Ghrelin suppresses migration of macrophages via inhibition of ROCK2 under chronic intermittent hypoxia.

OBJECTIVES: Migration of macrophages and atherosclerosis result in various diseases, including coronary heart disease. This study aimed to clarify the roles that ghrelin and Rho-associated coiled-coil-containing protein kinase 2 (ROCK2) play in migration of macrophages under chronic intermittent hypoxia (CIH). METHODS: A rat model of CIH was constructed and changes in ghrelin and ROCK2 protein expression were measured by western blot assay. The migratory ability of macrophages was determined by the transwell assay. Hematoxylin and eosin staining was applied to detect the changes in intima-media thickness. RESULTS: We found that CIH enhanced migration of macrophages, and this effect was attenuated by exogenous ghrelin. Additionally, the facilitative effect of CIH on migration of macrophages was strengthened or decreased by upregulation or downregulation of ROCK2, respectively. This phenomenon indicated that ROCK2 was involved in CIH-induced migration in macrophages. Furthermore, western blot and transwell assays showed that ghrelin inhibited CIH-induced migration via ROCK2 suppression in macrophages. CONCLUSIONS: In summary, the present study shows that ghrelin inhibits CIH-induced migration via ROCK2 suppression in macrophages. Our research may help lead to identifying a new molecular mechanism for targeted therapy of atherosclerosis and its associated coronary artery diseases under intermittent hypoxia.

Tissue factor mediates microvesicles shedding from MDA-MB-231 breast cancer cells.

Extracellular vesicles, such as microvesicles (MVs), were identified as important players in tumor progression and acquisition of an aggressive phenotype. Tissue factor (TF) is a transmembrane protein that initiates the blood coagulation cascade. In tumor cells, TF has been associated with aggressiveness and cancer progression. Previous studies demonstrate that TF is incorporated into MVs secreted by tumor cells; however, it is unknown whether TF is actively involved in the release of MVs. Here, we investigated the influence of TF expression on the release of MVs. TF silencing was achieved through CRISPR/Cas9 approaches in the human breast cancer cell line, MDA-MB-231. TF knockout in MDA-MB-231 cells efficiently reduced TF-dependent signaling and procoagulant activity. Remarkably, silencing of TF caused a significant decrease in the number of MVs released by MDA-MB-231 cells. We also observed an increase in actin-positive membrane projections in TF knockout cells and a reduction in RhoA expression when compared to TF-expressing cells. Treatment of MDA-MB-231 cells with the RhoA-ROCK signaling pathway inhibitor, fasudil, significantly reduced the release of MVs. Taken together, our results suggest a novel and relevant role for TF in tumor biology by playing an active role in the MVs secretion.

Acute hypobaric hypoxia augments ROCK2 protein level and activity.

AIM: Rho kinase activation plays an important role in hypoxic pulmonary vasoconstriction and increased vascular resistance. The present study investigated changes in the level and activity of rho kinase isoform 2 (ROCK2) under acute hypobaric hypoxia exposure. For this, Wistar rats were taken as the model organism. MATERIALS AND METHODS: Fifteen male Wistar rats (4-6 week old, 250 grams) were normalized with the surrounding environment by providing a 12/12 hour day and night acclimatization cycle. The rats were divided into 3 groups: (a) control group (no exposure, n = 5), (b) Group 1 (12 hour hypobaric-hypoxia exposure, n = 5) and (c) Group 2 (12 hour hypobaric hypoxia and 12 hour normobaric normoxia exposure, n = 5). A change in behavior of the animals was noted before sacrifice. Blood was collected from the beating heart of the anesthetized animal. Lungs were dissected out and used to estimate the levels and activity of ROCK2 in different groups using commercially available kits. Lung histology was evaluated by immunohistochemistry. ROCK2 gene expression was studied in the blood and lungs using quantitative PCR. RESULTS AND CONCLUSIONS: Control and Group 2 animals had an active movement while the Group 1 animals were sluggish before the sacrifice. Formation of a large perivascular edema cuff and collagen deposition in lungs of Group 1 and a reduction in Group 2 was observed. The protein levels and activities of ROCK2 were increased in Group 1 (p < 0.05) and became normal in Group 2 which was akin to control group animals. ROCK2 expression in PBMCs was increased in Group 1 (10.7 fold, p = 0.005) and was decreased in Group 2 (5.5 fold, p = 0.02). The outcomes establish that acute hypobaric hypoxia augments ROCK2 protein level and activity.

[Effect of antepartum taurine supplementation in regulating the activity of Rho family factors and promoting the proliferation of neural stem cells in neonatal rats with fetal growth restriction].

OBJECTIVE: To study the possible effect of antepartum taurine supplementation in regulating the activity of Rho family factors and promoting the proliferation of neural stem cells in neonatal rats with fetal growth restriction (FGR), and to provide a basis for antepartum taurine supplementation to promote brain development in children with FGR. METHODS: A total of 24 pregnant Sprague-Dawley rats were randomly divided into three groups: control, FGR, and taurine (n=8 each ). A rat model of FGR was established by food restriction throughout pregnancy. RT-PCR, immunohistochemistry, and Western blot were used to measure the expression of the specific intracellular markers for neural stem cells fatty acid binding protein 7 (FABP7), Rho-associated coiled-coil containing protein kinase 2 (ROCK2), ras homolog gene family, member A (RhoA), and Ras-related C3 botulinum toxin substrate (Rac). RESULTS: The FGR group had significantly lower OD value of FABP7-positive cells and mRNA and protein expression of FABP7 than the control group, and the taurine group had significantly higher OD value of FABP7-positive cells and mRNA and protein expression of FABP7 than the FGR group (P<0.05). The FGR group had significantly higher mRNA expression of RhoA and ROCK2 than the control group. The taurine group had significantly higher mRNA expression of RhoA and ROCK2 than the control group and significantly lower expression than the FGR group (P<0.05). The FGR group had significantly lower mRNA expression of Rac than the control group. The taurine group had significantly higher mRNA expression of Rac than the FGR and control groups (P<0.05). The FGR group had significantly higher protein expression of RhoA and ROCK2 than the control group. The taurine group had significantly lower protein expression of RhoA and ROCK2 than the FGR group (P<0.05). CONCLUSIONS: Antepartum taurine supplementation can promote the proliferation of neural stem cells in rats with FGR, and its mechanism may be related to the regulation of the activity of Rho family factors.

DLC-1 is an independent prognostic marker and potential therapeutic target in hepatocellular cancer.

BACKGROUND: The 5-year survival rate of patients with hepatocellular cancer (HCC) was very low because of invasion and metastasis in the early stage. Biomarkers might help predict early occurrence of invasion and metastasis. Accumulating evidence has shown that deleted in liver cancer-1 (DLC1) may be considered as a metastasis suppressor gene in numerous solid and hematological cancers. However, its prognostic role and mechanisms that regulate and coordinate these activities remain poorly understood. METHODS: With the method of immunohistochemistry, the expression of DLC-1 as well as Rho A, ROCK2, moesin had been characterized in 80 HCC tissues and adjacent noncancerous tissues. The correlation between their expression and their relationships with clinicopathological characteristics of HCC were also investigated. In addition, the prognostic value of DLC1 expression within the tumor tissues was assessed by Cox regression and Kaplan-Meier analysis. RESULTS: DLC1 expression was significantly lower in HCC tissues than in adjacent noncancerous tissues, and DLC-1 expression was found to be negatively correlated with tumor differentiation, TNM stage and lymph node metastasis. Furthermore, DLC-1 expression was found to inversely correlate with Rho A, ROCK2 and moesin which were all highly expressed in HCC tissues. Kaplan-Meier analysis showed that significantly longer 5-year survival rate was seen in HCC patients with higher DLC1 expression, compared to those with lower expression of DLC1. Multivariate Cox proportional hazard analyses revealed that DLC1 was an independent factor affecting the overall survival probability. CONCLUSION: DLC1 could be served as a tumor suppressor gene in the progression especially in the invasion and metastasis of HCC. DLC1 perhaps played its role by regulating the expression of Rho A, ROCK2 and moesin. Evaluation of the expression of DLC-1 might be a good prognostic marker for patients with HCC.

A real-time, fluorescence-based assay for Rho-associated protein kinase activity.

Inhibitors of Rho-associated protein kinase (ROCK) enzymatic activity have been shown to reduce the invasive phenotype observed in metastatic hepatocellular carcinoma (HCC). We describe the design, synthesis, and evaluation of a direct probe for ROCK activity utilizing a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox. The Sox fluorophore undergoes an increase in fluorescence upon phosphorylation of a proximal amino acid via chelation-enhanced fluorescence (CHEF, ex. = 360 nm and em. = 485 nm), allowing for the direct visualization of the rate of phosphate addition to a peptide substrate over time. Our optimal probe design, ROCK-S1, is capable of sensitively reporting ROCK activity with a limit of detection of 10 pM and a high degree of reproducibility (Z'-factor = 0.6 at 100 pM ROCK2). As a proof-of-principle for high-throughput screening (HTS) we demonstrate the ability to rapidly assess the efficacy of a 78 member, small molecule library against ROCK2 using a robotics platform. We identify two previously unreported ROCK2 inhibitor scaffolds, PHA665752 and IKK16, with IC50 values of 3.6 µM and 247 nM respectively. Lastly, we define conditions for selectively monitoring ROCK activity in the presence of potential off-target enzymes (PKCα, PKA, and PAK) with similar substrate specificities.

Expression of Wnt11 and Rock2 protein with clinical characteristics of esophageal squamous cell carcinoma in Kazakh and Han patients.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most malignancies with a very poor outcome in China. Wnt11 and Rock2, new identified proteins highly associated with metastasis of many cancers, which were never reported in esophageal squamous cell carcinoma (ESCC). Here we measured the expression levels of Wnt11 and Rock2 in tissues from 265 patients with ESCC. Immunohistochemical staining was employed to detect the correlation of Wnt11 and Rock2 expression with clinicopathological features. METHODS: The expression of Wnt11 and Rock2 was detected by immunohistochemistry in esophageal squamous cell carcinomas and normal esophageal tissues. A chi-square test was used to assess the statistical significance of the correlations between Wnt11, Rock2 expression and different clinicopathological parameters, respectively. RESULTS: The high-expression of Wnt11 and Rock2 was observed in ESCCs. Seventy-five cases of ESCC (51.7%) showed a positive expression of Wnt11, which indicated a significant association with the AJCC stage (P=0.007). Ninety-eight cases of ESCC (65.5%) showed a positive expression of Rock2, which indicated a significant association with ethnic background. There were no close correlations between Rock2 expression and gender, tumor location, AJCC stage, lymph node metastasis. Specifically, the expression of Rock2 was significantly different between Hans and Kazaks ethnicities (P=0.000). In Kaplan-Meier curve analysis, no significant correlation was observed between the expression of Wnt11, Rock-2 and the poor prognosis of ESCCs. CONCLUSION: Our finding suggests that the over-expression of Rock2 may play an important role in the carcinogenesis and progression, and may become a new underlying molecular marker in the diagnosis and treatment in ESCC.

Using carboxyfluorescein diacetate succinimidyl ester to monitor intracellular protein glycation.

Protein glycation is a ubiquitous process involved in vascular complications observed in diabetes. Glyoxal (GO), an intracellular reactive oxoaldehyde that is one of the most potent glycation agents, readily reacts with amines present on proteins to produce the lysine-derived adduct carboxymethyllysine, which is a prevalent advanced glycation end-product (AGE). Our group previously showed that cell exposure to GO leads to an alteration in the cell contractile activity that could occur as a result of the glycation of various proteins regulating the cell contractile machinery. Here, we measured the extent of glycation on three functionally distinct proteins known to participate in cell contraction and cytoskeletal organization-Rho-kinase (ROCK), actin, and gelsolin (GSN)-using an assay based on the reaction of the cell membrane-permeable fluorescent probe carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), which reacts with primary amine groups of proteins. By combining CFDA-SE fluorescence and Western blot detection, we observed (following GO incubation) increased glycation of actin and ROCK as well as an increased interaction between actin and GSN as observed by co-immunoprecipitation. Thus, we conclude that the use of the fluorescent probe CFDA-SE offers an interesting alternative to perform a comparative analysis of the extent of intracellular protein glycation in live cells.

Expression of vasoactive proteins in gastric antral mucosa reflects vascular dysfunction in patients with cirrhosis and portal hypertension.

BACKGROUND & AIMS: Patients with cirrhosis display hypocontractility of splanchnic vessels because of dysregulation of vasoactive proteins, such as decreased effect of RhoA/ROCK and increased activity of ß-Arrestin-2 and eNOS. However, it is unknown whether the dysregulation of vasoactive proteins is displayed in other vessels. We investigated whether expression of vasoactive proteins can be evaluated in gastric mucosa vessels. METHODS: Biopsies from the gastric mucosa of 111 patients with cirrhosis were collected at three different centres and from 13 controls. Forty-nine patients had received TIPS. Portal pressure gradient was measured in 49 patients with TIPS and in 16 patients without TIPS. Biopsies from the antrum were conserved in formaldehyde for immunohistochemistry or shock-frozen for PCR and Western blot. RESULTS: The mucosal transcription of vascular markers (αSMA, CD31) was higher in cirrhotic patients than controls, which was confirmed by immunohistochemistry. On average, relative mucosal levels of RhoA and ROCK were lower, while ß-Arrestin-2 levels were higher in cirrhotic patients compared to controls. Transcriptional levels of eNOS increased with presence of ascites and grade of oesophageal varices. Patients with TIPS showed less pronounced markers of vascular dysfunction in gastric mucosa. CONCLUSION: This is the first evidence that the expression of vasoactive proteins in mucosa from the gastric antrum of patients with cirrhosis reflects their vascular dysfunction and possibly changes after therapeutic interventions.

Estudo de genes diferencialmente expressos em câncer de vulva: validações a partir de resultados de CGH array / Study of differentially expressed genes in vulvar cancer: results from the validation of array CGH

Introdução: O carcinoma de vulva é um tumor de baixa ocorrência, sendo responsável por menos de 3% de todos os tumores malignos que acometem mulheres. Em mulheres jovens a ocorrência da doença está atrelada a fatores de risco como tabagismo e infecção por HPV, entretanto em mulheres acima dos 50 anos ocorre por mecanismos genéticos ainda pouco elucidados. Nos últimos anos poucos autores estudaram alterações genômicas em carcinomas vulvares, contudo nenhum deles determinou um fator prognóstico definitivo, nem tampouco correlacionou esses achados com a expressão gênica, mesmo representando pontos-chave na compreensão do processo de carcinogênese. A partir da análise de arranjos de hibridação genômica comparativa (CGH-array) realizada previamente em nosso laboratório, dois genes candidatos, ROCK1 e RhoD, localizados em regiões com alta frequência de ganhos em nossas amostras de neoplasias vulvares, foram selecionados para este estudo. Objetivo: Validar a expressão dos genes candidatos, ROCK1 e RhoD, que foram identificados em regiões com ganho de cópias nas amostras de carcinoma vulvar pelo método de CGHarray, a fim de determinar melhores e mais acurados valores prognósticos no carcinoma vulvar...

Introduction: The vulvar carcinoma is a low occurrence tumor, accounting for less than 3% of all malignant tumors that affect women. In young women the occurrence of the disease is linked to risk factors such as smoking and HPV infection, but the majority of cases of vulvar cancer occurs in women over 50 years by genetic mechanisms still poorly understood. Recently, few authors studied genomic changes in vulvar carcinomas, however none of them determined on prognostic factor, nor correlate these findings with gene expression, even representing key points in understanding the carcinogenesis process. From the analysis of comparative genomic hybridization arrays (array CGH) previously performed in our laboratory, two candidate genes, ROCK1 and Rhod, located in regions with high frequency gains in our samples of vulvar cancer were selected for this study. Objective: To validate the expression of the candidate genes, RhoD and ROCK1, which have been identified in regions with a gain of copies in vulvar carcinoma samples by CGH-array method, in order to determine the best and most accurate prognostic values in vulvar carcinoma. Methods: 16 cases of vulvar cancer were rescued from AC Camargo Cancer Center’s Biobank...

Clinical significance of mTOR, ZEB1, ROCK1 expression in lung tissues of pulmonary fibrosis patients.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown causes. Three proteins (mammalian target of rapamycin, mTOR; zinc finger E-box-binding homeobox 1, ZEB1; Rho-associated, coiled-coil containing protein kinase 1, ROCK1) may be related to pulmonary fibrosis. However, they have not been assessed in human pulmonary fibrosis. We assessed the clinical significance of mTOR, ZEB1, and ROCK1 expression in human pulmonary fibrosis of usual interstitial pneumonia (UIP) pattern. METHODS: The mTOR, ZEB1, and ROCK1 expression was evaluated by immunohistochemical staining of 30 surgical lung biopsy tissues from 26 IPF and 4 UIP pattern connective tissue disease related interstitial lung disease (CTD-ILD) patients. The expression scores correlated with the clinical features. RESULTS: The mTOR, ZEB1 and ROCK1 mainly expressed in alveolar epithelial cells of UIP lungs. The histological fibrosis scores and lung function decline in the strong mTOR expression group were higher than those in the weak and intermediate expression group. Patients with positive ZEB1 expression had higher fibrosis scores and greater decline in carbon monoxide diffusion capacity (DLCO) than patients with negative ZEB1 expression. Patients with positive mTOR or ZEB1 expression had poorer prognosis than that of patients with negative mTOR or ZEB1 expression, although it was not statistically significant. ROCK1 was not associated with the studied clinicopathological features. CONCLUSIONS: The mTOR and ZEB1 expression in pulmonary fibrosis patients significantly correlated with the fibrosis score and lung function decline, indicating that it may be related to the prognosis of pulmonary fibrosis. Further studies involving large numbers of homogeneous IPF patients are warranted.

Antitumoral effect of a selective Rho-kinase inhibitor Y-27632 against Ehrlich ascites carcinoma in mice.

BACKGROUND: The Rho proteins and Rho-kinase (ROCK) enzymes are responsible for signal transduction, and cause cell permeability, contractility, differentiation, migration, proliferation or apoptosis depending on cell types. All of these functions are vital for cancer initiation and progression. In this study, the preventive and protective effects of a selective ROCK inhibitor Y-27632 against Ehrlich ascites carcinoma in Swiss albino mice were investigated. METHODS: Adult male albino mice were divided into five equal groups, and Y-27632 (0.1, 1, and 10 mg/kg) was given to groups as two steps; before (pre-carcinoma) and after inoculation of carcinoma cell suspensions (post-carcinoma). At the end of the experiments (at day 15), cardiac blood samples, the ascitic fluid, and intestinal specimens were collected for histopathology and biochemical investigation. RESULTS: Significant decreases in the body weight and immunostaining scores in small and large intestine for ROCK2, preservation of serum glutathione (GSH) levels, and an increase in tumor level of nitric oxide were recorded in groups pretreated with Y-27632. However, treatment with Y-27632 after tumor inoculation did not affect body weight and ROCK2 immunostaining scores, increased serum MDA levels, and decreased GSH levels. CONCLUSIONS: This is the first study on the effectiveness of Y-27632 in this experimental tumor model. Our findings provided direct evidence for ROCK involvement in tumor development. These data suggest that pretreatment with Y-27632 has a protective effect against tumor formation.

Investigation of the esophageal Rho-kinase expression in patients with Barrett's esophagus.

The mechanisms responsible for the malignant transformation in Barrett's esophagus (BE) are still poorly understood. The authors have evaluated the role of Rho-kinase (ROCK1 and ROCK2) expressions in patients with BE. All patients underwent upper gastrointestinal system endoscopy, which was confirmed histologically. Real-time PCR revealed no marked change in gene expressions of ROCK1 and ROCK2 at mRNA levels in BE when compared to controls. Immunohistochemical and western blot analyses showed no change in ROCK1 and ROCK2 protein expressions in BE. This study demonstrates that Rho-kinase gene and protein expressions are not modified in BE.

Time-dependent changes in the activation of RhoA/ROCK and ERM/p-ERM in the increased expression of intestinal P-glycoprotein by repeated oral treatment with etoposide.

Previously, we reported that repeated oral treatment with etoposide (ETP) increased ileal membrane localization of P-glycoprotein (P-gp) and that this was possibly mediated by increased expression of the ezrin/radixin/moesin (ERM)/phosphorylated ERM (p-ERM) via activation of RhoA/ROCK. These changes caused decreases in the analgesia of oral morphine (substrate drug for P-gp). However, there are no reports indicating the temporal changes in the above-mentioned factors after initiation and cessation of repeated treatment with the substrate drugs for P-gp including ETP. We examined the relationships between time-dependent changes in protein expressions of ileal P-gp and those of RhoA, ROCK, ERM, and p-ERM, and in oral morphine analgesia after initiation or cessation of repeated oral treatment with ETP. Ileal membrane localization of RhoA was increased 3 days after initiating ETP treatment; on 5 or 7 days, that of ROCK, ERM, and p-ERM was increased along with increments in P-gp expression, leading to decreases in oral morphine analgesia. All these changes returned toward normal levels 3 days after cessation. These data suggest that regulating the active state of the above-mentioned proteins during cancer chemotherapy or creating a timeframe of discontinuation a few days after cessation may enable effective palliative care using oral opioids.

Activation of ERM-family proteins via RhoA-ROCK signaling increases intestinal P-gp expression and leads to attenuation of oral morphine analgesia.

Previously, we reported that repeated oral treatment with etoposide (ETP) causes attenuation of oral morphine analgesia through upregulation of ileal P-glycoprotein (P-gp) mediated by Ras homolog gene family, member A (RhoA) activation. However, the detailed mechanism of the increase in ileal P-gp via RhoA activation remains unknown. Recently, it has been reported that ezrin-radixin-moesin (ERM) proteins, linking several plasma-membrane proteins to the actin cytoskeleton, are involved in the membrane localization and functional activity of P-gp. Moreover, the cross-linking activities of ERM are known to be regulated by RhoA and Rho-associated coiled-coil containing kinase (ROCK). Here, we examined the involvement of ERM in the changes in expression of P-gp via RhoA and ROCK in ileal membrane induced by ETP. Repeated oral treatment with ETP significantly increased the ileal membrane localization of ERM and phosphorylated ERM (p-ERM) in association with upregulation of P-gp and activation of RhoA and ROCK. Interestingly, coadministration of rosuvastatin (inhibitor of RhoA activation) and fasudil (ROCK inhibitor) prevented increments in the activation and phosphorylation of ERM, respectively. In conclusion, upregulation of ileal membrane localization of ERM and p-ERM via activation of RhoA/ROCK induced by ETP treatment may be involved in the regulation of ileal membrane localization of P-gp.

Regulation of ROCK activity in cancer.

Cancer-associated changes in cellular behavior, such as modified cell-cell contact, increased migratory potential, and generation of cellular force, all require alteration of the cytoskeleton. Two homologous mammalian serine/threonine kinases, Rho-associated protein kinases (ROCK I and II), are key regulators of the actin cytoskeleton acting downstream of the small GTPase Rho. ROCK is associated with cancer progression, and ROCK protein expression is elevated in several types of cancer. ROCKs exist in a closed, inactive conformation under quiescent conditions, which is changed to an open, active conformation by the direct binding of guanosine triphosphate (GTP)-loaded Rho. In recent years, a number of ROCK isoform-specific binding partners have been found to modulate the kinase activity through direct interactions with the catalytic domain or via altered cellular localization of the kinases. Thus, these findings demonstrate additional modes to regulate ROCK activity. This review describes the molecular mechanisms of ROCK activity regulation in cancer, with emphasis on ROCK isoform-specific regulation and interaction partners, and discusses the potential of ROCKs as therapeutic targets in cancer.

The Rho kinase inhibitor fasudil is involved in p53-mediated apoptosis in human hepatocellular carcinoma cells.

PURPOSE: Rho kinase is an important factor in tumor progression. We demonstrated that Rho kinase-associated coil-containing protein kinase (ROCK) is expressed in hepatic tissues in hepatocellular carcinoma (HCC) and confirmed its roles in cell survival in HCC cells using the ROCK inhibitor, fasudil. METHODS: ROCK protein levels were estimated in hepatic tissues with HCC compared with healthy liver tissues or hepatic hemangioma tissues using immunohistochemistry. Furthermore, HepG2 and Huh7 cells were cultured with ROCK inhibitor, fasudil for 24 h in vitro. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay, and apoptotic cells were detected by cell death ELISA. The expression apoptosis-related proteins were analyzed using Western blotting. RESULTS: Fasudil significantly decreased cell proliferation and induced apoptosis mediated by increases in p53, Bax, caspase-9, and caspase-3 in HepG2 and Huh7 cells. The induction of apoptosis was inhibited in HCC cells precultured with p53 decoy oligodeoxynucleotide. CONCLUSION: These results suggest that ROCK inhibits the p53-mediated apoptosis pathway in HCC. Fasudil may thus be a beneficial approach to HCC therapy.

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.

The defective protein level of myosin light chain phosphatase (MLCP) in the isolated saphenous vein, as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM).

We examined the contractile reactivity to 5-hydroxytryptamine (5-HT) in isolated human saphenous vein (SV), as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM) and non-DM (NDM). Vascular rings of endothelium-denuded SV were used for functional and biochemical experiments. The vasoconstrictions caused by 5-HT were significantly greater (hyperreactivity) in the DM group than in the NDM group. RhoA/ROCK pathway is activated by various G-protein-coupled receptor agonists and consequently induces phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin light chain phosphatase (MLCP), which inhibits MLCP activity. In the resting state of the vessels, total tissue protein levels of 5-HT(2A) receptor, 5-HT(1B) receptor, RhoA, ROCK1, and ROCK2 did not differ between NDM and DM groups. However, the total protein level of MYPT1 was significantly lower in the DM group than in the NDM group. Furthermore, the ratio of P(Thr(696))-MYPT1 to total MYPT1 was significantly higher in the DM group than in the NDM group. These results suggest that the hyperreactivity to 5-HT in the SV smooth muscle of patients with DM is due to not only enhanced phosphorylation of MLCP but also defective protein level of MLCP. Thus, we reveal for the first time that the defective protein level of MLCP in the DM group can partially explain the poor patency of SV graft harvested from patients with DM.

Actomyosin-mediated cellular tension drives increased tissue stiffness and ß-catenin activation to induce epidermal hyperplasia and tumor growth.

Tumors and associated stroma manifest mechanical properties that promote cancer. Mechanosensation of tissue stiffness activates the Rho/ROCK pathway to increase actomyosin-mediated cellular tension to re-establish force equilibrium. To determine how actomyosin tension affects tissue homeostasis and tumor development, we expressed conditionally active ROCK2 in mouse skin. ROCK activation elevated tissue stiffness via increased collagen. ß-catenin, a key element of mechanotranscription pathways, was stabilized by ROCK activation leading to nuclear accumulation, transcriptional activation, and consequent hyperproliferation and skin thickening. Inhibiting actomyosin contractility by blocking LIMK or myosin ATPase attenuated these responses, as did FAK inhibition. Tumor number, growth, and progression were increased by ROCK activation, while ROCK blockade was inhibitory, implicating actomyosin-mediated cellular tension and consequent collagen deposition as significant tumor promoters.