ADAMTS4 is involved in the production of the Alzheimer disease amyloid biomarker APP669-711.

Amyloid-ß (Aß) deposition in the brain parenchyma is one of the pathological hallmarks of Alzheimer disease (AD). We have previously identified amyloid precursor protein (APP)669-711 (a.k.a. Aß(-3)-40) in human plasma using immunoprecipitation combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (IP-MALDI-MS). Furthermore, we found that the level of a composite biomarker, i.e., a combination of APP669-711/Aß1-42 ratio and Aß1-40/Aß1-42 ratio in human plasma, correlates with the amyloid PET status of AD patients. However, the production mechanism of APP669-711 has remained unclear. Using in vitro and in vivo assays, we identified A Disintegrin and Metalloproteinase with a Thrombospondin type 1 motif, type 4 (ADAMTS4) as a responsible enzyme for APP669-711 production. ADAMTS4 cleaves APP directly to generate the C-terminal stub c102, which is subsequently proteolyzed by γ-secretase to release APP669-711. Genetic knockout of ADAMTS4 reduced the production of endogenous APP669-711 by 30% to 40% in cultured cells as well as mouse plasma, irrespectively of Aß levels. Finally, we found that the endogenous murine APP669-711/Aß1-42 ratio was increased in aged AD model mice, which shows Aß deposition as observed in human patients. These data suggest that ADAMTS4 is involved in the production of APP669-711, and a plasma biomarker determined by IP-MALDI-MS can be used to estimate the level of Aß deposition in the brain of mouse models.

Therapeutic effect of ouabagenin, a novel liver X receptor agonist, on atherosclerosis in nonalcoholic steatohepatitis in SHRSP5/Dmcr rat model.

The liver X receptor (LXR) can enhance cholesterol transporters, which could remove excess cholesterol from foam cells in atheromas. LXR has two subtypes: LXRα, which aggravates hepatic lipid accumulation, and LXRß, which does not. In 2018, ouabagenin (OBG) was reported as a potential LXRß-specific agonist. We aimed to examine whether OBG specifically affects LXRß in nonalcoholic steatohepatitis (NASH); it did not aggravate hepatic steatosis and can suppress the development of atherosclerosis. SHRSP5/Dmcr rats fed a high-fat and high-cholesterol diet were divided into four groups as follows: (I) L-NAME group, (II) L-NAME/OBG group, (III) OBG (-) group, and (IV) OBG (+) group. All groups' rats were intraperitoneally administered L-NAME. The L-NAME/OBG group's rats were intraperitoneally administered OBG and L-NAME simultaneously. After L-NAME administration, the OBG (+) group's rats were administered OBG, while the OBG (-) group's rats were not. Although all rats developed NASH, OBG did not exacerbate steatosis (L-NAME/OBG and OBG (+) groups). In addition, endothelial cells were protected in the L-NAME/OBG group and foam cells in the atheroma were reduced in the OBG (+) group. OBG is an LXRß-specific agonist and has a potential therapeutic effect on atherosclerosis without developing lipid accumulation in the liver.

Increased Glycine-conjugated and Unconjugated Bile Acid Levels Associated with Aggravation of Nonalcoholic Steatohepatitis and Cardiovascular Disease in SHRSP5/Dmcr Rat.

The SHRSP5/Dmcr is a useful animal model for the development of nonalcoholic steatohepatitis (NASH) pathology when fed a high-fat, high-cholesterol diet, and further drug interventions can lead to concomitant cardiovascular disease. While SHRSP5/Dmcr rats have been used for basic research related to NASH, details of their bile acid metabolism in this condition are unknown. In this study, we aimed to clarify the changes in the serum bile acid (BA) fractions associated with NASH and found that glycine-conjugated and unconjugated bile acid increased with worsening NASH and cardiovascular disease while taurine-conjugated BA relatively decreased.

Antioxidant action of xanthine oxidase inhibitor febuxostat protects the liver and blood vasculature in SHRSP5/Dmcr rats.

BACKGROUND: Xanthine oxidase (XO) generates reactive oxygen species during uric acid production. Therefore, XO inhibitors, which suppress oxidative stress, may effectively treat non-alcoholic steatohepatitis (NASH) and atherosclerosis via uric acid reduction. In this study, we examined the antioxidant effect of the XO inhibitor febuxostat on NASH and atherosclerosis in stroke-prone spontaneously hypertensive 5 (SHRSP5/Dmcr) rats. METHODS: SHRSP5/Dmcr rats were divided into three groups: SHRSP5/Dmcr + high-fat and high-cholesterol (HFC) diet [control group, n = 5], SHRSP5/Dmcr + HFC diet + 10% fructose (40 ml/day) [fructose group, n = 5], and SHRSP5/Dmcr + HFC diet + 10% fructose (40 ml/day) + febuxostat (1.0 mg/kg/day) [febuxostat group, n = 5]. Glucose and insulin resistance, blood biochemistry, histopathological staining, endothelial function, and oxidative stress markers were evaluated. RESULTS: Febuxostat reduced the plasma uric acid levels. Oxidative stress-related genes were downregulated, whereas antioxidant factor-related genes were upregulated in the febuxostat group compared with those in the fructose group. Febuxostat also ameliorated inflammation, fibrosis, and lipid accumulation in the liver. Mesenteric lipid deposition decreased in the arteries, and aortic endothelial function improved in the febuxostat group. CONCLUSIONS: Overall, the XO inhibitor febuxostat exerted protective effects against NASH and atherosclerosis in SHRSP5/Dmcr rats.

Potential of a Novel Chemical Compound Targeting Matrix Metalloprotease-13 for Early Osteoarthritis: An In Vitro Study.

Osteoarthritis is a progressive disease characterized by cartilage destruction in the joints. Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) play key roles in osteoarthritis progression. In this study, we screened a chemical compound library to identify new drug candidates that target MMP and ADAMTS using a cytokine-stimulated OUMS-27 chondrosarcoma cells. By screening PCR-based mRNA expression, we selected 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide as a potential candidate. We found that 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide attenuated IL-1ß-induced MMP13 mRNA expression in a dose-dependent manner, without causing serious cytotoxicity. Signaling pathway analysis revealed that 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide attenuated ERK- and p-38-phosphorylation as well as JNK phosphorylation. We then examined the additive effect of 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide in combination with low-dose betamethasone on IL-1ß-stimulated cells. Combined treatment with 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide and betamethasone significantly attenuated MMP13 and ADAMTS9 mRNA expression. In conclusion, we identified a potential compound of interest that may help attenuate matrix-degrading enzymes in the early osteoarthritis-affected joints.

Bile acids aggravate nonalcoholic steatohepatitis and cardiovascular disease in SHRSP5/Dmcr rat model.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is linked to an increased risk of cardiovascular disease, regardless of the risk factors in metabolic syndrome. However, the intermediary factors between NASH and cardiovascular disease are still unknown. A previous study revealed that serum and hepatic bile acid (BA) levels are increased in some NASH patients. We aimed to examine whether NASH and cardiovascular disease were aggravated by BA using an animal model. METHOD AND RESULTS: From 10 to 18 weeks of age, SHRSP5/Dmcr rats divided into 3 groups were fed 3 types of high-fat and high-cholesterol (HFC) diets which were changed in the cholic acid (CA) concentration (0%, 2%, or 4%). The nitro oxide synthase inhibition (L-NAME) was administered intraperitoneally from 16 to 18 weeks of age. The 4% CA groups showed the worst LV dysfunction and myocardial fibrosis, and demonstrated severe hepatic fibrosis and lipid depositions. In addition, a large amount of lipid accumulation was observed in the aortas of the 4% CA group, and NFκB and VCAM-1 gene expression levels were increased. These findings were not seen in the 0% CA group. CONCLUSION: In the SHRSP5/Dmcr rat model, NASH and cardiovascular disease were aggravated with increasing BAs concentrations in an HFC diet.

Mechanical strain attenuates cytokine-induced ADAMTS9 expression via transient receptor potential vanilloid type 1.

The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1ß and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.

Induction of CEMIP in Chondrocytes by Inflammatory Cytokines: Underlying Mechanisms and Potential Involvement in Osteoarthritis.

In patients with osteoarthritis (OA), there is a decrease in both the concentration and molecular size of hyaluronan (HA) in the synovial fluid and cartilage. Cell migration-inducing hyaluronidase 1 (CEMIP), also known as hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID), was recently reported as an HA depolymerization-related molecule expressed in the cartilage of patients with OA. However, the underlying mechanism of CEMIP regulation is not well understood. We found that CEMIP expression was transiently increased by interleukine-1ß (IL-1ß) stimulation in chondrocytic cells. We also observed that ERK activation and NF-κB nuclear translocation were involved in the induction of CEMIP by IL-1ß. In addition, both administration of HA and mechanical strain attenuated the CEMIP induction in IL-1ß-stimulated chondrocytes. In conclusion, we clarified the regulatory mechanism of CEMIP in chondrocytes by inflammatory cytokines and suggested the potential involvement in osteoarthritis development.

Activated clotting time on the day of atrial fibrillation ablation for minimally interrupted and uninterrupted direct oral anticoagulation therapy: Sequential changes, differences among direct oral anticoagulants, and ablation safety outcomes.

BACKGROUND: Activated clotting time (ACT)-guided heparinization is used during atrial fibrillation (AF) ablation. Differences in sensitivity to ACT assays have been identified among different direct oral anticoagulants (DOACs). OBJECTIVE: We aimed to examine ACT just before ablation (pre-ACT) for different ablation start times (9:00, 11:00, 13:00, or 15:00) and ablation safety outcomes in minimally interrupted (min-Int) and uninterrupted (Unint) DOAC regimens and examine differences in pre-ACT values among four DOACs. METHODS: Consecutive patients were randomized into the min-Int (n = 307) or Unint (n = 277) groups. DOACs examined were apixaban, dabigatran, edoxaban, and rivaroxaban. RESULTS: No sequential changes in pre-ACT values were observed for each DOAC used and for all four DOACs combined in the min-Int and Unint groups. There was no meaningful difference in pre-ACT at each ablation start time between the groups. Clinically significant differences in overall pre-ACT were not obtained between the groups (138 ± 24 vs 142 ± 23 seconds). The pre-ACT (baseline) value for dabigatran was on average 29 seconds higher than that for the other three DOACs. The min-Int and Unint groups showed similar thromboembolic (0% vs 0%) and bleeding event rates (major, 1% vs 0%; all, 3.5% vs 2.5%). CONCLUSION: The pre-ACT did not show a sequential change in the min-Int and Unint groups. No notable differences in the time-dependent change in pre-ACT between the groups were observed. Variations in baseline ACT suggest the need for moderate adjustment of ACT for adequate modification of heparin dose for the other three DOACs. Both regimens provided similar acceptable AF ablation safety outcomes.

Effects of Oral Anticoagulants on Patients With Atrial Fibrillation Aged 90 Years and Older: Comparison Among Direct Oral Anticoagulant, Warfarin Anticoagulant, and Nonanticoagulation.

This study aimed to investigate the effects of anticoagulants on ultra-aged patients with nonvalvular atrial fibrillation (AF). We retrospectively studied 320 consecutive patients with AF (median age, 91 years; range 90-100.1 years). Patients were categorized as follows: patients taking direct oral anticoagulant (DOAC group, n = 93), those taking warfarin (warfarin group, n = 147), and those not taking oral anticoagulants (non-OAC group, n = 80). During the follow-up periods (median 3.00 years; first and fourth quantiles, 1.13 and 4.56 years, respectively), in thromboembolic events, the DOAC, warfarin, and non-OAC groups showed the lowest (0%, 0/93; 0%/year), intermediate (4.7%, 7/149; 1.43%/year), and highest (5%, 4/80; 2.65%/year) incidence rates, respectively. In major bleeding events, the DOAC, warfarin, and non-OAC groups showed the highest (9.67%, 9/96; 5.00%/year), intermediate (8.1%, 12/149; 2.46%/year), and lowest (0%, 0/80; 0%/year) incidence rates, respectively. These differences in the relationships of the 3 groups were statistically significant. Confounding factors did not affect these results. Bruises associated with impairment of motor function with aging caused major bleeding in approximately 60% of major bleeding cases. The Cox proportional hazards model revealed that warfarin decreased mortality, whereas antiplatelet drugs increased mortality. In conclusion, DOACs had considerably high incidence of major bleeding events, whereas absence of OAC treatment was associated with substantially high thromboembolic events. Warfarin showed acceptable incidence ratios of both events. At present, warfarin is thus believed to be adequate for ultra-aged (≥90 years) patients with nonvalvular AF. Avoidance of bruises was important to prevent major bleeding events. Antiplatelet drugs were suggested not to be adequate for these patients.

Adjunctive left anterior line ablation induced left atrial dysfunction and dyssynchrony in atrial fibrillation ablation.

We evaluated the effects of adjunctive left anterior line (LAL) ablation on LA dyssynchrony and function using real-time three-dimensional echocardiography (3DE) in connection with thromboembolic complications and tachyarrhythmia recurrence in patients with persistent atrial fibrillation (AF). We randomly and prospectively assigned consecutive persistent AF patients to the LAL (n = 52, 65 ± 7 years) and control groups (n = 50, 64 ± 10 years). In the LAL group, extensive encircling pulmonary vein isolation (EEPVI), roof line ablation, and LAL ablation regardless of the extent of the low-voltage area (LVA) were performed. The control group underwent EEPVI and roof line ablation. After ablation, 3DE demonstrated LA dyssynchrony in 23 (46%) and 4 patients (8%, P < 0.001) of the LAL and control groups, respectively. Baseline LA LVAs were relatively small in most patients and there were no significant differences in extent of LVA between control and LAL groups or between patients with and without dyssynchrony. During the follow-up periods (771 ± 121 days), patients with LA dyssynchrony in the LAL group did not show significant differences in symptomatic thromboembolic events (0%) and atrial tachyarrhythmia recurrence (39%) from patients without LA dyssynchrony in the LAL (0% and 30%) and control groups (0% and 32%, respectively). LA ejection fraction and active emptying fraction were lower by 9% on average in the LAL group than in the control group (P < 0.0001). Similarly, in the LAL group, LA ejection fraction, active emptying fraction, and expansion index were significantly lower by approximately 7%, 8%, and 15%, respectively, in LA with dyssynchrony than those in LA without dyssynchrony. In conclusion, LA dyssynchrony and LA hypofunction were induced by LAL ablation in patients with persistent AF and relatively mild LVA. LAL ablation with or without LA dyssynchrony is thought not to affect thromboembolic complications or atrial tachyarrhythmia recurrence.

Non-alcoholic steatohepatitis aggravates nitric oxide synthase inhibition-induced arteriosclerosis in SHRSP5/Dmcr rat model.

Non-alcoholic steatohepatitis (NASH) is linked to increased cardiovascular risk, independent of the broad spectrum of metabolic syndrome risk factors. Stroke-prone (SP) spontaneously hypertensive rats (SHRSP5/Dmcr) fed a high-fat and high-cholesterol (HFC) diet developed hepatic lesions similar to those in human NASH pathology. These rats simultaneously developed lipid deposits in the mesenteric arteries, cardiac fibrosis, endothelial dysfunction and left ventricle (LV) diastolic dysfunction. However, the intermediary factors between NASH and cardiovascular disease are still unknown. We investigated whether NASH aggravates nitric oxide (NO) synthase inhibition-induced arteriosclerosis in SHRSP5/Dmcr rats. Wistar Kyoto and SHRSP5/Dmcr rats were divided into 4 groups of 5 and fed the stroke-prone (SP) or HFC diets for 8 weeks. To induce NO synthase inhibition, Nω -nitro-L-arginine methyl ester hydrochloride (L-NAME) mixed with drinking water was administered in the final 2 weeks. The NASH+L-NAME group demonstrated the following characteristics related to arteriosclerosis and myocardial ischaemia: (a) LV systolic dysfunction with asynergy, (b) replacement fibrosis caused by the shedding of cardiomyocytes and (c) arterial lipid deposition and coronary occlusion secondary to endothelial dysfunction. These characteristics were not observed in the NASH or non-NASH+L-NAME groups. The SHRSP5/Dmcr rat model demonstrates that NASH significantly aggravates cardiovascular risk.

Differences in activated clotting time and initial heparin dosage during atrial fibrillation ablation for patients with edoxaban compared with warfarin.

BACKGROUND: Different target activated clotting times (ACTs) during atrial fibrillation (AF) ablation have been proposed. Moreover, relationships between initial bolus dose of heparin at the start of AF ablation in patients receiving edoxaban anticoagulation therapy and ACT are unclear. METHODS: Patients who received anticoagulation with uninterrupted warfarin (control; n = 120) or interrupted edoxaban (n = 120) on the morning of day of ablation were studied. An initial dose of 100 U/kg heparin was administered as a reliable control for warfarin. Initial heparin doses of 120, 130, 140, or 150 U/kg were randomly administered to the edoxaban group. RESULTS: Edoxaban group showed shorter baseline ACT before the procedure (130 ± 16 seconds) than the warfarin group (152 ± 26 seconds, P < 0.0001). In the warfarin group, 100 U/kg heparin showed 361 ± 48 seconds 15-minute ACT. In the edoxaban group, an increase in initial dose induced prolongation of 15-minute ACT (i.e., 15-minute ACTs of 293 ± 56, 306 ± 39, 311 ± 45, and 319 ± 45 seconds for 120, 130, 140, and 150 U/kg initial doses, respectively). The total heparin required during the procedure was higher in the edoxaban group than in the warfarin group (109 ± 37 vs. 77 ± 21 U/kg/h, P < 0.0001). The 120-150 U/kg dose of heparin in edoxaban group did not cause thromboembolic or major bleeding complications. CONCLUSION: Edoxaban interrupted on the day of ablation showed a shorter baseline ACT than uninterrupted warfarin. Edoxaban required a higher initial heparin dose to achieve a similar 15-minute ACT to warfarin. These results are useful for determining the initial heparin dose required to achieve variable target ACTs.

Host-produced ADAMTS4 Inhibits Early-Stage Tumor Growth.

Several research groups demonstrated that 'a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs (ADAMTS)'-family proteases play roles in cancer progression. However, the origins and contributions of these proteases are not known. Here, we demonstrate an association between host-produced ADAMTS4 and early-stage tumor growth. Murine Lewis lung carcinoma (LLC) tumors showed marked expressions of Adamts4 and Adamts5. We examined the contributions and distributions of host-derived Adamts4 and Adamts5 on tumor growth, using Adamts4LacZ/LacZ and Adamts5LacZ/LacZ knockout mice. Interestingly, the Adamts4LacZ/LacZ mice showed enhanced tumor growth compared to wild-type mice at 5-, 10- and 12-days post-inoculation, whereas the Adamts5LacZ/LacZ mice did not show significant differences in tumor growth. We next examined LacZ distribution in LLC tumor-bearing Adamts4LacZ/LacZ mice by ß-galactosidase (ß-gal) staining. We found that the ß-gal-positive signals were strictly localized at the interior areas of the tumor at 10 days post-inoculation. Multiple staining demonstrated that most of the ß-gal-positive cells were localized at the tumor vasculature in Adamts4LacZ/LacZ mice. Interestingly, ß-gal-positive signals were not co-localized with biglycan after 10 days post-inoculation, excluding the biglycan cleavage by host-derived ADAMTS4. Taken together, these findings illustrate that host-derived ADAMTS4 was expressed at the tumor vessels and was associated with early-stage tumor growth.

Circulating adipocyte fatty acid-binding protein is a predictor of cardiovascular events in patients with stable angina undergoing percutaneous coronary intervention.

BACKGROUND: Adipocyte fatty acid-binding protein (A-FABP) is expressed in both adipocytes and macrophages. Recent studies have shown that A-FABP is secreted by adipocytes and that the A-FABP concentration is associated with obesity, insulin resistance, and atherosclerosis. We have reported that the coronary atherosclerotic burden is associated with the serum A-FABP concentration. In the present study, we investigated whether the serum A-FABP concentration is associated with prognosis in patients with stable angina pectoris who have undergone percutaneous coronary intervention (PCI). METHODS: This was a prospective single-center trial. In total, 130 patients with stable angina pectoris undergoing their first PCI were enrolled from August 2008 to July 2010 at Kagawa Prefectural Central Hospital. The primary endpoints were cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, revascularization, and hospitalization for heart failure. RESULTS: During the follow-up (median, 50 months; interquartile range, 23-66 months), 49 cardiovascular events occurred. Kaplan-Meier analysis showed that the cumulative incidence of the primary endpoints in the high A-FABP group (median A-FABP concentration of ≥ 18.6 ng/ml) was greater than that in the low A-FABP group. Cox analysis showed that the A-FABP concentration was an independent predictor of cardiovascular events adjusted for age and the presence of multi-vessel disease (hazard ratio, 1.03; 95% confidence interval, 1.01-1.04; p = 0.01). CONCLUSION: The serum A-FABP concentration is associated with prognosis in patients with stable angina undergoing PCI, suggesting that the serum A-FABP concentration could be useful for risk assessment of secondary prevention. TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000029283 (registration date: September 25, 2017), retrospectively registered.

ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor.

Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy.

Eosinophil Cationic Protein Shows Survival Effect on H9c2 Cardiac Myoblast Cells with Enhanced Phosphorylation of ERK and Akt/GSK-3ß under Oxidative Stress.

Eosinophil cationic protein (ECP) is well known as a cationic protein contained in the basic granules of activated eosinophils. Recent studies have reported that ECP exhibits novel activities on various types of cells, including rat neonatal cardiomyocytes. Here we evaluated the effects of ECP on rat cardiac myoblast H9c2 cells. Our results showed that ECP enhanced the survival of the cells, in part by promoting the ERK and Akt/GSK-3ß signaling pathways. ECP attenuated the cytotoxic effects of H2O2 on H9c2 cells as well as the production of reactive oxygen species, the number of apoptotic cells and caspase 3/7 activity in the cells. In conclusion, ECP activated the ERK and Akt/GSK-3ß pathways, resulting in anti-oxidative effects on H9c2 cells that attenuated apoptosis.

Adaptive-servo ventilation combined with deep sedation is an effective strategy during pulmonary vein isolation.

AIMS: Pulmonary vein isolation (PVI) by catheter ablation for atrial fibrillation (AF) requires suppression of patient restlessness by sufficient sedation in addition to maintaining stable respiration. We applied adaptive-servo ventilation (ASV) and examined the effects of ASV combined with deep propofol sedation on PVI using a NavX. METHODS AND RESULTS: We analysed 75 paroxysmal AF (PAF) patients (62 ± 11 years; 53 men and 22 women) who underwent PVI for treatment of PAF using an ASV system combined with deep sedation (ASV group). Control patients included 75 consecutive PAF patients (62 ± 11 years; 51 men and 24 women) who underwent PVI just before introduction of the ASV system. Deep sedation was defined as a Ramsay sedation score of 6. The ASV group had a lower frequency of restless body movements compared with the control group during PVI (1.5 ± 0.7 vs. 7.8 ± 1.4 times, P < 0.01). The frequency of respiratory compensation and EnGuide alignment of catheter position by the NavX was lower in the ASV (4.2 ± 3.3 and 8.8 ± 7.1 times) than control group (7.1 ± 5.1 and 15.2 ± 10.0 times, P < 0.05 and <0.01, respectively). Consequently, significantly lower total electrical energy supply (48.7 ± 6.0 KJ) was required in the ASV than control group (64.5 ± 24.9 KJ, P < 0.01). Further, significantly shorter fluoroscopy and procedural times were observed in the ASV (28 ± 5 and 109 ± 25 min) than the control group (33 ± 6 and 141 ± 38 min, respectively, P < 0.01) and the AF recurrence rate was significantly lower in the ASV than the control group (12 vs. 25%, P < 0.01). CONCLUSION: ASV combined with deep sedation is an effective strategy during PVI using the NavX in patients with PAF.

Eosinophil cationic protein enhances stabilization of ß-catenin during cardiomyocyte differentiation in P19CL6 embryonal carcinoma cells.

Prior to gastrulation, the Wnt signaling pathway through stabilized ß-catenin enhances the differentiation of mouse ES cell into cardiomyocytes. We have recently shown that cardiomyocyte differentiation is enhanced by eosinophil cationic protein (ECP) through accelerated expression of marker genes of early cardiac differentiation. Furthermore, ECP enhanced the expression of Wnt3a in P19CL6 cells which were stimulated to differentiate into cardiomyocytes by DMSO. Following these findings, we evaluated in this study the potential of ECP to activate the Wnt/ß-catenin signaling pathway during cardiomyocyte differentiation. Analysis by real time qPCR revealed that ECP increased the expression of Frizzled genes such as Frizzled-1, -2, -4 and -10 in P19CL6 cells in the presence of DMSO. The increased expression of those Wnt receptors was found to inhibit the phosphorylation of ß-catenin resulting in the stabilization and translocation of ß-catenin into the nucleus of P19CL6 cells during the early stages of cardiomyocyte differentiation. When assessed for ß-catenin/TCF transcriptional activity with a TCF-luciferase (TOP/FOP) assay, ECP enhanced luciferase activity in P19CL6 cells during 48 h after transfection with TOP/FOP flash reporter in a stoichiometric manner. Collectively, this suggests that ECP can activate a canonical Wnt/ß-catenin signaling pathway by enhancing the stabilization of ß-catenin during cardiomyocyte differentiation.

Augmentation of ADAMTS9 gene expression by IL-1ß is reversed by NFκB and MAPK inhibitors, but not PI3 kinase inhibitors.

The pathways involved in the regulation of a disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS9) expression have not yet been elucidated. Therefore, the aim of this study was to investigate the involvement of nuclear factor-κB (NF-κB), mitogen activated protein kinases (MAPK) and Phosphatidylinositol 3-kinase (PI3 kinase) in ADAMTS9 gene regulation, with special focus on the involvement of NF-κB in IL-1ß-induced ADAMTS9 expression. The OUMS-27 chondrosarcoma cells were exposed to IL-1ß. They were pretreated with 20 µM PD98059 (specific inhibitor of p44/42 kinase), 10 µM SB203580 (specific inhibitor of p38 kinase), 20 µM SB600125 (MAPK inhibitor), and 1 µM Wortmannin and 10 µM LY294002 (specific inhibitors of PI3 kinase) for 30 min and subsequently incubated with IL-1ß. For the effects of NF-κB and IκB inhibitors, cells were pretreated with curcumin or BAY117085 for 30 min and subsequently incubated with IL-1ß. BAY117085 and different concentrations of curcumin were applied to the cells just after the first experiment to determine their concentration effect on ADAMTS9 gene expression. After total RNA was extracted, they were reversely transcribed with random primers and then real-time polymerase chain reaction (PCR) was performed on cDNA samples. There was a significant difference between control and stimulated cells in terms of ADAMTS9/ß-actin ratio. Wortmannin and LY294002 did not have any repressive effect on the OUMS-27 whereas SB203580 and SP600125 were found to decrease the expression of ADAMTS9 gene. BAY 117085 and curcumin, which are two NF-κB inhibitors, led to a decrease in the ratio of ADAMTS9/ß-actin. As a conclusion, the pathways MAPK and NF-κB were thought to be responsible pathways for the induction of ADAMTS9 gene.