The potential of the transformer-based survival analysis model, SurvTrace, for predicting recurrent cardiovascular events and stratifying high-risk patients with ischemic heart disease.

INTRODUCTION: Ischemic heart disease is a leading cause of death worldwide, and its importance is increasing with the aging population. The aim of this study was to evaluate the accuracy of SurvTrace, a survival analysis model using the Transformer-a state-of-the-art deep learning method-for predicting recurrent cardiovascular events and stratifying high-risk patients. The model's performance was compared to that of a conventional scoring system utilizing real-world data from cardiovascular patients. METHODS: This study consecutively enrolled patients who underwent percutaneous coronary intervention (PCI) at the Department of Cardiovascular Medicine, University of Tokyo Hospital, between 2005 and 2019. Each patient's initial PCI at our hospital was designated as the index procedure, and a composite of major adverse cardiovascular events (MACE) was monitored for up to two years post-index event. Data regarding patient background, clinical presentation, medical history, medications, and perioperative complications were collected to predict MACE. The performance of two models-a conventional scoring system proposed by Wilson et al. and the Transformer-based model SurvTrace-was evaluated using Harrell's c-index, Kaplan-Meier curves, and log-rank tests. RESULTS: A total of 3938 cases were included in the study, with 394 used as the test dataset and the remaining 3544 used for model training. SurvTrace exhibited a mean c-index of 0.72 (95% confidence intervals (CI): 0.69-0.76), which indicated higher prognostic accuracy compared with the conventional scoring system's 0.64 (95% CI: 0.64-0.64). Moreover, SurvTrace demonstrated superior risk stratification ability, effectively distinguishing between the high-risk group and other risk categories in terms of event occurrence. In contrast, the conventional system only showed a significant difference between the low-risk and high-risk groups. CONCLUSION: This study based on real-world cardiovascular patient data underscores the potential of the Transformer-based survival analysis model, SurvTrace, for predicting recurrent cardiovascular events and stratifying high-risk patients.

Multimodality Risk Assessment of Patients with Ischemic Heart Disease Using Deep Learning Models Applied to Electrocardiograms and Chest X-rays.

Comprehensive management approaches for patients with ischemic heart disease (IHD) are important aids for prognostication and treatment planning. While single-modality deep neural networks (DNNs) have shown promising performance for detecting cardiac abnormalities, the potential benefits of using DNNs for multimodality risk assessment in patients with IHD have not been reported. The purpose of this study was to investigate the effectiveness of multimodality risk assessment in patients with IHD using a DNN that utilizes 12-lead electrocardiograms (ECGs) and chest X-rays (CXRs), with the prediction of major adverse cardiovascular events (MACEs) being of particular concern.DNN models were applied to detection of left ventricular systolic dysfunction (LVSD) on ECGs and identification of cardiomegaly findings on CXRs. A total of 2107 patients who underwent elective percutaneous coronary intervention were categorized into 4 groups according to the models' outputs: Dual-modality high-risk (n = 105), ECG high-risk (n = 181), CXR high-risk (n = 392), and No-risk (n = 1,429).A total of 342 MACEs were observed. The incidence of a MACE was the highest in the Dual-modality high-risk group (P < 0.001). Multivariate Cox hazards analysis for predicting MACE revealed that the Dual-modality high-risk group had a significantly higher risk of MACE than the No-risk group (hazard ratio (HR): 2.370, P < 0.001), the ECG high-risk group (HR: 1.906, P = 0.010), and the CXR high-risk group (HR: 1.624, P = 0.018), after controlling for confounding factors.The results suggest the usefulness of multimodality risk assessment using DNN models applied to 12-lead ECG and CXR data from patients with IHD.

Age prediction from coronary angiography using a deep neural network: Age as a potential label to extract prognosis-related imaging features.

Coronary angiography (CAG) is still considered the reference standard for coronary artery assessment, especially in the treatment of acute coronary syndrome (ACS). Although aging causes changes in coronary arteries, the age-related imaging features on CAG and their prognostic relevance have not been fully characterized. We hypothesized that a deep neural network (DNN) model could be trained to estimate vascular age only using CAG and that this age prediction from CAG could show significant associations with clinical outcomes of ACS. A DNN was trained to estimate vascular age using ten separate frames from each of 5,923 CAG videos from 572 patients. It was then tested on 1,437 CAG videos from 144 patients. Subsequently, 298 ACS patients who underwent percutaneous coronary intervention (PCI) were analysed to assess whether predicted age by DNN was associated with clinical outcomes. Age predicted as a continuous variable showed mean absolute error of 4 years with R squared of 0.72 (r = 0.856). Among the ACS patients stratified by predicted age from CAG images before PCI, major adverse cardiovascular events (MACE) were more frequently observed in the older vascular age group than in the younger vascular age group (p = 0.017). Furthermore, after controlling for actual age, gender, peak creatine kinase, and history of heart failure, the older vascular age group independently suffered from more MACE (hazard ratio 2.14, 95% CI 1.07 to 4.29, p = 0.032). The vascular age estimated based on CAG imaging by DNN showed high predictive value. The age predicted from CAG images by DNN could have significant associations with clinical outcomes in patients with ACS.

Agreement between single plane and biplane derived angiographic fractional flow reserve in patients with intermediate coronary artery stenosis.

Fractional flow reserve (FFR) is often used to evaluate the physiological severity of intermediate coronary stenoses, but less-invasive assessment methods are desirable. We evaluated the feasibility of angiographic FFR (angioFFR) calculated from two projections acquired simultaneously by a biplane C-arm system and angioFFR calculated from two projections acquired independently by one plane of the same biplane C-arm system. AngioFFR was validated against FFR in terms of detection of hemodynamically relevant coronary artery stenoses. Twenty-two Patients who underwent angiography and FFR for coronary artery disease were included. We used a non-commercial prototype to calculate biplane angioFFR for 22 vessels (19 LAD, 1 LCx, 2 RCA) and single plane angioFFR for 17 of the same 22 vessels. FFR < 0.8 was measured in 8 vessels. The Pearson correlation coefficients with FFR were 0.55 for single plane angioFFR and 0.61 for biplane angioFFR and the diagnostic accuracies were 88% (95% CI 73-100%) for single plane angioFFR and 86% (95% CI 72-100%) for biplane angioFFR. Bland-Altman plots revealed that compared with FFR, the limits of agreement for single plane angioFFR were - 0.07 to 0.19 (mean difference 0.06, p = 0.002) and the limits of agreement for biplane FFR were - 0.09 to 0.15 (mean difference 0.03, p = 0.03). In conclusion, angioFFR calculated from single or biplane acquisitions by a biplane C-arm is feasible and may evolve to a tool for less invasive imaging-based assessment of myocardial ischemia.

Potential value of saline-induced Pd/Pa ratio in patients with coronary artery stenosis.

Background: Fractional flow reserve (FFR) is the current gold standard for identifying myocardial ischemia in individuals with coronary artery stenosis. However, FFR is not penetrated as much worldwide due to time consumption, costs associated with adenosine, FFR-related discomfort, and complications. Resting physiological indexes may be widely accepted alternatives to FFR, while the discrepancies with FFR were found in up to 20% of lesions. The saline-induced Pd/Pa ratio (SPR) is a new simplified option for evaluating coronary stenosis. However, the clinical implication of SPR remains unclear. Objectives: In the present study, we aimed to compare the accuracies of SPR and resting full-cycle ratio (RFR) and to investigate the incremental value of SPR in clinical practice. Methods: In this multicenter prospective study, 112 coronary lesions (105 patients) were evaluated by SPR, RFR, and FFR. Results: The overall median age was 71 years, and 84.8% were men. SPR was correlated more strongly with FFR than with RFR (r = 0.874 vs. 0.713, respectively; p < 0.001). Using FFR < 0.80 as the reference standard variable, the area under the receiver-operating characteristic (ROC) curve for SPR was superior to that of RFR (0.932 vs. 0.840, respectively; p = 0.009). Conclusion: Saline-induced Pd/Pa ratio predicted FFR more accurately than RFR. SPR could be an alternative method for evaluating coronary artery stenosis and further investigation including elucidation of the mechanism of SPR is needed (225 words).

Repeated Resection for Recurrent Metastatic Solid Pseudopapillary Neoplasm of the Pancreas.

BACKGROUND Solid pseudopapillary neoplasm (SPN) accounts for 1.0% to 2.0% of all pancreatic neoplasms. SPN generally has good prognosis after surgery; however, 10% to 15% of patients have local recurrence or distant metastasis. There have been a few reports of successful surgical resection of isolated recurrent tumors after radical resection and sporadic reports of multiple metastasectomies. Herein, we present a case of recurrent SPN treated by repeated surgeries. CASE REPORT A 49-year-old woman was referred to our hospital with jaundice and right upper abdominal pain. Computed tomography (CT) scanning revealed a 73×43-mm heterogeneous mass in the pancreatic head. We performed a pancreatoduodenectomy and diagnosed SPN. The patient was discharged without any complications and was followed up by CT once every 6 to 12 months. Six years later, a 15×15-mm tumor was detected in Couinaud segment VI of the liver. A liver biopsy showed a pathological match to the pancreatic tumor. We performed a partial hepatectomy, and the pathology report confirmed metastatic SPN. At 8 and 10 years after the initial surgery, the patient underwent further partial hepatectomies for confirmed solitary liver metastases of SPN. The Ki-67 index increased for each metastasis identified (initial tumor, 1.88%; 6 years, 7.38%; 8 years, 5.53%; 10 years, 11.22%). No further masses were detected, and the patient survived more than 10 years following surgery. CONCLUSIONS Despite histological transformation to high-grade malignant disease, repeated aggressive surgical resection led to long-term survival in our patient with SPN.

Automatic detection of vessel structure by deep learning using intravascular ultrasound images of the coronary arteries.

Intravascular ultrasound (IVUS) is a diagnostic modality used during percutaneous coronary intervention. However, specialist skills are required to interpret IVUS images. To address this issue, we developed a new artificial intelligence (AI) program that categorizes vessel components, including calcification and stents, seen in IVUS images of complex lesions. When developing our AI using U-Net, IVUS images were taken from patients with angina pectoris and were manually segmented into the following categories: lumen area, medial plus plaque area, calcification, and stent. To evaluate our AI's performance, we calculated the classification accuracy of vessel components in IVUS images of vessels with clinically significantly narrowed lumina (< 4 mm2) and those with severe calcification. Additionally, we assessed the correlation between lumen areas in manually-labeled ground truth images and those in AI-predicted images, the mean intersection over union (IoU) of a test set, and the recall score for detecting stent struts in each IVUS image in which a stent was present in the test set. Among 3738 labeled images, 323 were randomly selected for use as a test set. The remaining 3415 images were used for training. The classification accuracies for vessels with significantly narrowed lumina and those with severe calcification were 0.97 and 0.98, respectively. Additionally, there was a significant correlation in the lumen area between the ground truth images and the predicted images (ρ = 0.97, R2 = 0.97, p < 0.001). However, the mean IoU of the test set was 0.66 and the recall score for detecting stent struts was 0.64. Our AI program accurately classified vessels requiring treatment and vessel components, except for stents in IVUS images of complex lesions. AI may be a powerful tool for assisting in the interpretation of IVUS imaging and could promote the popularization of IVUS-guided percutaneous coronary intervention in a clinical setting.

Factors associated with left ventricular reverse remodelling after percutaneous coronary intervention in patients with left ventricular systolic dysfunction.

Percutaneous coronary intervention (PCI) is sometimes considered as an alternative therapeutic strategy to surgical revascularization in patients with coronary artery disease (CAD) and reduced left ventricular ejection fraction (LVEF). However, the types or conditions of patients that receive the clinical benefit of left ventricular reverse remodelling (LVRR) remain unknown. The purpose of this study was to investigate the determinants of LVRR following PCI in CAD patients with reduced LVEF. From 4394 consecutive patients who underwent PCI, a total of 286 patients with reduced LV systolic function (LVEF < 50% at initial left ventriculography) were included in the analysis. LVRR was defined as LV end-systolic volume reduction ≥ 15% and improvement of LVEF ≥ 10% at 6 months follow-up left ventriculography. Patients were divided into LVRR (n = 63) and non-LVRR (n = 223) groups. Multivariate logistic regression analysis revealed that unprotected left main coronary artery (LMCA) intervention was significantly associated with LVRR (P = 0.007, odds ratios [OR] 4.70, 95% confidence interval [CI] 1.54-14.38), while prior PCI (P = 0.001, OR 0.35, 95% CI 0.19-0.66), presence of in-stent restenosis (P = 0.016, OR 0.32, 95% CI 0.12-0.81), and presence of de-novo stenosis (P = 0.038, OR 0.36, 95% CI 0.14-0.95) were negatively associated with LVRR. These data suggest the potential prognostic benefit of unprotected LMCA intervention for LVRR and importance of angiographic follow-up in patients with CAD and LV systolic dysfunction.

Allyl isothiocyanate (AITC) activates nonselective cation currents in human cardiac fibroblasts: possible involvement of TRPA1.

The effects of allyl isothiocyanate (AITC), transient receptor potential ankyrin 1 (TRPA1) agonist, on cultured human cardiac fibroblasts were examined by measuring intracellular Ca2+ concentration [Ca2+]i and whole-cell voltage clamp techniques. AITC (200 µM) increased Ca2+ entry in the presence of [Ca2+]i. Ruthenium red (RR) (30 µM), and La3+ (0.5 mM), a general cation channel blocker, inhibited AITC-induced Ca2+ entry. Under the patch pipette filled with Cs+- and EGTA-solution, AITC induced the current of a reversal potential (Er) of approximately +0 mV. When extracellular Na+ ion was changed by NMDG+, the inward current activated by AITC was markedly reduced. La3+ and RR inhibited the AITC-induced current. The conventional RT-PCR analysis, Western blot, and immunocytochemical studies showed TRPA1 mRNA and protein expression. The present study shows the first evidence for functional Ca2+-permeable nonselective cation currents induced by AITC, possibly via TRPA1 in human cardiac fibroblast.

Short-Term and Long-Term Efficacy of Drug-Coated Balloon for In-Stent Restenosis in Hemodialysis Patients with Coronary Artery Disease.

The efficacy of drug-coated balloons (DCB) for in-stent restenosis (ISR) in hemodialysis (HD) patients remains unclear.We retrospectively evaluated 153 consecutive patients who underwent DCB for ISR with follow-ups for up to 3 years after the procedure between February 2014 and June 2017. Patients were divided into an HD group (n = 39) and a non-HD group (n = 114). The primary endpoint was target lesion revascularization (TLR). The secondary endpoints were all revascularizations and major adverse cardiac events (MACE) defined as cardiac death, myocardial infarction and cerebral infarction. Kaplan-Meier curves of survival free from TLR were compared between the two groups. We also performed propensity score matching and then compared the two matched groups (n = 27 in each group). The acute procedure success rate was similar for the two groups (100% versus 99.1%, P = 0.56). The incidence of TLR was higher in the HD group than in the non-HD group (41.0% versus 9.6%, P < 0.0001). The rate of revascularizations and MACE combined was significantly higher in the HD group than in the non-HD group (64.1% versus 17.5%, P < 0.0001). Kaplan-Meier analyses showed that survival free from TLR was significantly lower in the HD group than in the non-HD group both before and after propensity score matching (P < 0.0001 and P = 0.005, respectively; log-rank test).Contrary to the similar acute procedure success, recurrent ISR and MACE occurred more frequently in HD patients than in non-HD patients after DCB, which indicates poorer long-term efficacy of DCB in HD patients.

Heat induces interleukin-6 in skeletal muscle cells via TRPV1/PKC/CREB pathways.

Interleukin-6 (IL-6) is released from skeletal muscle cells and induced by exercise, heat, catecholamine, glucose, lipopolysaccharide, reactive oxygen species, and inflammation. However, the mechanism that induces release of IL-6 from skeletal muscle cells remains unknown. Thermosensitive transient receptor potential (TRP) proteins such as TRPV1-4 play vital roles in cellular functions. In this study we hypothesized that TRPV1 senses heat, transmits a signal into the nucleus, and produces IL-6. The purpose of the present study is to investigate the underlying mechanisms whereby skeletal muscle cells sense and respond to heat. When mouse myoblast cells were exposed to 37-42°C for 2 h, mRNA expression of IL-6 increased in a temperature-dependent manner. Heat also increased IL-6 secretion in myoblast cells. A fura 2 fluorescence dual-wavelength excitation method showed that heat increased intracellular calcium flux in a temperature-dependent manner. Intracellular calcium flux and IL-6 mRNA expression were increased by the TRPV1 agonists capsaicin and N-arachidonoyldopamine and decreased by the TRPV1 antagonists AMG9810 and SB366791 and siRNA-mediated knockdown of TRPV1. TRPV2, 3, and 4 agonists did not change intracellular calcium flux. Western blotting with inhibitors demonstrated that heat increased phosphorylation levels of TRPV1, followed by PKC and cAMP response element-binding protein (CREB). PKC inhibitors, Gö6983 and staurosporine, CREB inhibitors, curcumin and naphthol AS-E, and knockdown of CREB suppressed the heat-induced increases in IL-6. These results indicate that heat increases IL-6 in skeletal muscle cells through the TRPV1, PKC, and CREB signal transduction pathway.NEW & NOTEWORTHY Heat increases the release of interleukin-6 (IL-6) from skeletal muscle cells. IL-6 has been shown to serve immune responses and metabolic functions in muscle. It can be anti-inflammatory as well as proinflammatory. However, the mechanism that induces release of IL-6 from skeletal muscle cells remains unknown. Here we show that heat increases IL-6 in skeletal muscle cells through the transient receptor potential vannilloid 1, PKC, and cAMP response element-binding protein signal transduction pathway.

Relationship between chronotropic incompetence and ß-blockers based on changes in chronotropic response during cardiopulmonary exercise testing.

BACKGROUND: Chronotropic incompetence (CI), an attenuated heart rate (HR) response to exercise, is common in patients with cardiovascular disease. The aim of this study was to assess changes in the chronotropic response (CR) during cardiopulmonary exercise testing (CPET) in patients undergoing cardiac rehabilitation and investigate the effects of ß-blockers. METHODS: Patients undergoing cardiac rehabilitation performed CPET. Failure to achieve 80% of the age-predicted maximal HR (APMHR) defined CI. Values of the metabolic chronotropic relationship (MCR) were calculated from the ratio of the HR reserve to metabolic reserve at 4 stages, warm-up (MCR-Wu), anaerobic threshold (MCR-AT), respiratory compensation (MCR-Rc), and peak point (MCR-Pk), using the Wilkoff model. In patients who showed an increase in MCR at ≥ 3 of the 4 exercise stages, CR was considered to have improved. RESULTS: Patients with high BNP levels (≥ 80 pg/ml) had a lower MCR at all stages compared with those with low BNP levels (< 80 pg/ml). Of the 80 patients, 47 showed an increase in both peak VO2 and AT, and of these 31 (66.0%) were taking ß-blockers. Improvement in CR was observed in 30 of 47 patients with CI, and 70% of these were taking ß-blockers. In patients not taking ß-blockers, MCR-AT was lower than MCR-Rc, whereas in those taking ß-blockers MCR-AT was higher than MCR-Rc. CONCLUSIONS: An attenuated HR response may occur during the early stages of exercise. The HR response according to the presence or absence of ß-blockers is clearly identifiable by comparing MCR-AT and MCR-Rc using the Wilkoff model.

Effects of methylglyoxal on human cardiac fibroblast: roles of transient receptor potential ankyrin 1 (TRPA1) channels.

Cardiac fibroblasts contribute to the pathogenesis of cardiac remodeling. Methylglyoxal (MG) is an endogenous carbonyl compound produced under hyperglycemic conditions, which may play a role in the development of pathophysiological conditions including diabetic cardiomyopathy. However, the mechanism by which this occurs and the molecular targets of MG are unclear. We investigated the effects of MG on Ca(2+) signals, its underlying mechanism, and cell cycle progression/cell differentiation in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, Western blot, immunocytochemical analysis, and intracellular Ca(2+) concentration [Ca(2+)]i measurement were applied. Cell cycle progression was assessed using the fluorescence activated cell sorting. MG induced Ca(2+) entry concentration dependently. Ruthenium red (RR), a general cation channel blocker, and HC030031, a selective transient receptor potential ankyrin 1 (TRPA1) antagonist, inhibited MG-induced Ca(2+) entry. Treatment with aminoguanidine, a MG scavenger, also inhibited it. Allyl isothiocyanate, a selective TRPA1 agonist, increased Ca(2+) entry. The use of small interfering RNA to knock down TRPA1 reduced the MG-induced Ca(2+) entry as well as TRPA1 mRNA expression. The quantitative real-time RT-PCR analysis showed the prominent existence of TRPA1 mRNA. Expression of TRPA1 protein was confirmed by Western blotting and immunocytochemical analyses. MG promoted cell cycle progression from G0/G1 to S/G2/M, which was suppressed by HC030031 or RR. MG also enhanced α-smooth muscle actin expression. The present results suggest that methylglyoxal activates TRPA1 and promotes cell cycle progression and differentiation in human cardiac fibroblasts. MG might participate the development of pathophysiological conditions including diabetic cardiomyopathy via activation of TRPA1.

Roles of transient receptor potential canonical (TRPC) channels and reverse-mode Na+/Ca2+ exchanger on cell proliferation in human cardiac fibroblasts: effects of transforming growth factor ß1.

Expression of transient receptor potential canonical channels (TRPC) and the effects of transforming growth factor-ß1 (TGF-ß1) on Ca2+ signals and fibroblast proliferation were investigated in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, western blot, immunocytochemical analysis, and intracellular Ca2+ concentration [Ca2+]i measurement were applied. Cell proliferation and cell cycle progression were assessed using MTT assays and fluorescence activated cell sorting. Human cardiac fibroblasts have the expression of TRPC1,3,4,6 mRNA and proteins. 1-oleoyl-2-acetyl-sn-glycerol (OAG) and thapsigargin induced extracellular Ca(2+)-mediated [Ca2+]i rise. siRNA for knock down of TRPC6 reduced OAG-induced Ca2+ entry. Hyperforin as well as angiotensin II (Ang II) induced Ca2+ entry. KB-R7943, a reverse-mode Na+/Ca2+ exchanger (NCX) inhibitor, and/or replacement of Na+ with NMDG+ inhibited thapsigargin-, OAG- and Ang II-induced Ca2+ entry. Treatment with TGF-ß1 increased thapsigargin-, OAG- and Ang II-induced Ca2+ entry with an enhancement of TRPC1,6 protein expression, suppressed by KB-R7943. TGF-ß1 and AngII promoted cell cycle progression from G0/G1 to S/G2/M and cell proliferation. A decrease of the extracellular Ca2+ and KB-R7943 suppressed it. Human cardiac fibroblasts contain several TRPC-mediated Ca2+ influx pathways, which activate the reverse-mode NCX. TGF-ß1 enhances the Ca2+ influx pathways requiring Ca2+ signals for its effect on fibroblast proliferation.

Use of potassium channel tetramerization domain-containing 12 as a biomarker for diagnosis and prognosis of gastrointestinal stromal tumor.

Previously, we showed that the expression of potassium channel tetramerization domain-containing 12 (KCTD12), which was discovered by a proteomics approach, is associated with high-risk behavior of gastrointestinal stromal tumors (GISTs). Here, we examined the distribution and expression of this protein by immunostaining with a commercially available polyclonal KCTD12 antibody in GISTs (n = 64) and other types of malignancy (n = 168) to clarify its diagnostic and clinical significance. Diffuse KCTD12 immunoreactivity was found in most GISTs (52 cases; 81%). KCTD12 expression was observed primarily in vascular endothelial cells, Purkinje cells of the cerebellum, and some neurons scattered throughout the cerebral cortex. KCTD12 was absent from not only the interstitial cells of Cajal but also interstitial cells of Cajal hyperplasia that was encountered incidentally in colon diverticulitis. KCTD12 immunostaining was also seen in malignant peripheral nerve sheath tumors (2/10 cases; 20%), synovial sarcomas (2/10; 20%), solitary fibrous tumor (1/8; 13%), angiosarcoma (1/7; 14%), and colon adenocarcinoma (1/24; 4%). In survival analyses, the 5-year recurrence-free survival rate of patients without KCTD12 expression was only 16.7% compared with 95.6% in those with KCTD12 expression (P < .0001). Ki-67 and KCTD12 were significant predictors of recurrence-free survival, and KCTD12 expression provided additional information about recurrence-free survival after accounting for Ki-67 status. Overall, KCTD12 expression was specific for GISTs from neoplastic and nonneoplastic adult tissues other than brain and served as a predictor of GIST recurrence. These findings suggest that KCTD12 is a useful and reliable biomarker for both the diagnosis and prognosis of GIST.

Amiodarone inhibits tissue factor expression in monocytic THP-1 cells.

There is a possibility thrombus formation is closely involved in sudden cardiac death. Amiodarone, a potassium channel inhibitor, is known to reduce mortality in patients with coronary artery disease or low ejection fraction, having antithrombotic actions. Using human monocytic THP-1 cells, we investigated the effects of amiodarone on tissue factor mRNA and protein expression. The involvement of the two main potassium channels existing in THP-1 cells was also investigated. Amiodarone (10µM) significantly and almost completely inhibited the increase of tissue factor mRNA and protein expression induced by tumor necrosis factor-α (100ng/ml). The inhibitory effects of amiodarone on tissue factor mRNA expression showed dose-dependency. Margatoxin (1nM), a selective blocker of voltage-dependent potassium channel Kv1.3, also inhibited tissue factor protein expression, but didn't significantly inhibit mRNA expression. Ba(2+), a blocker of inwardly rectifying potassium channel Kir2.1, partly inhibited the increase of tissue factor mRNA and protein expression. This is the first study that shows amiodarone inhibits tissue factor expression in monocytic cells, by inhibiting mRNA transcription. The result may correlate with the facts amiodarone has antithrombotic actions in patients under extraordinary conditions where thrombus formation is enhanced. The inhibitory effects of amiodarone on tissue factor expression are drastic, different from those of margatoxin and Ba(2+). The result suggests amiodarone has an underlying mechanism to intensely inhibit tissue factor expression other than blocking Kv1.3 and Kir2.1.

Effects of serum amyloid a and lysophosphatidylcholine on intracellular calcium concentration in human coronary artery smooth muscle cells.

Serum amyloid A (SAA), an acute-phase protein, and lysophosphatidylcholine (LPC), an oxidized LDL component, contribute to the physiological processes of atherosclerosis and cardiovascular disease. However, the effects of SAA/LPC on human coronary artery smooth muscle cells (hCASMCs) have not been fully investigated. Therefore, we examined the effects of SAA/LPC on Ca(2+)/Mg(2+) mobilization and its underlying mechanisms in hCASMCs. Intracellular Ca(2+)/Mg(2+) concentration ([Ca(2+)](i) / [Mg(2+)](i)) was measured with fura-2 AM/mag-fura-2 AM. Conventional RT-PCR analysis was also performed. Both SAA and LPC increased [Ca(2+)](i) by Ca(2+) entry. The SAA-induced Ca(2+) entry was inhibited by Gd(3+), SKF96365, and 2-aminoethoxydiphenyl borate (2-APB), a nonselective transient receptor potential (TRP) channel blocker, but not nifedipine. The LPC-induced Ca(2+) entry was blocked by Gd(3+), but not nifedipine, SKF96365 and 2-APB. U-73122 and PTX prevented the activation of SAA-, but not LPC-induced Ca(2+) influx. LPC, but not SAA, increased [Mg(2+)](i) as well as [Ca(2+)](i). The RT-PCR analysis revealed the expression of TRPC1/4, TRPV1/2/4, and TRPM7/8 mRNA. These results suggest that SAA/LPC activate Ca(2+) influx in hCASMCs; SAA activates it via PTX-sensitive G-protein, PLC and TRPC pathways, while LPC activates it independently of these pathways, where TRPM7 may be partly involved. Thus, TRP protein appears to be a target molecule of Ca(2+) signaling in hCASMCs elicited by SAA/LPC, which may play roles in coronary muscle dysfunction under pathophysiological and inflammatory conditions such as atherosclerosis.

NFBD1/MDC1 stabilizes oncogenic MDM2 to contribute to cell fate determination in response to DNA damage.

In response to DNA damage, NFBD1/MDC1 induces the accumulation of DNA repair machinery such as MRN complex at the sites of damaged DNA to form nuclear foci. In this study, we found that NFBD1 directly interacts with MDM2 and increases its stability. During adriamycin (ADR)-mediated apoptosis, expression levels of NFBD1 reduced in association with the down-regulation of MDM2. Enforced expression of NFBD1 resulted in a significant stabilization of MDM2. Consistent with these observations, siRNA-mediated knockdown of the endogenous NFBD1 decreased the amounts of the endogenous MDM2. Immunoprecipitation and in vitro pull-down assays demonstrated that NFBD1 interacts with MDM2 through its COOH-terminal BRCT domains. In accordance with our recent results, enforced expression of NFBD1 rendered cells resistant to DNA damage. Similar results were also obtained in cells expressing exogenous MDM2. Taken together, our present findings suggest that NFBD1-mediated stabilization contributes to cell survival in response to DNA damage.

NFBD1/MDC1 associates with p53 and regulates its function at the crossroad between cell survival and death in response to DNA damage.

NFBD1/MDC1, which belongs to the BRCT superfamily, has an anti-apoptotic activity and contributes to the early cellular responses to DNA damage. Here we found that NFBD1 protects cells from apoptotic cell death by inhibiting phosphorylation of p53 at Ser-15 under steady state as well as early phase of DNA damage, thereby blocking its transcriptional and pro-apoptotic activities. During late phase of DNA damage, a remarkable reduction of NFBD1 was observed in dying but not in surviving A549 cells bearing wild-type p53. Small interference RNA-mediated knockdown of the endogenous NFBD1 resulted in an increase in sensitivity to adriamycin in A549 cells but not in p53-deficient H1299 cells. Immunoprecipitation and luciferase reporter analyses demonstrated that NFBD1 binds to the NH(2)-terminal region of p53 and strongly inhibits its transcriptional activity. Additionally, BRCT domains, which can interact with p53, reduced the adriamycin-induced phosphorylation levels of p53 at Ser-15 and also suppressed the transcriptional activity of p53. Thus, our present findings strongly suggest that NFBD1 plays an important role in the decision of cell survival and death after DNA damage through the regulation of p53.

Oxidative stress induces p53-dependent apoptosis in hepatoblastoma cell through its nuclear translocation.

Hepatoblastoma (HBL) is the most common malignant liver tumor in children. Since tumor suppressor p53 is rarely mutated in HBL, it remains unknown whether p53 could contribute to the hepatocarcinogenesis. In the present study, we have found for the first time that, like neuroblastoma (NBL), wild-type p53 was abnormally accumulated in the cytoplasm of the human HBL-derived Huh6 cells. In accordance with this notion, immunohistochemical analysis demonstrated that p53 is largely expressed in cytoplasm of human primary HBLs. In response to the oxidative stress, Huh6 cells underwent apoptotic cell death in association with the nuclear translocation of p53 and the transactivation of its target gene implicated in apoptotic cell death. siRNA-mediated knockdown of the endogenous p53 conferred the resistance of Huh6 cells to oxidative stress. Intriguingly, histone deacetylase inhibitor (nicotinamide) treatment strongly inhibited the oxidative stress-induced nuclear translocation of p53 as well as the p53-dependent apoptosis in Huh6 cells. In contrast to the previous observations, the cytoplasmic anchor protein for p53 termed Parc had undetectable effect on the cytoplasmic retention of p53. Collectively, our present results suggest that the abnormal cytoplasmic localization of p53 might contribute at least in part to the development of HBL.