Underlying pathways were examined by qrtPCR. ResultsROCK inhibition leads to a significant enhancement of fiber growth in vitro. Sholl analysis revealed a higher degree of branching of treated fibers. Cytotoxicity assay showed no influence of Y27632 on cellular survival. In vivo measurement revealed significant enhanced regeneration after injury in the treated group. QrtPCR of trigeminal ganglia confirmed ROCK knock-down as well as altered pathways. ConclusionThe inhibition of ROCK after corneal nerve injury resulted in an enhanced regrowth of fibers in vitro and in vivo. This might be a step towards a new therapeutic concept for the treatment of impaired corneal nerves in diseases such as neurotrophic keratopathy.

Ispitivanje neuroprotektivnog efekta inhibicije Ro-kinaze u ćelijskom modelu neurodegeneracije izazvane 1-metil-4-fenilpiridinijumom (MPP+)

Introduction: The 1-methyl 4-phenyl 1,2,3,6-tetrahydropiridium (MPTP) induced model of neurodegeneration in Parkinson's disease (PD) is one of the most commonly used experimental models. This neurotoxic agent , or rather its metabolite MPP+, leads to inhibition of mitochondrial complex I, an increase in free radicals' production and ATP depletion, all resulting in cellular demise and death. Rho-kinase is an enzyme involved with numerous cellregulatory mechanisms, such as cytoskeleton organization, axonogenesis, vesicular transport regulation and apoptosis regulation, which are all important for cell survival. Aim: Our aim was to investigate the effects of Rho-kinase inhibition on the MPP+ induced model of neurodegeneration and the role of Akt and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways in this process. Material and methods: The experiments were performed on the human neuroblastoma SHSY5Y cell line. The MTT test was used to measure the viability of the cells after the MPP+ and/ or Rho-kinase inhibitor, fasudil, treatments. Changes in activation levels, or expression of pAMPK, pAkt, AMPK and Akt, were measured using the immunoblotting method, and the protein levels were quantified by densitometry. Results: The MPP+ caused a dose-dependent decrease in cellular viability, compared to the control group (untreated cells), while fasudil treatment, prior to MPP+ exposure, improved cell viability in a dose dependant manner, compared to MPP+ treatment. Analysis of activation status of target proteins showed an increase in Akt activation after the fasudil treatment, while the AMPK activation was not significantly changed. Conclusion: Inhibition of Rho-kinase using fasudil causes a decrease in MPP+ induced cell death, which is possibly mediated by an activation of the Akt/PI3K signaling pathway.

Cytoprotective Effect of Rho Kinase Inhibitors against Oxidative Stress inTrabecular Meshwork Cells

Cytoprotective Effect of Rho Kinase Inhibitors against Oxidative Stress inTrabecular Meshwork Cells

Short-term Effect of Rho-kinase Inhibitor on Retinal Circulation

Short-term Effect of Rho-kinase Inhibitor on Retinal Circulation

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

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

Lentiviral Vector-Mediated Expression of Exoenzyme C3 Transferase Lowers Intraocular Pressure in Monkeys.

Primary open-angle glaucoma (POAG) is considered a lifelong disease characterized by optic nerve deterioration and visual field damage. Although the disease progression can usually be controlled by lowering the intraocular pressure (IOP), therapeutic effects of current approaches do not last long. Gene therapy could be a promising method for persistent treatment of the disease. Our previous study demonstrated that gene transfer of exoenzyme C3 transferase (C3) to the trabecular meshwork (TM) to inhibit Rho GTPase (Rho), the upstream signal molecule of Rho-associated kinase (ROCK), resulted in lowered IOP in normal rodent eyes. In the present study, we show that the lentiviral vector (LV)-mediated C3 expression inactivates RhoA in human TM cells by ADP ribosylation, resulting indisruption of the actin cytoskeleton and altered cell morphology. In addition, intracameral delivery of the C3 vector to monkey eyes leads to persistently lowered IOP without obvious signs of inflammation. This is the first report of using a vector to transduce the TM of an alive non-human primate with a gene that alters cellular machinery and physiology. Our results in non-human primates support that LV-mediated C3 expression in the TM may have therapeutic potential forglaucoma, the leading cause of irreversible blindness in humans.

329 Effect of Rho kinase inhibitor on culture of primary mouse keratinocytes

329 Effect of Rho kinase inhibitor on culture of primary mouse keratinocytes

Evaluation of the Rho A/Rho-kinase pathway in the uterus of the rat model of polycystic ovary syndrome

The aim of this study was to investigate the expression of RhoA/Rho-kinase in the uterus and the effect of Rho-kinase inhibitors on uterine contractions of dehydroepiandrosterone (DHEA) induced polycystic ovary syndrome (PCOS) rats. Forty-four female Sprague-Dawley (21 days old) rats divided into three groups: The control group (n = 14, any procedure was not performed), vehicle group (n = 14, 0.2 ml of sesame oil, subcutaneous injection, 20 days) and PCOS group (n = 16, DHEA 6 mg/100 g in 0.2 ml of sesame oil, subcutaneous injection, 20 days). The myometrium thickness and uterine wet weight were assessed. The mRNA and protein expressions of Rho A, the effect of Rho-kinase inhibitors (fasudil and Y-27632) on KCl, carbachol, and PGF2α induced contractions were evaluated in the uterus. In the PCOS group, the myometrium thickness and uterine wet weight significantly increased compared to the control group and vehicle group. The mRNA expression level and the immunoreactive score of Rho A, ROCK 1, ROCK 2 were similar in all groups. In the PCOS group, KCl, carbachol, and PGF2α induced uterine contractions significantly increased compared to the control group and vehicle group. Fasudil and Y-27632 significantly inhibited KCl, carbachol, and PGF2α induced uterine contractions in all groups. In conclusion, the expression of Rho A, ROCK 1, ROCK 2 not changed although myometrium thickness, uterine wet weight and the contractile responses of uterus increased in the PCOS group. The results suggest that the Rho-kinase inhibitors effectively suppressed increased contractions in the PCOS group they might be potential therapeutic agents.

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

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

Therapeutic effect of Rho kinase inhibitor FSD‑C10 in a mouse model of Alzheimer's disease

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.

Treatment with low dose fasudil for acute ischemic stroke in chronic hypertension

We investigated the effect of Rho kinase inhibition on changes in cerebral blood flow (CBF), brain injury and vascular function after ischemic stroke in spontaneously hypertensive rats (SHR). Changes in core MCA and collateral perfusion were measured by a validated laser Doppler method. Animals underwent 2 h tMCAO and 2 h reperfusion. Fasudil (0.1 mg/kg, i.v.) or vehicle was given at 30 min ischemia (n = 9/group; mean (SD)). Brain injury was determined by 2,3,5-triphenyltetrazolium chloride staining. To determine the effect of fasudil on vascular function, fasudil was given 10 min before reperfusion and parenchymal arterioles studied isolated (n = 6/group; mean(SD)). Collateral perfusion was low in vehicle-treated SHR (−8(32)%) that changed minimally with fasudil (6(24)%, p > 0.05, effect size: 0.47;95% CI−0.49–1.39). Reperfusion CBF was below baseline in vehicle (−27(26)%) and fasudil (−32(25)%, p > 0.05, effect size: 0.19; 95% CI−0.74–1.11) groups, suggesting incomplete reperfusion in both groups. Fasudil had little effect on brain injury volume (28(13)% vs. 36(7)% in vehicle, p > 0.05, effect size: 0.75; 95% CI−0.24–1.66). In isolated parenchymal arterioles, myogenic tone was similar between groups (37(6)% vs. 38(10)% in vehicle, p > 0.05, effect size: 0.09; 95% CI−1.05–1.21). There were no differences with fasudil treatment vs. vehicle in perfusion, brain injury and vascular function that may be related to the low dose that had minimal blood pressure lowering effect.

Effects of Rho kinase inhibitor fasudil on paraquat-induced pulmonary fibrosis in rats

Objective To investigate the effect of Rho kinase inhibitor, fasudil, on pulmonary fibrosis induced by paraquat in rats in order to elucidate the underlying mechanisms. Methods A total of 72 Sprague-Dawley male rats of specific pathogen free (SPF) were randomly(random number) divided into four groups: the normal control group (NS group, n=18), fasudil control group (FS control group, n=18), paraquat poisoning group (PQ poisoning group, n=18) and fasudil intervention group (FS intervention group, n=18). On days 7, 14, 28 after paraquat exposure, six rats were respectively selected from each group. These rats were anesthetized and sacrificed immediately, and their lung tissues were collected. The hydroxyproline (HYP) in the lung tissue was detected by using alkaline hydrolysis. The expressions of type Ⅰ, Ⅲ collagen protein, connective tissue growth factor (CTGF) and ROCK1 mRNA in Rho/ROCK signaling pathway were assayed by using the real-time quantitative PCR (RT-PCR), and the levels of type Ⅰ, Ⅲ collagen protein, connective tissue growth factor (CTGF) and Rho / ROCK signaling pathway ROCK1 protein were measured by using Western blotting. The pathological changes of lung tissue were observed under light microscope. Results There were no significant differences in the observed biomarkers between FS control group and NS group (P>0.05). While in PQ poisoning group and FS intervention group on days 7, 14, 28 (all P<0.05), the amount of HYP increased obviously (P<0.05), the expressions of type Ⅰ, Ⅲ collagen protein, CTGF, ROCK1 mRNA and protein levels were increased significantly (P<0.05). Compared with the PQ poisoning group, the amount of HYP decreased significantly, and the expressions of type Ⅰ, Ⅲ collagen protein, CTGF, ROCK1 mRNA and protein levels were decreased significantly in FS intervention group on days 7, 14, 28 (all P<0.05). The pathological changes of lung tissue revealed that the degree of pulmonary fibrosis in the PQ poisoning group were most serious on 28 d after paraquat exposure, and the degree of pulmonary fibrosis were lessened in FS intervention group on days 7, 14, 28. Conclusions ROCK inhibitor, fasudil, has obvious therapeutic effects on paraquat-induced lung fibrosis, by regulating Rho / ROCK signaling pathway with down-regulated expression of CTGF, and decrease in the levels of type Ⅰ, Ⅲ collagen protein, thus reducing protein deposition. Key words: Paraquat; Pulmonary fibrosis; Fasudil; Rho / ROCK signaling pathway

MP42-10 AUGMENTED BLADDER SMOOTH MUSCLE RESPONSIVENESS TO HYPERGLYCEMIA THROUGH A CAVEOLAE-DEPENDENT ACTIVATION OF RHO KINASE PATHWAY

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2017MP42-10 AUGMENTED BLADDER SMOOTH MUSCLE RESPONSIVENESS TO HYPERGLYCEMIA THROUGH A CAVEOLAE-DEPENDENT ACTIVATION OF RHO KINASE PATHWAY Vivian Cristofaro, Josephine A. Carew, Suhas P. Dasari, Raj K. Goyal, and Maryrose P. Sullivan Vivian CristofaroVivian Cristofaro More articles by this author , Josephine A. CarewJosephine A. Carew More articles by this author , Suhas P. DasariSuhas P. Dasari More articles by this author , Raj K. GoyalRaj K. Goyal More articles by this author , and Maryrose P. SullivanMaryrose P. Sullivan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1299AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Diabetic bladder is characterized by an early compensatory phase in which bladder smooth muscle (BSM) exhibits augmented contractility. While little is known about the causes of this response, an upregulation of Rho-dependent signaling in response to hyperglycemia has been suggested. In other smooth muscle systems, RhoA-ROCK signaling has been shown to be dependent on caveolae, membrane invaginations involved in the regulation of a myriad of signaling pathways. Since alterations in caveolar elements have been reported in animal models of diabetes, this study investigated the involvement of these membrane microdomains in the Rho-dependent changes in contractility induced by hyperglycemia. METHODS Longitudinal mouse bladder strips without mucosa were mounted in organ baths for isometric tension studies. Changes in the amplitude of bladder contractions in response to exogenous carbachol (1μM) were evaluated under euglycemia (11.5mM) as well as after exposure of BSM tissue to high glucose Krebs (23mM) for up to 8 hours. Separate experiments were similarly performed in Krebs supplemented with mannitol (11.5mM) to control for increased osmotic conditions. The effect of high glucose on contractile responses to CCh was investigated in the presence of Rho kinase inhibitor Y27632 (1μM), as well as after the depletion of BSM caveolae, achieved by incubation with mβCD (10mM, 1 hour). RESULTS The amplitude of contractile responses to CCh measured in BSM were significantly higher after 2 hours of hyperglycemia compared to those measured under euglycemic conditions and remained elevated for 8 hours. Contractile responses to CCh remained unchanged over the same time period in the presence of mannitol. The augmented CCh responses induced by high glucose were completely prevented in the presence of Rho kinase inhibitor used at a dose that did not affected CCh responses under euglycemia. Similarly, augmented CCh responses induced by hyperglycemia were prevented after BSM caveolae were depleted by exposure to mβCD. CONCLUSIONS The increased detrusor responses to CCh during exposure to hyperglycemia is consistent with BSM hypercontractility reported in the compensated phase of diabetes. The effects of Rho kinase inhibitor and mβCD in preventing the increase in BSM responsiveness under hyperglycemic conditions suggest the involvement of Rho-mediated pathway and the requirement of intact caveolae in this process. These findings might provide a potential link between the diabetes-induced BSM hyperreactivity and the caveolae-mediated regulation of Rho signaling. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e548 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Vivian Cristofaro More articles by this author Josephine A. Carew More articles by this author Suhas P. Dasari More articles by this author Raj K. Goyal More articles by this author Maryrose P. Sullivan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Inhibition of Rho Kinase Induces Antioxidative Molecules and Suppresses Reactive Oxidative Species in Trabecular Meshwork Cells.

Purpose To investigate the effect of rho kinase inhibitors on oxidative stress in trabecular meshwork (TM) cells. Methods TM cells were isolated from the eyes of cynomolgus monkeys. Y-27632 and menadione were used to inhibit rho kinase and induce production of reactive oxygen species (ROS), respectively. The cynomolgus monkey array and 12,613 probes were used in DNA microarray analysis, and the affected genes were categorized using gene ontology analysis. The mRNA levels of the target genes were confirmed by real-time RT-PCR. Intracellular oxidative stress was detected using a fluorescent reagent sensitive to ROS. Cell viability was assessed by the WST-8 assay. Results Gene ontology analysis revealed upregulation of genes involved in antioxidant activity, and upregulation of catalase was confirmed by real-time RT-PCR after 30 min treatment with Y-27632. Production of ROS was increased by menadione, and the effect was partly suppressed by pretreatment with Y-27632. At a lower dose of menadione, Y-27632 stimulated TM cells and significantly increased their viability following menadione treatment compared to control cells. Conclusion Using microarray analysis, Y-27632 was shown to upregulate antioxidative genes including catalase and partially reduce ROS production and cell death by oxidative stress caused by menadione.

Effects of Rho Kinase Inhibitors on Intraocular Pressure and Aqueous Humor Dynamics in Nonhuman Primates and Rabbits.

This study examines the effects of 2 Rho kinase inhibitors on intraocular pressure (IOP) and aqueous humor dynamics. IOPs of New Zealand albino rabbits with ocular normotension and cynomolgus macaques (nonhuman primate, NHP) with chronic unilateral laser-induced glaucoma were measured at baseline and periodically after a 9 a.m. dose of H-1152, Y-27632, or vehicle. In a separate group of NHPs, aqueous flow, outflow facility, uveoscleral outflow, and IOP were determined after treatment with Y-27632 or vehicle control. Decreases in IOP were found in rabbits (n = 5) at 6 h after one dose of 2% Y-27632 (29%, P = 0.0002) or 1% H-1152 (35%, P = 0.0001), and in hypertensive eyes of NHPs (n = 7-9) at 3 h after one dose of 2% Y-27632 (35%, P = 0.005) or 1% H-1152 (51%, P = 0.0003). With 2 doses of 1% Y-27632 or vehicle in NHP hypertensive eyes (n = 12), significant drug effects were IOP reduction of 28% (P = 0.05) at 2.5 h after the second dose and increases in aqueous flow (36%; P = 0.013), uveoscleral outflow (59%, P = 0.008), and outflow facility (40%; P = 0.01). In normotensive eyes of the same animals, aqueous flow increased by 21% (P = 0.03). No significant change was found in any of the other parameters. Y-27632 and H-1152 lower IOP in rabbits and hypertensive eyes of NHPs for at least 6 h after single doses. The Y-27632 effect on IOP in hypertensive NHP eyes is caused by increases in outflow facility and uveoscleral outflow. An increase in aqueous humor formation attenuates but does not prevent an IOP decrease.

Effects of Rho Kinase Inhibitors on Grafts of Dopaminergic Cell Precursors in a Rat Model of Parkinson's Disease.

In models of Parkinson's disease (PD), Rho kinase (ROCK) inhibitors have antiapoptotic and axon-stabilizing effects on damaged neurons, decrease the neuroinflammatory response, and protect against dopaminergic neuron death and axonal retraction. ROCK inhibitors have also shown protective effects against apoptosis induced by handling and dissociation of several types of stem cells. However, the effect of ROCK inhibitors on dopaminergic cell grafts has not been investigated. In the present study, treatment of dopaminergic cell suspension with ROCK inhibitors yielded significant decreases in the number of surviving dopaminergic neurons, in the density of graft-derived dopaminergic fibers, and in graft vascularization. Dopaminergic neuron death also markedly increased in primary mesencephalic cultures when the cell suspension was treated with ROCK inhibitors before plating, which suggests that decreased angiogenesis is not the only factor leading to cell death in grafts. Interestingly, treatment of the host 6-hydroxydopamine-lesioned rats with ROCK inhibitors induced a slight, nonsignificant increase in the number of surviving neurons, as well as marked increases in the density of graft-derived dopaminergic fibers and the size of the striatal reinnervated area. The study findings discourage treatment of cell suspensions before grafting. However, treatment of the host induces a marked increase in graft-derived striatal reinnervation. Because ROCK inhibitors have also exerted neuroprotective effects in several models of PD, treatment of the host with ROCK inhibitors, currently used against vascular diseases in clinical practice, before and after grafting may be a useful adjuvant to cell therapy in PD. Cell-replacement therapy is one promising therapy for Parkinson's disease (PD). However, many questions must be addressed before widespread application. Rho kinase (ROCK) inhibitors have been used in a variety of applications associated with stem cell research and may be an excellent strategy for improving survival of grafted neurons and graft-derived dopaminergic innervation. The present results discourage the treatment of suspensions of dopaminergic precursors with ROCK inhibitors in the pregrafting period. However, treatment of the host (patients with PD) with ROCK inhibitors, currently used against vascular diseases, may be a useful adjuvant to cell therapy in PD.