Increased Rho Kinase Activity Research Articles

Inhibition of IL-6 signaling prevents serum-induced umbilical cord artery dysfunction from patients with severe COVID-19.

Coronavirus disease 2019 (COVID-19) infection has a negative impact on the cytokine profile of pregnant women. Increased levels of proinflammatory cytokines seem to be correlated with the severity of the disease, in addition to predisposing to miscarriage or premature birth. Proinflammatory cytokines increase the generation of reactive oxygen species (ROS). It is unclear how interleukin-6 (IL-6) found in the circulation of patients with severe COVID-19 might affect gestational health, particularly concerning umbilical cord function. This study tested the hypothesis that IL-6 present in the circulation of women with severe COVID-19 causes umbilical cord artery dysfunction by increasing ROS generation and activating redox-sensitive proteins. Umbilical cord arteries were incubated with serum from healthy women and women with severe COVID-19. Vascular function was assessed using concentration-effect curves to serotonin in the presence or absence of pharmacological agents, such as tocilizumab (antibody against the IL-6 receptor), tiron (ROS scavenger), ML171 (Nox1 inhibitor), and Y27632 (Rho kinase inhibitor). ROS generation was assessed by the dihydroethidine probe and Rho kinase activity by an enzymatic assay. Umbilical arteries exposed to serum from women with severe COVID-19 were hyperreactive to serotonin. This effect was abolished in the presence of tocilizumab, tiron, ML171, and Y27632. In addition, serum from women with severe COVID-19 increased Nox1-dependent ROS generation and Rho kinase activity. Increased Rho kinase activity was abolished by tocilizumab and tiron. Serum cytokines in women with severe COVID-19 promote umbilical artery dysfunction. IL-6 is key to Nox-linked vascular oxidative stress and activation of the Rho kinase pathway.

Rho-kinase pathway activation and apoptosis in circulating leucocytes in patients with heart failure with reduced ejection fraction.

BackgroundIncreased Rho‐kinase activity in circulating leucocytes is observed in heart failure with reduced ejection fraction (HFrEF). However, there is little information in HFrEF regarding other Rho‐kinase pathway components an on the relationship between Rho‐kinase and apoptosis. Here, Rho‐kinase activation levels and phosphorylation of major downstream molecules and apoptosis levels were measured for the first time both in HFrEF patients and healthy individuals.MethodsCross‐sectional study comparing HFrEF patients (n = 20) and healthy controls (n = 19). Rho‐kinase activity in circulating leucocytes (peripheral blood mononuclear cells, PBMCs) was determined by myosin light chain phosphatase 1 (MYPT1) and ezrin‐radixin‐moesin (ERM) phosphorylation. Rho‐kinase cascade proteins phosphorylation p38‐MAPK, myosin light chain‐2, JAK and JNK were also analysed along with apoptosis.ResultsMYPT1 and ERM phosphorylation were significantly elevated in HFrEF patients, (3.9‐ and 4.8‐fold higher than in controls, respectively). JAK phosphorylation was significantly increased by 300% over controls. Phosphorylation of downstream molecules p38‐MAPK and myosin light chain‐2 was significantly higher by 360% and 490%, respectively, while JNK phosphorylation was reduced by 60%. Catecholamine and angiotensin II levels were significantly higher in HFrEF patients, while angiotensin‐(1‐9) levels were lower. Apoptosis in circulating leucocytes was significantly increased in HFrEF patients by 2.8‐fold compared with controls and significantly correlated with Rho‐kinase activation.ConclusionRho‐kinase pathway is activated in PMBCs from HFrEF patients despite optimal treatment, and it is closely associated with neurohormonal activation and with apoptosis. ROCK cascade inhibition might induce clinical benefits in HFrEF patients, and its assessment in PMBCs could be useful to evaluate reverse remodelling and disease regression.

Adipose cell size changes are associated with a drastic actin remodeling

Adipose tissue plays a major role in regulating whole-body insulin sensitivity and energy metabolism. To accommodate surplus energy, the tissue rapidly expands by increasing adipose cell size (hypertrophy) and cell number (hyperplasia). Previous studies have shown that enlarged, hypertrophic adipocytes are less responsive to insulin, and that adipocyte size could serve as a predictor for the development of type 2 diabetes. In the present study, we demonstrate that changes in adipocyte size correlate with a drastic remodeling of the actin cytoskeleton. Expansion of primary adipocytes following 2 weeks of high-fat diet (HFD)-feeding in C57BL6/J mice was associated with a drastic increase in filamentous (F)-actin as assessed by fluorescence microscopy, increased Rho-kinase activity, and changed expression of actin-regulating proteins, favoring actin polymerization. At the same time, increased cell size was associated with impaired insulin response, while the interaction between the cytoskeletal scaffolding protein IQGAP1 and insulin receptor substrate (IRS)-1 remained intact. Reversed feeding from HFD to chow restored cell size, insulin response, expression of actin-regulatory proteins and decreased the amount of F-actin filaments. Together, we report a drastic cytoskeletal remodeling during adipocyte expansion, a process which could contribute to deteriorating adipocyte function.

Increased Rho kinase activity in patients with heart ischemia/reperfusion.

Rho kinase is a downstream effector of Rho GTPase that is known to regulate various pathological processes. The aim of this study was to evaluate the regulation of Rho kinase activity in leukocytes in patients with ischemia/reperfusion (I/R) injury. We investigated 38 patients with acute ST-segment elevation myocardial infarction (STEMI), 26 patients with atherosclerosis (AS) and 22 normal subjects. All patients underwent coronary angiography (CAG) and all STEMI patients received primary percutaneous coronary intervention (PPCI) of the left anterior descending artery (LAD) within 12 h after chest pain on-set. Blood samples for leukocyte Rho kinase activity were obtained before CAG and 3 and 24 hours after CAG/PCI. Rho kinase activity increased in the I/R and AS groups. Compared with the AS group, Rho kinase activity was significantly higher in peripheral blood leukocytes in STEMI/PPCI. Furthermore, there was no correlation between changes in Rho kinase activity and changes in high-sensitivity troponin I (hs-TnI) and C-reactive protein (CRP). There was a negative correlation between Rho kinase activity and IL-6. Rho kinase is involved in the pathogenesis of heart I/R injury in patients. Inhibition of Rho kinase may be an additional therapeutic intervention for the treatment of I/R.

ROCK2 Regulates the Expression of Cell Adhesion Molecules and Cell-to-Cell Adhesion in Vascular Endothelial Cells

A pro-inflammatory lipid mediator, lysophosphatidic acid (LPA), is a potent activator of the RhoA/Rho kinase signaling pathway and has been shown to induce the expression of cell adhesion molecules (CAMs). LPA levels are elevated in the circulation of patients with acute coronary syndrome and has also been described inside human atherosclerotic specimens. However, the involvement of Rho-kinase isoforms (i.e., ROCK1, ROCK2) in LPA-mediated expression of CAMs in endothelial cells is not fully understood. In this study, we investigated isoform-specific roles of Rho-kinase in LPA-mediated E-selectin expression using human aortic endothelial cells (HAEC). Exposure of HAEC to LPA resulted in an increase of E-selectin expression at both mRNA (real-time PCR) and protein (Western blot) levels. We confirmed increased Rho-kinase activity in the LPA-stimulated cells. Y-27632, a specific Rho-kinase inhibitor, completely abolished E-selectin protein expression, indicating that LPA induces E-selectin expression via Rho-kinase activation. LPA-induced E-selectin gene expression was strongly suppressed by a chemical inhibitor of NF-κB, indicating the contribution of NF-κB transactivation in this pathway. Intriguingly, phosphorylation of NF-κB RelA/p65 was decreased by Rho-kinase inhibition in the LPA-stimulated cells. Furthermore, using isoform specific siRNAs, we found that Rho-kinase isoform ROCK2 is the key mediator of LPA-induced E-selectin expression and cell adhesion between HAECs and human monocytic cells (THP-1). In the present study, we demonstrate that ROCK2 mediates LPA-induced expression of E-selectin via NF-κB signaling in endothelial cells. ROCK2 could be an important therapeutic target against atherosclerosis. Disclosure Y. Takeda: None. K. Matoba: None. D. Kawanami: None. Y. Nagai: None. T. Akamine: None. S. Ishizawa: None. Y. Kanazawa: None. T. Yokota: None. K. Utsunomiya: None.

Altered ureteral contractility with ageing: Role of the rho-kinase pathway

This study investigated the role of calcium sensitisation in the regulation of ureteral contractility with ageing. Isolated ureteral strips from young (6-month old) and older (3-year old) pigs were mounted in Krebs bicarbonate solution and contractility induced by the α1-adrenoceptor agonist phenylephrine (30 and 300 μM) and 5-HT (10 and 100 μM), recorded in the absence and presence of the rho-kinase inhibitors Y-27632 (10 μM) and fasudil (30 μM). Ureteral strips developed bursts of contractile activity which was measured as area under the curve (AUC) and frequency. Maximum contraction to phenylephrine was significantly enhanced in tissues from older animals compared to younger animals (p < 0.001) while maximum contraction to 5-HT was greater in tissues from younger animals (p < 0.001). Both inhibitors significantly depressed AUC and frequency responses to both agonists in ureters from both age groups (p < 0.05). Inhibition by Y-27632 of phenylephrine (300 μM)- and 5-HT(100 μM)-induced contractions was greater in tissues from older animals than young (p < 0.05). Rho-kinase activity was also assayed in ureteral tissues, and basal activity was similar in ureters from both age groups. Neither phenylephrine nor 5-HT increased rho-kinase activity over basal levels. These data demonstrate the significant role rho-kinase plays in ureteral contractility and possible alterations with age.

Effects of chronic Δ9-tetrahydrocannabinol treatment on Rho/Rho-kinase signalization pathway in mouse brain

Δ9-Tetrahydrocannabinol (Δ9-THC) shows its effects by activating cannabinoid receptors which are on some tissues and neurons. Cannabinoid systems have role on cell proliferation and development of neurons. Furthermore, it is interesting that cannabinoid system and rho/rho-kinase signalization pathway, which have important role on cell development and proliferation, may have role on neuron proliferation and development together. Thus, a study is planned to investigate rhoA and rho-kinase enzyme expressions and their activities in the brain of chronic Δ9-THC treated mice. One group of mice are treated with Δ9-THC once to see effects of acute treatment. Another group of mice are treated with Δ9-THC three times per day for one month. After this period, rhoA and rho-kinase enzyme expressions and their activities in mice brains are analyzed by ELISA method. Chronic administration of Δ9-THC decreased the expression of rhoA while acute treatment has no meaningful effect on it. Administration of Δ9-THC did not affect expression of rho-kinase on both chronic and acute treatment. Administration of Δ9-THC increased rho-kinase activity on both chronic and acute treatment, however, chronic treatment decreased its activity with respect to acute treatment. This study showed that chronic Δ9-THC treatment down-regulated rhoA expression and did not change the expression level of rho-kinase which is downstream effector of rhoA. However, it elevated the rho-kinase activity. Δ9-THC induced down-regulation of rhoA may cause elevation of cypin expression and may have benefit on cypin related diseases. Furthermore, use of rho-kinase inhibitors and Δ9-THC together can be useful on rho-kinase related diseases.

Increased Rho kinase activity predicts worse cardiovascular outcome in ST-segment elevation myocardial infarction patients.

Few clinical studies have assessed Rho kinase (ROCK) in patients with dia-betes mellitus (DM) and ST-segment elevation myocardial infarction (STEMI). This study aimed to determine whether ROCK activity in circulating leukocytes is increased in STEMI patients with DM and whether ROCK activity predicts the onset of cardiovascular events. Blood samples were collected from 60 STEMI patients, divided into non-diabetes mellitus (NDM) and DM groups. Main outcome measures were all-cause mortality, readmission for acute coronary syndrome (ACS), congestive heart failure (CHF), or stroke from pres-entation to approximately 5 years (mean: 41.3 ± 19.6 months; range: 3-60 months). Compared with the NDM group (n = 34), ROCK1 activity was greater in the DM group (n = 26) (33.14 ± 11.31 vs. 26.24 ± 11.06, p = 0.021), while ROCK2 activity was not different between the groups. There occurred 3 deaths, and 10 readmissions with ACS, 4 with CHF and 2 with stroke during the follow-up period. Patients with a high ROCK1 activity on admission had a 3-fold risk of cardiovascular events (RR 3.15, 95% CI 1.04-9.58) compared with those with low ROCK1 activity. Patients with history of stroke had almost a 4-fold risk of cardiovascular events (RR 3.74; 95% CI 1.02-13.80). ROCK1 activity was increased in STEMI patients with DM, which suggests that ROCK1 activity may be a useful diagnostic and prognostic marker of cardiovascular events for these patients. ROCK1 activity might help identify a subset of STEMI patients at particularly high risk.

Changes of Rho kinase activity in peripheral blood T lymphocytes from patients with atopic dermatitis and their significance

Objective To evaluate changes of Rho kinase (ROK) activity in peripheral blood T lymphocytes from patients with atopic dermatitis (AD), and to analyze their clinical significance. Methods Eight milliliters of heparin-anticoagulated blood samples were collected from 60 patients with AD and 60 healthy human controls followed by separation of T lymphocytes and sera from these blood samples as well as culture of isolated T lymphocytes with 10% fetal bovine serum for 24 hours. Both patient- and control-derived T lymphocytes were classified into two groups to be cultured with patient- or control-derived sera. In addition, some patient-derived T lymphocytes were classified into 4 groups: Y27632 group treated with the Rho kinase-specific inhibitor Y2763, CD3/CD28 group treated with anti-CD3/anti-CD28 monoclonal antibodies, Y27632 + CD3/CD28 group treated with Y27632 and anti-CD3/anti-CD28 monoclonal antibodies, and control group treated with patient-derived sera. Subsequently, Western-blot analysis was performed to evaluate ROK activity in cells, methyl thiazolyl tetrazolium (MTT) assay to evaluate proliferative activity of T lymphocytes, and ELISA to measure interleukin 6 (IL-6) and IL-10 levels in supernatants of T lymphocytes. Results ROK activity was significantly lower in fresh T lymphocytes from patients than in those from healthy controls (2.47% ± 0.89% vs. 0.65% ± 0.35%, t = 2.729, P 0.05). Compared with T lymphocytes cultured with control-derived sera, those cultured with patient-derived sera showed significantly increased ROK activity (F= 8.22, P< 0.001). Concretely speaking, ROK activity was significantly higher in patient-derived T lymphocytes cultured with patient-derived sera than in those cultured with control-derived sera (2.41% ± 0.87% vs. 0.76% ± 0.41%, P< 0.05), and higher in control-derived T lymphocytes cultured with patient-derived sera than in those cultured with control-derived sera (2.17% ± 0.85% vs. 0.64% ± 0.33%, P< 0.05) at 24 hours. Y27632 could significantly inhibit the proliferation of as well as secretion of IL-6 (F = 18.68, 22.95, respectively, both P< 0.001) by patient - derived T lymphocytes, but had insignificant effects on secretion of IL-10. The cellular proliferative activity and IL-6 supernatant level were significantly lower in the Y27632 group than in the control group, and lower in the Y27632 + CD3/CD28 group than in the CD3/CD28 group (all P< 0.05). Conclusion Aberrant activation of ROK exists in T lymphocytes from patients with AD, which may play a certain role in the pathogenesis of AD. Key words: Dermatitis, atopic; T-lymphocytes; rho-Associated kinases; Signal transduction; Interleukin-6; Interleukin-10

Leptin augments coronary vasoconstriction and smooth muscle proliferation via a Rho-kinase-dependent pathway.

Leptin has been implicated as a key upstream mediator of pathways associated with coronary vascular dysfunction and disease. The purpose of this investigation was to test the hypothesis that leptin modifies the coronary artery proteome and promotes increases in coronary smooth muscle contraction and proliferation via influences on Rho kinase signaling. Global proteomic assessment of coronary arteries from lean swine cultured with obese concentrations of leptin (30 ng/mL) for 3 days revealed significant alterations in the coronary artery proteome (68 proteins) and identified an association between leptin treatment and calcium signaling/contraction (four proteins) and cellular growth and proliferation (35 proteins). Isometric tension studies demonstrated that both acute (30 min) and chronic (3 days, serum-free media) exposure to obese concentrations of leptin potentiated depolarization-induced contraction of coronary arteries. Inhibition of Rho kinase significantly reduced leptin-mediated increases in coronary artery contractions. The effects of leptin on the functional expression of Rho kinase were time-dependent, as acute treatment increased Rho kinase activity while chronic (3 day) exposure was associated with increases in Rho kinase protein abundance. Proliferation assays following chronic leptin administration (8 day, serum-containing media) demonstrated that leptin augmented coronary vascular smooth muscle proliferation and increased Rho kinase activity. Inhibition of Rho kinase significantly reduced these effects of leptin. Taken together, these findings demonstrate that leptin promotes increases in coronary vasoconstriction and smooth muscle proliferation and indicate that these phenotypic effects are associated with alterations in the coronary artery proteome and dynamic effects on the Rho kinase pathway.

Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells.

Mechanisms of the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.

Increased rho kinase activity in mononuclear cells of dialysis and stage 3–4 chronic kidney disease patients with left ventricular hypertrophy: Cardiovascular risk implications

AimsCardiovascular disease (CVD) is the leading cause of excess mortality in chronic kidney disease (CKD) and dialysis patients (DP) who have higher prevalence of left ventricular hypertrophy (LVH), the strongest predictor of CV events. Rho kinase (ROCK) activation is linked in hypertensive patients to cardiac remodeling while ROCK inhibition suppresses cardiomyocyte hypertrophy and, in a human clinical condition opposite to hypertension, its downregulation associates with lack of CV remodeling. Information on ROCK activation-LVH link in CKD and DP is lacking. Materials and methodsMononuclear cells (PBMCs) MYPT-1 phosphorylation, a marker of ROCK activity, and the effect of fasudil, a ROCK inhibitor, on MYPT-1 phosphorylation were assessed in 23 DPs, 13 stage 3–4 CKD and 36 healthy subjects (HS) by Western blot. LV mass was assessed by M-mode echocardiography. Key findingsDP and CKD had higher MYPT-1 phosphorylation compared to HS (p<0.001 and p=0.003). Fasudil (500 and 1000μM) dose dependently reduced MYPT-1 phosphorylation in DP (p<0.01). DP had higher LV mass than CKD (p<0.001). MYPT-1 phosphorylation was higher in patients with LVH (p=0.009) and correlated with LV mass both in DP and CKD with LVH (p<0.001 and p=0.006). SignificanceIn DP and CKD, ROCK activity tracks with LVH. This ROCK activation-LVH link provided in these CVD high-risk patients along with similar findings in hypertensive patients and added to opposite findings in a human model opposite to hypertension and in type 2 diabetic patients, identify ROCK activation as a potential LVH marker and provide further rationale for ROCK activation inhibition as target of therapy in CVD high-risk patients.

Aging-related Increase in Rho Kinase Activity in the Nigral Region Is Counteracted by Physical Exercise.

Abnormal activation of the RhoA/Rho kinase (ROCK) pathway plays a pivotal role in neuroinflammatory and pro-oxidative responses, axonal retraction, and apoptosis. We observed increased expression of RhoA, ROCK II, and ROCK activity in the brain of aged rats, particularly in the substantia nigra. Increased ROCK activity may enhance major mechanisms responsible for aging-related neurodegeneration, thus representing a major factor in the vulnerability of dopaminergic neurons to damage. We also observed that physical exercise decreased ROCK activation in aged rats. This suggests that decreased ROCK activation plays an important role in the neuroprotective effects of exercise observed in several previous studies. Furthermore, the present results suggest that ROCK inhibitors may constitute an effective neuroprotective strategy against aging-related risk of dopaminergic degeneration and possibly against other aging-related neurodegenerative processes.

Inverse relationship of Rho kinase and myosin-light chain kinase expression in the aging human detrusor smooth muscle.

BackgroundRho kinase (ROCK) and myosin-light chain kinase (MLCK) are key enzymes in smooth muscle contraction. Previous data have suggested that ROCK contribution to human detrusor contraction is increasing with age. Here, we have analyzed the transcriptional expression of Rho kinase isoforms (ROCK1 and ROCK2) as well as MLCK in the aging human detrusor smooth muscle obtained from resected tissue.MethodsSmall pieces of macroscopically healthy human detrusor smooth muscle (urothelium-free) were prepared for quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Transcript expression (mRNA level) of the target genes ROCK1, ROCK2 and MLCK was normalized to three common reference genes (glyceraldehyde-3-phosphate dehydrogenase, β-actin, phosphoglycerate kinase 1).ResultsWe found that across all ages the expression level of ROCK (i.e. ROCK1 and ROCK2 together) was almost equal to that of MLCK in the human bladder. Further, ROCK2 showed a significantly higher expression level than ROCK1. Among all subjects, there was no significant correlation of any single target gene to age, but expression levels of ROCK and MLCK were inversely correlated. Moreover, the within-subject analysis revealed that the ROCK-to-MLCK ratio showed a significantly negative correlation to age. Thus, within a given subject, there is a relative ROCK down-regulation and concomitant MLCK up-regulation.ConclusionsTogether with previous data in human detrusor specimens showing increased ROCK contribution to detrusor contraction, we speculate that the drop of the ROCK-to-MLCK ratio may occur as an attempt to compensate for the increased Rho kinase activity.

Tu1906 Direct Activation of MEK1 Kinase by Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Barrett's Associated Esophageal Adenocarcinoma Cells

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. We have shown that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression is markedly increased in human Barrett's mucosal biopsies with dysplasia and in EA tissues, suggesting that NOX5-S may play an important role in this progression. In this study we examined the signal-transduction pathway of acid-induced NOX5-S expression in FLO EA cells. Acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of ROCK2 with ROCK2 siRNA and MEK1 with MEK1 siRNA, but not by knockdown of ROCK1 and MEK2. Conversely, overexpression of constitutively active ROCK2, but not constitutively active ROCK1, significantly enhanced NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased the phosphorylation of MEK1 at threonine 292 (T292), but not at Serine 298 (S298). Overexpression of WT MEK1 and constitutively active MEK1 significantly increased NOX5-S promoter activity. Mutation of Threonine at 292 to Alanine abolished NOX5-S promoter activity and H2O2 production induced by overexpression of constitutively active MEK1. Acid-induced increase in ERK-2 phosphorylation was significantly reduced by Rho kinase inhibitor Y27632. Acidinduced increase in Rho kinase activity was not affected by knockdown of MEK1. In an in vitro kinase assay, we found that active ROCK2 increased MEK1 phosphorylation at T292. To test whether Rho kinases directly activate MEK1, we used synthesized peptides containing the flanking sequence of T292 and T292A in the ROCK2 kinase assay. We found that ROCK2 phosphorylated WT peptide DAAETPPRPRTPGRPLSSYGM, but did not phosphorylate T292A mutation peptide DAAETPPRPRAPGRPLSSYGM. Acid-induced increase in MEK1 T292 phosphorylation was significantly decreased by knockdown of ROCK2, but not of ROCK1. Overexpression of constitutively active ROCK2 remarkably increased MEK1 T292 phosphorylation, but not MEK1 S298 phosphorylation, in FLO EA cells. We conclude that acid-induced increase in NOX5-S expression and H2O2 production is mediated by sequential activation of ROCK2 and MEK1 in FLO EA cells. Activation of MEK1 depends on direct phosphorylation of MEK1 T292 by ROCK2. Supported by NIH NIDDK R01 DK080703.

Tu1911 A Novel Mechanism for Radiocontrast-Induced Pancreatic NF-KB Translocation and Injury Through Triggering Calcium and Calcineurin Activation

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. We have shown that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression is markedly increased in human Barrett's mucosal biopsies with dysplasia and in EA tissues, suggesting that NOX5-S may play an important role in this progression. In this study we examined the signal-transduction pathway of acid-induced NOX5-S expression in FLO EA cells. Acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of ROCK2 with ROCK2 siRNA and MEK1 with MEK1 siRNA, but not by knockdown of ROCK1 and MEK2. Conversely, overexpression of constitutively active ROCK2, but not constitutively active ROCK1, significantly enhanced NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased the phosphorylation of MEK1 at threonine 292 (T292), but not at Serine 298 (S298). Overexpression of WT MEK1 and constitutively active MEK1 significantly increased NOX5-S promoter activity. Mutation of Threonine at 292 to Alanine abolished NOX5-S promoter activity and H2O2 production induced by overexpression of constitutively active MEK1. Acid-induced increase in ERK-2 phosphorylation was significantly reduced by Rho kinase inhibitor Y27632. Acidinduced increase in Rho kinase activity was not affected by knockdown of MEK1. In an in vitro kinase assay, we found that active ROCK2 increased MEK1 phosphorylation at T292. To test whether Rho kinases directly activate MEK1, we used synthesized peptides containing the flanking sequence of T292 and T292A in the ROCK2 kinase assay. We found that ROCK2 phosphorylated WT peptide DAAETPPRPRTPGRPLSSYGM, but did not phosphorylate T292A mutation peptide DAAETPPRPRAPGRPLSSYGM. Acid-induced increase in MEK1 T292 phosphorylation was significantly decreased by knockdown of ROCK2, but not of ROCK1. Overexpression of constitutively active ROCK2 remarkably increased MEK1 T292 phosphorylation, but not MEK1 S298 phosphorylation, in FLO EA cells. We conclude that acid-induced increase in NOX5-S expression and H2O2 production is mediated by sequential activation of ROCK2 and MEK1 in FLO EA cells. Activation of MEK1 depends on direct phosphorylation of MEK1 T292 by ROCK2. Supported by NIH NIDDK R01 DK080703.

Tu1907 Curcumin Reduces the Inflammatory Factors of Reflux Esophagitis Through Non PKC Pathway in Part in Rat Model

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. We have shown that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression is markedly increased in human Barrett's mucosal biopsies with dysplasia and in EA tissues, suggesting that NOX5-S may play an important role in this progression. In this study we examined the signal-transduction pathway of acid-induced NOX5-S expression in FLO EA cells. Acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of ROCK2 with ROCK2 siRNA and MEK1 with MEK1 siRNA, but not by knockdown of ROCK1 and MEK2. Conversely, overexpression of constitutively active ROCK2, but not constitutively active ROCK1, significantly enhanced NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased the phosphorylation of MEK1 at threonine 292 (T292), but not at Serine 298 (S298). Overexpression of WT MEK1 and constitutively active MEK1 significantly increased NOX5-S promoter activity. Mutation of Threonine at 292 to Alanine abolished NOX5-S promoter activity and H2O2 production induced by overexpression of constitutively active MEK1. Acid-induced increase in ERK-2 phosphorylation was significantly reduced by Rho kinase inhibitor Y27632. Acidinduced increase in Rho kinase activity was not affected by knockdown of MEK1. In an in vitro kinase assay, we found that active ROCK2 increased MEK1 phosphorylation at T292. To test whether Rho kinases directly activate MEK1, we used synthesized peptides containing the flanking sequence of T292 and T292A in the ROCK2 kinase assay. We found that ROCK2 phosphorylated WT peptide DAAETPPRPRTPGRPLSSYGM, but did not phosphorylate T292A mutation peptide DAAETPPRPRAPGRPLSSYGM. Acid-induced increase in MEK1 T292 phosphorylation was significantly decreased by knockdown of ROCK2, but not of ROCK1. Overexpression of constitutively active ROCK2 remarkably increased MEK1 T292 phosphorylation, but not MEK1 S298 phosphorylation, in FLO EA cells. We conclude that acid-induced increase in NOX5-S expression and H2O2 production is mediated by sequential activation of ROCK2 and MEK1 in FLO EA cells. Activation of MEK1 depends on direct phosphorylation of MEK1 T292 by ROCK2. Supported by NIH NIDDK R01 DK080703.

Abstract 14056: Prognostic Impact of Rho-kinase Activity in Circulating Leukocytes of Patients with Vasospastic Angina

Background: Rho-kinase plays a central role in the pathogenesis of coronary artery spasm. We have recently demonstrated that Rho-kinase activity in circulating leukocytes is a useful biomarker for diagnosis and disease activity assessment of vasospastic angina (VSA). However, its prognostic impact in VSA patients remains to be examined. Methods: We prospectively enrolled 174 consecutive VSA patients (M/F 116/58, 62±12 [SD] yrs) and 53 non-VSA patients (M/F 32/21, 62±15 yrs) and followed them for 14±9 months. At the enrollment, Rho-kinase activity in circulating leukocytes (the ratio of phosphorylated/total form of myosin binding subunit, a Rho-kinase substrate) was measured. The primary endpoint was major adverse cardiac events (MACE). Results: Baseline Rho-kinase activity was significantly higher in VSA than in non-VSA patients (1.20±0.33 vs. 1.07±0.21, P&lt;0.01) and all VSA patients were treated with calcium channel blockers. During the follow-up period, MACE occurred in 9 VSA patients (5.2%) but none in non-VSA patients. Baseline Rho-kinase activity was significantly higher in VSA patients with MACE than in those without it (1.33±0.13 vs. 1.20±0.33, P&lt;0.001). Importantly, when we divided VSA patients into 2 groups by a median value of baseline Rho-kinase activity, Kaplan-Meier survival analysis showed a significantly worse prognosis in VSA patients with high Rho-kinase activity (p-MBS/t-MBS ≥1.20) ( Figure ). Univariate linear regression models showed that there was no association between established prognostic factors of VSA (e.g. history of out-of-hospital cardiac arrest, current smoking, angina at rest, significant organic stenosis and β-blocker use) and baseline increased levels of Rho-kinase activity. Conclusions: These results indicate that increased Rho-kinase activity in circulating leukocytes may be a useful and independent biomarker for long-term prognosis of VSA patients.

GW25-e1716 Effect of Atorvastatin on Endothelial Function and Rho Kinase Activity in Patients with Stable Coronary Heart Disease

In addition to inhibiting cholesterol synthesis, statins decrease the formation of isoprenoid intermediates required for the activation of RhoARho kinase pathway. Increased Rho kinase activity has been implicated in endothelial dysfunction and plays pivotal roles in cardiovascular diseases including

N-[11C]-methyl-hydroxyfasudil is a potential biomarker of cardiac hypertrophy

IntroductionPathologic cardiac hypertrophy is one of the leading causes of sudden death from cardiac disease and involves a complex network of bio-signaling mechanisms. To date, the clinical detection and pathologic progression of hypertrophy remains elusive. Here we tested whether imaging Rho kinase activity would serve an accurate proxy for detecting hypertrophy. Specifically, we examine the use of the N-[11C]-methylated derivative of hydroxyfasudil, a Rho kinase inhibitor, as a biomarker for accurate identification of cardiomyocyte hypertrophy. MethodsBoth transformed and primary neonatal cardiomyocytes were treated with isoproterenol to induce β-adrenergic receptor stimulation and hypertrophy. Phenotypic hypertrophy was verified using cytochemical evaluation of cell and nuclear size. Western blot and activity assays were used to detect ERK 1/2 mTOR and Rho kinase activation. N-[11C]-methyl-hydroxyfasudil binding was verified using in vitro binding assays with isoproterenol stimulated cells. ResultsIsoproterenol induced a rapid and distinct activation of ERK 1/2, mTOR and Rho kinase with negligible cytotoxicity. Subsequent expansion in cell and nuclear size that is typically associated with hypertrophy was also observed. Enhanced retention of N-[11C]-methyl-hydroxyfasudil observed after ISO-induced Rho kinase activation in hypertrophic cells was prevented by pre-treatment with unlabeled hydroxyfasudil. ConclusionsN-[11C]-methyl-hydroxyfasudil is able to measure increased Rho kinase activity via specific binding in hypertrophied cardiomyocytes and demonstrates the potential for molecular imaging of altered Rho kinase activity in diseases such as cardiac hypertrophy.