The Assessment of the Efficacy of Imperatorin in Reducing Overactive Bladder Symptoms.

Overactive bladder syndrome (OAB) is a prevalent condition that affects the elderly population in particular and significantly impairs quality of life. Imperatorin, a naturally occurring furocoumarin, possesses diverse pharmacological properties that warrant consideration for drug development. The aim of this study was to investigate the potential of imperatorin (IMP) to attenuate the cystometric and biochemical changes typically associated with retinyl acetate-induced overactive bladder (OAB) and to assess its viability as a pharmacological intervention for OAB patients. A total of 60 rats were divided into four groups: I-control, II-rats with rapamycin (RA)-induced OAB, III-rats administered IMP at a dose of 10 mg/kg/day, and IV-rats with RA-induced OAB treated with IMP. IMP or vehicle were injected intraperitoneally for 14 days. The cystometry and assessment of bladder blood flow were performed two days after the last dose of IMP. The rats were then placed in metabolic cages for 24 h. Urothelial thickness measurements and biochemical analyses were performed. Intravesical infusion of RA induced OAB. Notably, intraperitoneal administration of imperatorin had no discernible effect on urinary bladder function and micturition cycles in normal rats. IMP attenuated the severity of RA-induced OAB. RA induced increases in urothelial ATP, calcitonin gene-related peptide (CGRP), organic cation transporter 3 (OCT3), and vesicular acetylcholine transporter (VAChT), as well as significant c-Fos expression in all micturition areas analyzed, which were attenuated by IMP. Furthermore, elevated levels of Rho kinase (ROCK1) and VAChT were observed in the detrusor, which were reversed by IMP in the context of RA-induced OAB in the urothelium, detrusor muscle, and urine. Imperatorin has a mitigating effect on detrusor overactivity. The mechanisms of action of IMP in the bladder appear to be diverse and complex. These findings suggest that IMP may provide protection against RA-induced OAB and could potentially develop into an innovative therapeutic strategy for the treatment of OAB.

Assessment of CIP2A and ROCK-I expression and their prognostic value in breast cancer.

Breast cancer is the most leading cause of cancer death in females worldwide. Identification of novel biomarkers for prognosis is required. Imunohistochemical evaluation of CIP2A and ROCK-1 expressions in 126 breast tissue specimens stratified as 21 ductal hyperplasias, 17 duct carcinoma in situ (DCIS) and 88 invasive carcinomas (56 invasive ductal carcinomas NST, 32 invasive lobular carcinomas) was studied. High CIP2A expression was detected in 48.9% of invasive carcinomas. CIP2A overexpression was significantly related to Nottingham prognostic index (NPI) (p = 0.011), stage (p = 0.01), ER negativity (p = 0.031), PR negativity (p = 0.048), and HER-2 positivity (p = 0.02). CIP2A was significantly overexpressed in triple-negative breast cancer (TNBC) (p = 0.004). ROCK-1 expression was detected in 54.5% of invasive carcinomas. Statistically significant associations were observed between ROCK-1 expression and NPI (p = 0.032), stage (p = 0.002), ER negativity (p = 0.012), PR negativity (p = 0.023), HER-2 positivity (p = 0.016), and TNBC subtype (p = 0.033). A positive association between CIP2A and ROCK-1 expressions (p < 0.0001) was documented. There was a significant association between shorter overall survival and high CIP2A and positive ROCK-1 expressions (p < 0.0001) and (p < 0.0001). CIP2A and ROCK-1 expressions could be used as markers for the poor prognosis of breast cancer.

One-Year Efficacy and Safety Assessment of Ripasudil, a Rho Kinase Inhibitor, in an Addition to or Replacing Existing Treatment Regimens: A Retrospective Study.

Purpose: To evaluate efficacy and safety of ripasudil for 1 year in addition to or replacing existing treatment regimens. Methods: We retrospectively reviewed the medical records for 128 eyes of 128 glaucoma patients who were prescribed ripasudil as an addition to or a switch from their preexisting antiglaucoma instillations. We investigated the rate and factors for discontinuation and intraocular pressure (IOP) reduction. Results: Almost half of the patients (60 eyes) discontinued ripasudil treatment before the 1 year mark, while remaining patients completed the treatment. The lack of efficacy and development of adverse effects were significantly correlated with discontinuation (P < 0.001) in the Cox proportional hazards model. In the Kaplan-Meier curve, adverse effects occurred in earlier phase and almost 60% dropped out within 3 months after ripasudil administration. However, adverse effects also occurred randomly throughout the study period. In patients who continued ripasudil, the mean IOPs (mmHg) at baseline, 6 and 12 months after treatment were 17.7 ± 5.1, 14.6 ± 5.0, and 14.8 ± 3.8 in the Addition group, and 17.8 ± 4.1, 15.4 ± 3.2, and 15.4 ± 5.0 in the Switch group, respectively (all P values <0.05). Conclusions: Almost half of the patients discontinued ripasudil owing to the lack of efficacy and the generation of adverse effects within the 1 year. In the remaining half, the addition and switching of ripasudil to the existing glaucoma treatment effectively reduced IOP for 1 year.

Development and design of novel cardiovascular therapeutics based on Rho kinase inhibition-In silico approach.

Rho kinases, one of the best-known members of the serine/threonine (Ser/Thr) protein kinase family, can be used as target enzymes for the treatment of many diseases such as cancer or multiple sclerosis, and especially for the treatment of cardiovascular diseases. This study presents QSAR modeling for a series of 41 chemical compounds as Rho kinase inhibitors based on the Monte Carlo method. QSAR models were developed for three random splits into the training and test set. Molecular descriptors used for QSAR modeling were based on the SMILES notation and local invariants of the molecular graph. The statistical quality of the developed model, including robustness and predictability, was tested with different statistical approaches and satisfying results were obtained. The best calculated QSAR model had the following statistical parameters: r2 = 0.8825 and q2 = 0.8626 for the training set and r2 = 0.9377 and q2 = 0.9124 for the test set. Novel statistical metric entitled as the index of ideality of correlation was used for the final model assessment, and the obtained results were 0.6631 for the training and 0.9683 for the test set. Molecular fragments responsible for the increases and decreases of the studied activity were defined and they were further used for the computer-aided design of new compounds as potential Rho kinase inhibitors. The final assessment of the developed QSAR model and designed inhibitors was achieved with the application of molecular docking. An excellent correlation between the results from QSAR and molecular docking studies was obtained.

Assessment of Basilar Artery Reactivity in Stroke and Subarachnoid Hemorrhage Using Wire Myograph.

Blood flow regulation of normal cerebral arteries is a critical and important factor to supply the brain tissue with nutrients and oxygen. Stroke insult results in a disruption or reduction in cerebral arteries' blood flow with subsequent brain tissue damage. Hemorrhagic stroke is one type of stroke and accounts for about 13 % of all of stroke insults. In this type of stroke, the cerebral artery breaks open and causes bleeding in or surrounding the brain. Subsequently, this bleeding causes blood vessels to constrict in a process called vasospasm, in which the vessels narrow and impede the blood flow to brain tissue. Hemorrhagic stroke is the major cause of prolonged constriction of cerebral arteries. This leads to partial brain damage and sometimes death in patients with aneurysmal subarachnoid hemorrhage. Among the key delicate techniques to assess small blood vessel functionality is the wire myograph, which can be utilized in several cerebral injury models including stroke. The wire myograph is a device that provides information about the reactivity, stiffness, and elasticity of small blood vessels under isometric conditions. In this book chapter, we describe the techniques involved in wire myography assessment and the different measures and parameters recorded; we describe the utility of this technique in evaluating the effects of subarachnoid hemorrhage on basilar artery sensitivity to different agonists.

Generation and Feasibility Assessment of a New Vehicle for Cell-Based Therapy for Treating Corneal Endothelial Dysfunction.

The corneal endothelium maintains corneal transparency by its pump and barrier functions; consequently, its decompensation due to any pathological reason causes severe vision loss due to corneal haziness. Corneal transplantation is the only therapeutic choice for treating corneal endothelial dysfunction, but associated problems, such as a shortages of donor corneas, the difficulty of the surgical procedure, and graft failure, still need to be resolved. Regenerative medicine is attractive to researchers as a means of providing innovative therapies for corneal endothelial dysfunction, as it now does for other diseases. We previously demonstrated the successful regeneration of corneal endothelium in animal models by injecting cultured corneal endothelial cells (CECs) in combination with a Rho kinase (ROCK) inhibitor. The purpose of the present study was to optimize the vehicle for clinical use in cell-based therapy. Our screening of cell culture media revealed that RELAR medium promoted CEC adhesion. We then modified RELAR medium by removing hormones, growth factors, and potentially toxic materials to generate a cell therapy vehicle (CTV) composed of amino acid, salts, glucose, and vitamins. Injection of CECs in CTV enabled efficient engraftment and regeneration of the corneal endothelium in the rabbit corneal endothelial dysfunction model, with restoration of a transparent cornea. The CECs retained >85% viability after a 24 hour preservation as a cell suspension in CTV at 4°C and maintained their potency to regenerate the corneal endothelium in vivo. The vehicle developed here is clinically applicable for cell-based therapy aimed at treating the corneal endothelium. Our strategy involves the generation of vehicle from a culture medium appropriate for a given cell type by removing materials that are not favorable for clinical use.

Assessment of response to beta-blockers by expression of ßArr2 and RhoA/ROCK2 in antrum mucosa in cirrhotic patients.

BACKGROUND & AIMS: Non-selective beta-blockers (NSBB) are first choice for prevention of variceal bleeding. But possible deleterious effects in refractory ascites and frequent non-response are clinical drawbacks. Since levels of vasoactive proteins in antrum mucosa reflect vascular dysfunction in cirrhosis, these expression levels might also reflect hemodynamic response to NSBB. METHODS: Biopsies from the gastric and duodenal mucosa of 25 patients with cirrhosis were collected and the hepatic venous pressure gradient (HVPG) was measured before and after an acute propranolol challenge. Transcription and protein expression of Ras homolog family member A (RhoA), Rho-kinase (ROCK)2, beta-arrestin2 (ßArr2), endothelial nitric oxide synthase (eNOS) and the phosphorylation of downstream effectors VASP and moesin were analyzed using PCR and Western blot. Further 21 patients on NSBB were evaluated on their follow up for events of variceal bleeding defined as non-response. RESULTS: Ten patients showed HVPG <10mmHg, further seven patients showed significant hemodynamic response to NSBB, whereas eight patients were non-responders. The mucosal transcription of vasoactive proteins was higher in antrum mucosa compared to corpus and duodenum. The transcriptional levels of vasoactive proteins were higher in patients with HVPG >10mmHg and HVPG >16mmHg. Interestingly, mRNA levels of RhoA and ROCK2 were lower in patients with large varices at endoscopy. Moreover, RhoA and ROCK2 transcription correlated with the decrease of HVPG after acute NSBB challenge. Finally, acute and long-term non-responders showed lower expression of ßArr2 in antrum mucosa. CONCLUSION: This study shows for the first time that the expression of ßArr2 in antrum mucosa biopsies might reflect the hemodynamic response to NSBB and their long-term protective effect. This finding might offer an easy approach at upper endoscopy to facilitate the decision to treat with NSBB if varices are present.

ROCK1 in AgRP neurons regulates energy expenditure and locomotor activity in male mice.

Normal leptin signaling is essential for the maintenance of body weight homeostasis. Proopiomelanocortin- and agouti-related peptide (AgRP)-producing neurons play critical roles in regulating energy metabolism. Our recent work demonstrates that deletion of Rho-kinase 1 (ROCK1) in the AgRP neurons of mice increased body weight and adiposity. Here, we report that selective loss of ROCK1 in AgRP neurons caused a significant decrease in energy expenditure and locomotor activity of mice. These effects were independent of any change in food intake. Furthermore, AgRP neuron-specific ROCK1-deficient mice displayed central leptin resistance, as evidenced by impaired Signal Transducer and Activator of Transcription 3 activation in response to leptin administration. Leptin's ability to hyperpolarize and decrease firing rate of AgRP neurons was also abolished in the absence of ROCK1. Moreover, diet-induced and genetic forms of obesity resulted in reduced ROCK1 activity in murine arcuate nucleus. Of note, high-fat diet also impaired leptin-stimulated ROCK1 activity in arcuate nucleus, suggesting that a defect in hypothalamic ROCK1 activity may contribute to the pathogenesis of central leptin resistance in obesity. Together, these data demonstrate that ROCK1 activation in hypothalamic AgRP neurons is required for the homeostatic regulation of energy expenditure and adiposity. These results further support previous work identifying ROCK1 as a key regulator of energy balance and suggest that targeting ROCK1 in the hypothalamus may lead to development of antiobesity therapeutics.

Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells.

Differentiated cells from human embryonic stem cells (hESCs) provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs) provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation.

Loss of gene function is a valuable tool for screening genes in cellular processes including stem cell differentiation differentiation. However, the criteria for evaluating gene knockdown are usually based on end-point analysis and real-time, dynamic information is lacking. To overcome these limitations, we engineered a shRNA encoding LentiViral Dual Promoter vector (shLVDP) that enabled real-time monitoring of mesenchymal stem (MSC) differentiation and simultaneous gene knockdown. In this vector, the activity of the alpha-smooth muscle actin (αSMA) promoter was measured by the expression of a destabilized green fluorescent protein, and was used as an indicator of myogenic differentiation; constitutive expression of discosoma red fluorescent protein was used to measure transduction efficiency and to normalize αSMA promoter activity; and shRNA was encoded by a doxycycline (Dox)-regulatable H1 promoter. Importantly, the normalized promoter activity was independent of lentivirus titer allowing quantitative assessment of gene knockdown. Using this vector, we evaluated 11 genes in the TGF-ß1 or Rho signaling pathway on SMC maturation and on MSC differentiation along the myogenic lineage. As expected, knockdown of genes such as Smad2/3 or RhoA inhibited myogenic differentiation, while knocking down the myogenic differentiation inhibitor, Klf4, increased αSMA promoter activity significantly. Notably, some genes for example, Smad7 or KLF4 showed differential regulation of myogenic differentiation in MSC from different anatomic locations such as bone marrow and hair follicles. Finally, Dox-regulatable shRNA expression enabled temporal control of gene knockdown and provided dynamic information on the effect of different genes on myogenic phenotype. Our data suggests that shLVDP may be ideal for development of lentiviral microarrays to decipher gene regulatory networks of complex biological processes such as stem cell differentiation or reprogramming.

Systematic assessment and meta-analysis of the efficacy and safety of fasudil in the treatment of cerebral vasospasm in patients with subarachnoid hemorrhage.

PURPOSE: Cerebral vasospasm (CVS) is a frequent and unpredictable complication in patients with subarachnoid hemorrhage (SAH) and often leads to poor outcomes. This study was aimed at evaluating the efficacy and safety of fasudil in the treatment of CVS in patients with SAH. METHODS: A search was conducted using the full-text database of Chinese scientific journals, the Wanfang Database (January 1999 to November 2010), the Chinese Medical Association Digital Journal Database, PubMed, the Cochrane library, OVID, and EMBase (searching through November 2010). RESULTS: A total of 8 studies met the inclusion criteria. The incidence rates of symptomatic CVS and CVS confirmed by angiography among the patients in the fasudil group were only 48% (odds ratio [OR] = 0.48, 95% confidence interval [CI]: 0.32-0.72, P = 0.0005) and 40% (OR = 0.40, 95% CI: 0.24-0-67, P = 0.0004) respectively of that of the control group. The odds ratios of cerebral infarction for all cases and cerebral infarction for CVS cases in the fasudil group were only 50% (OR = 0.50, 95% CI: 0.34-0.76, P = 0.0009) and 43% (OR = 0.43, 95% CI: 0.26-0.70, P = 0.0008) respectively of that of the control group. CONCLUSIONS: Fasudil greatly reduces the occurrence of CVS and cerebral infarction in SAH patients, significantly improves the clinical outcomes of the patients (as assessed by the Glasgow Outcome Scale). Because of the limited number of trials and samples available for analysis, the conclusions from the present study still need to be validated with results from large randomized, controlled clinical trials.

Enhanced Rho-kinase activity in circulating neutrophils of patients with vasospastic angina: a possible biomarker for diagnosis and disease activity assessment.

OBJECTIVES: The aim of this study was to examine whether Rho-kinase activity is systemically enhanced in patients with vasospastic angina (VSA) and, if so, whether a noninvasive diagnostic method could be developed to improve practice. BACKGROUND: The activated Rho-kinase pathway plays a central role in the molecular mechanism of coronary vasospasm in animal models and patients with VSA. Recently, it has been reported that Rho-kinase activity in circulating leukocytes is associated with various diseases. METHODS: Fifty-three consecutive patients with chest pain who underwent acetylcholine provocation testing for coronary spasm were examined. Patients were divided into 2 groups depending on their response to the test: VSA (n = 33) and non-VSA (n = 20) groups. Venous blood samples were collected to measure Rho-kinase activity in circulating neutrophils, determined by the extent of phosphorylation of myosin-binding subunit (MBS), a substrate of Rho-kinase. RESULTS: Rho-kinase activity was significantly higher in the VSA group than in the non-VSA group (phosphorylated MBS/total MBS ratio 1.33 ± 0.37 vs. 0.95 ± 0.22, p < 0.001). In the VSA group, no correlation was noted between Rho-kinase activity and high-sensitivity C-reactive protein, smoking, or accumulated number of coronary risk factors. After the 3-month medical treatment, Rho-kinase activity in the VSA group was significantly decreased to 1.08 ± 0.31 (p < 0.001). On receiver-operating characteristic curve analysis, a phosphorylated MBS ratio of 1.18 was identified as the best cutoff level to predict the diagnosis of VSA. CONCLUSIONS: These results indicate that Rho-kinase activity in circulating neutrophils is enhanced in patients with VSA and may be a useful biomarker for diagnosis and disease activity assessment of the vasospastic disorder.

A model of NMDA receptor control of F-actin treadmilling in synaptic spines and their growth.

Synaptic spines grow as a consequence of the formation of F-actin filaments at the spine head. The dynamics of F-actin in the spine head upon excitation of N-methy-D-aspartate (NMDA) receptors has recently been investigated experimentally, but there is no quantitative account of how these dynamic changes occur upon activation of these receptors; this we now supply. Dynamics of F-actin at the apex of lamellipodia have been investigated in detail, giving rise to the treadmilling theory of F-actin dynamics, involving catalysis by profilin, for which quantitative models are now available. Here, we adapt such a model to describe the dynamics of F-actin in the synaptic-spine head and show that it gives quantitative descriptions of this treadmilling phenomena which are well fitted by Monte Carlo simulations. Next, the means by which excitation of NMDA receptors enhances the activity of profilin through activity of the Rho small GTPase RhoA and the specific kinase ROCK is discussed. This is then used to model the NMDA receptor excitatory enhancement of profilin and so the treadmilling process of F-actin dynamics in spine growth. Such modelling provides a quantitative description of the synaptic-spine dynamics of the filamentous to globular actin ratio that is observed experimentally.

LowModeMD--implicit low-mode velocity filtering applied to conformational search of macrocycles and protein loops.

We present a method for conformational search of complex molecular systems such as macrocycles and protein loops. The method is based on perturbing an existing conformation along a molecular dynamics trajectory using initial atomic velocities with kinetic energy concentrated on the low-frequency vibrational modes, followed by energy minimization. A novel Chebyshev polynomial filter is used to heavily dampen the high-frequency components of a randomly generated Maxwell-Boltzmann velocity vector. The method is very efficient, even for large systems; it is straightforward to implement and requires only standard force-field energy and gradient evaluations. The results of several computational experiments suggest that the method is capable of efficiently sampling low-strain energy conformations of complex systems with nontrivial nonbonded interaction networks.

Quantitative assessment of local collagen matrix remodeling in 3-D culture: the role of Rho kinase.

The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling. Human corneal fibroblasts were plated inside 100-microm thick fibrillar collagen matrices and cultured for 24 h in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph. Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p<0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced (p<0.01). Overall, the data suggests that Rho kinase-dependent contractile force generation leads to co-alignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction.

Thiazide-like diuretics attenuate agonist-induced vasoconstriction by calcium desensitization linked to Rho kinase.

Lowering blood pressure using thiazide-like diuretics, including chlorthalidone and hydrochlorothiazide, has been proven to be effective in clinical studies. However, the mechanisms by which thiazide-like diuretics lower blood pressure are still poorly understood. To evaluate whether thiazide-like diuretics cause calcium desensitization in smooth muscle cells, we measured their effects on agonist-induced increase of blood pressure in Wistar rats in vivo and on agonist-induced vasoconstriction of aortic rings, DNA synthesis, and protein synthesis, RhoA, Rho kinase, and intracellular calcium in vascular smooth muscle cells in vitro. Thiazide-like diuretics significantly attenuated angiotensin II-induced or norepinephrine-induced increase of systolic blood pressure in rats. Thiazide-like diuretics inhibited agonist-induced vasoconstriction of aortic rings in a concentration-dependent manner in the presence and absence of endothelium. The inhibitory effects of thiazide-like diuretics were similar to that of the specific Rho kinase inhibitor Y27632. RT-PCR and immunoblotting showed that RhoA and Rho kinase were significantly reduced in vascular smooth muscle cells after administration of thiazide-like diuretics. In contrast, thiazide-like diuretics did not affect protein tyrosine phosphatase-2 (SHP-2) expression. Agonist-induced changes of intracellular calcium were not affected by thiazide-like diuretics. The study indicates that thiazide-like diuretics inhibit agonist-induced vasoconstriction by calcium desensitization in smooth muscle cells linked to the Rho-Rho kinase pathway.

Rapid actin transport during cell protrusion.

Transformed rat fibroblasts expressing two variants of green fluorescent protein, each fused to beta-actin, were used to study actin dynamics during cell protrusion. The recently developed FLAP (fluorescence localization after photobleaching) method permits the tracking of one fluorophore after localized photobleaching by using the other as a colocalized reference. Here, by visualizing the ratio of bleached to total molecules, we found that actin was delivered to protruding zones of the leading edge of the cell at speeds that exceeded 5 micrometers per second. Monte Carlo modeling confirmed that this flow cannot be explained by diffusion and may involve active transport.