Modifiable lifestyle factors and lifetime risk of atrial fibrillation: longitudinal data from the Korea NHIS-HealS and UK Biobank cohorts.

BACKGROUND: The reason for higher incidence of atrial fibrillation (AF) in Europe compared with East Asia is unclear. We aimed to investigate the association between modifiable lifestyle factors and lifetime risk of AF in Europe and East Asia, along with race/ethnic similarities and disparities. METHODS: 1:1 propensity score matched pairs of 242,763 East Asians and 242,763 White Europeans without AF were analyzed. Modifiable lifestyle factors considered were blood pressure, body mass index, cigarette smoking, diabetes, alcohol consumption, and physical activity, categorized as non-adverse or adverse levels. Lifetime risk of AF was estimated from the index age of 45 years to the attained age of 85 years, accounting for the competing risk of death. RESULTS: The overall lifetime risk of AF was higher in White Europeans than East Asians (20.9% vs 15.4%, p < 0.001). The lifetime risk of AF was similar between the two races in individuals with non-adverse lifestyle factor profiles (13.4% vs 12.9%, p = 0.575), whereas it was higher in White Europeans with adverse lifestyle factor profiles (22.1% vs 15.8%, p < 0.001). The difference in the lifetime risk of AF between the two races increased as the burden of adverse lifestyle factors worsened (1 adverse lifestyle factor; 4.3% to ≥ 3 adverse lifestyle factors; 11.2%). Compared with East Asians, the relative risk of AF in White Europeans was 23% and 62% higher for one (hazard ratio [HR] 1.23, 95% confidence interval [CI] 1.16-1.29) and ≥ 3 adverse lifestyle factors (HR 1.62, 95% CI 1.51-1.75), respectively. CONCLUSIONS: The overall higher lifetime risk of AF in White Europeans compared with East Asians might be attributable to adverse lifestyle factors. Adherence to healthy lifestyle factors was associated with the lifetime risk of AF of about 1 in 8 regardless of race/ethnicity.

Pulmonary fibrosis model using micro-CT analyzable human PSC-derived alveolar organoids containing alveolar macrophage-like cells.

Human lung organoids (hLOs) are useful for disease modelling and drug screening. However, a lack of immune cells in hLOs limits the recapitulation of in vivo cellular physiology. Here, we generated hLOs containing alveolar macrophage (AMφ)-like cells derived from pluripotent stem cells (PSC). To bridge hLOs with advanced human lung high-resolution X-ray computed tomography (CT), we acquired quantitative micro-CT images. Three hLO types were observed during differentiation. Among them, alveolar hLOs highly expressed not only lung epithelial cell markers but also AMφ-specific markers. Furthermore, CD68+ AMφ-like cells were spatially organized on the luminal epithelial surface of alveolar hLOs. Bleomycin-treated alveolar hLOs showed upregulated expression of fibrosis-related markers and extracellular matrix deposits in the alveolar sacs. Alveolar hLOs also showed structural alterations such as excessive tissue fraction under bleomycin treatment. Therefore, we suggest that micro-CT analyzable PSC-derived alveolar hLOs are a promising in vitro model to predict lung toxicity manifestations, including fibrosis.

Polyhexamethylene guanidine phosphate increases stress granule formation in human 3D lung organoids under respiratory syncytial virus infection.

Polyhexamethylene guanidine phosphate (PHMG-p), a humidifier disinfectant, is known to cause lung toxicity, including inflammation and pulmonary fibrosis. In this study, we aimed to investigate the effect of PHMG-p on human lung tissue models (2D epithelial cells and 3D organoids) under conditions of oxidative stress and viral infection. The effect of PHMG-p was studied by evaluating the formation of stress granules (SGs), which play a pivotal role in cellular adaptation to various stress conditions. Under oxidative stress and respiratory syncytial virus (RSV) infection, exposure to PHMG-p remarkably increased eIF2α phosphorylation, which is essential for SG-related signalling, and significantly increased SG formation. Furthermore, PHMG-p induced fibrotic gene expression and caused cell death due to severe DNA damage, which was further increased under oxidative stress and RSV infection, indicating that PHMG-p induces severe lung toxicity under stress conditions. Taken together, toxicity evaluation under various stressful conditions is necessary to accurately predict potential lung toxicity of chemicals affecting the respiratory tract.

Live-cell screening platform using human-induced pluripotent stem cells expressing fluorescence-tagged cytochrome P450 1A1.

Human-induced pluripotent stem cells (hiPSCs) are invaluable sources for drug screening and toxicity tests because of their differentiation potential and proliferative capacity. Recently, the CRISPR-Cas9-mediated homologous recombination system has enabled reporter knock-ins at desired loci in hiPSCs, and here, we generated a hiPSC reporter line expressing mCherry-tagged cytochrome P450 1A1 (CYP1A1), which can be utilized to screen for the modulators of aryl hydrocarbon receptor (AHR) in live cells. CYP1A1-mCherry hiPSCs exhibited typical characteristics of pluripotent stem cells such as marker expression, differentiation potential, and normal karyotype. After differentiation into hepatocyte-like cells (HLCs), CYP1A1-mCherry fusion protein was expressed and localized at the endoplasmic reticulum, and induced by AHR agonists. We obtained 23 hits modulating CYP1A1 expression from high-content screening with 241 hepatotoxicity chemicals and nuclear receptor ligands, and identified three upregulating chemicals and two downregulating compounds. Responses of hiPSC-HLCs against an AHR agonist were more similar to human primary hepatocytes than of HepG2 hepatocellular carcinoma cells. This platform has the advantages of live-cell screening without sacrificing cells (unlike previously available CYP1A1 reporter cell lines), as well as an indefinite supply of cells, and can be utilized in a wide range of screening related to AHR- and CYP1A1-associated diseases in desired cell types.

Prediction of hepatotoxicity for drugs using human pluripotent stem cell-derived hepatocytes.

Drug-induced liver toxicity is a main reason for withdrawals of new drugs in late clinical phases and post-launch of the drugs. Thus, hepatotoxicity screening of drug candidates in pre-clinical stage is important for reducing drug attrition rates during the clinical development process. Here, we show commercially available hepatocytes that could be used for early toxicity evaluation of drug candidates. From our hepatic differentiation technology, we obtained highly pure (≥98%) hepatocytes from human embryonic stem cells (hESCs) having mature phenotypes and similar gene expression profiles with those of primary human tissues. Furthermore, we optimized 96-well culture condition of hESC-derived hepatocytes suitable for toxicity tests in vitro. To this end, we demonstrated the efficacy of our optimized hepatocyte model for predicting hepatotoxicity against the Chinese herbal medicines and showed that toxicity patterns from our hepatocyte model was similar to those of human primary cultured hepatocytes. We conclude that toxicity test using our hepatocyte model could be a good alternative cell source for pre-clinical study to predict potential hepatotoxicity in drug discovery industries.

Hepatic esterase activity is increased in hepatocyte-like cells derived from human embryonic stem cells using a 3D culture system.

OBJECTIVES: The aim of the study is to generate a spherical three-dimensional (3D) aggregate of hepatocyte-like cells (HLCs) differentiated from human embryonic stem cells and to investigate the effect of the 3D environment on hepatic maturation and drug metabolism. RESULTS: Quantitative real-time PCR analysis indicated that gene expression of mature hepatocyte markers, drug-metabolizing enzymes, and hepatic transporters was significantly higher in HLCs cultured in the 3D system than in those cultured in a two-dimensional system (p < 0.001). Moreover, hepatocyte-specific functions, including albumin secretion and bile canaliculi formation, were increased in HLCs cultured in the 3D system. In particular, 3D spheroidal culture increased expression of CES1 and BCHE, which encode hepatic esterases (p < 0.001). The enhanced activities of these hepatic esterases were confirmed by the cholinesterase activity assay and the increased susceptibility of HLCs to oseltamivir, which is metabolized by CES1. CONCLUSIONS: 3D spheroidal culture enhances the maturation and drug metabolism of stem cell-derived HLCs, and this may help to optimize hepatic differentiation protocols for hepatotoxicity testing.

Two zinc-aminoclays' in-vitro cytotoxicity assessment in HeLa cells and in-vivo embryotoxicity assay in zebrafish.

Two zinc-aminoclays [ZnACs] with functionalized primary amines [(-CH2)3NH2] were prepared by a simple sol-gel reaction using cationic metal precursors of ZnCl2 and Zn(NO3)2 with 3-aminopropyl triethoxysilane [APTES] under ambient conditions. Due to the facile interaction of heavy metals with primary amine sites and Zn-related intrinsic antimicrobial activity, toxicity assays of ZnACs nanoparticles (NPs) prior to their environmental and human-health applications are essential. However, such reports remain rare. Thus, in the present study, a cell viability assay of in-vitro HeLa cells comparing ZnCl2, Zn(NO3)2 salts, and ZnO (~50nm average diameter) NPs was performed. Interestingly, compared with the ZnCl2, and Zn(NO3)2 salts, and ZnO NPs (18.73/18.12/51.49µg/mL and 18.12/15.19/46.10µg/mL of IC50 values for 24 and 48h), the two ZnACs NPs exhibited the highest toxicity (IC50 values of 21.18/18.36µg/mL and 18.37/17.09µg/mL for 24 and 48h, respectively), whose concentrations were calculated on Zn elemental composition. This might be due to the enhanced bioavailability and uptake into cells of ZnAC NPs themselves and their positively charged hydrophilicity by reactive oxygen species (ROS) generation, particularly as ZnACs exist in cationic NP's form, not in released Zn2+ ionic form (i.e., dissolved nanometal). However, in an in-vivo embryotoxicity assay in zebrafish, ZnACs and ZnO NPs showed toxic effects at 50-100µg/mL (corresponding to 37.88-75.76 of Zn wt% µg/mL). The hatching rate (%) of zebrafish was lowest for the ZnO NPs, particularly where ZnAC-[(NO3)2] is slightly more toxic than ZnAC-[Cl2]. These results are all very pertinent to the issue of ZnACs' potential applications in the environmental and biomedical fields.

Nonylphenol-induced apoptotic cell death in mouse TM4 Sertoli cells via the generation of reactive oxygen species and activation of the ERK signaling pathway.

Nonylphenol (NP), a representative endocrine disruptor, interferes with reproductive function in aquatic organisms and animals. Although many previous studies have focused on apoptotic cell death by NP, the fundamental mechanism of NP on apoptosis remains poorly understood. Here, we investigated the molecular mechanism on NP-induced apoptotic cell death in mouse TM4 Sertoli cells. To evaluate NP treatment on cell viability, formazan and lactate dehydrogenase (LDH) assays were performed. Results indicate that NP reduced cell viability and increased the release of LDH in dose- and time-dependent manners. The reduction of cell viability by NP treatment appeared to involve necrosis as well as apoptosis based on nuclear fragmentation, an increase in the sub G1 population, and the detection of poly(ADP ribose) polymerase and caspase-3 cleavage. Additionally, the anti-apoptotic protein Bcl-2 diminished, whereas the pro-apoptotic protein Bax increased in a time-dependent manner. Note that NP-induced apoptotic cell death was enhanced by the generation of reactive oxygen species (ROS) and activation of extracellular signal-regulated kinase (ERK) signaling. Pretreatment with N-acetylcysteine, an antioxidant, attenuated NP-induced apoptotic cell death. Moreover, NP caused a transient activation of the MAPK pathway. In particular, NP-induced cell death was significantly suppressed by U0126, a specific inhibitor of ERK. Taken together, our results suggest that NP induces apoptosis in mouse TM4 Sertoli cells via ROS generation and ERK activation.

Connectivity study on resting-state EEG between motor imagery BCI-literate and BCI-illiterate groups.

Objective.Although motor imagery-based brain-computer interface (MI-BCI) holds significant potential, its practical application faces challenges such as BCI-illiteracy. To mitigate this issue, researchers have attempted to predict BCI-illiteracy by using the resting state, as this was found to be associated with BCI performance. As connectivity's significance in neuroscience has grown, BCI researchers have applied connectivity to it. However, the issues of connectivity have not been considered fully. First, although various connectivity metrics exist, only some have been used to predict BCI-illiteracy. This is problematic because each metric has a distinct hypothesis and perspective to estimate connectivity, resulting in different outcomes according to the metric. Second, the frequency range affects the connectivity estimation. In addition, it is still unknown whether each metric has its own optimal frequency range. Third, the way that estimating connectivity may vary depending upon the dataset has not been investigated. Meanwhile, we still do not know a great deal about how the resting state electroencephalography (EEG) network differs between BCI-literacy and -illiteracy.Approach.To address the issues above, we analyzed three large public EEG datasets using three functional connectivity and three effective connectivity metrics by employing diverse graph theory measures. Our analysis revealed that the appropriate frequency range to predict BCI-illiteracy varies depending upon the metric. The alpha range was found to be suitable for the metrics of the frequency domain, while alpha + theta were found to be appropriate for multivariate Granger causality. The difference in network efficiency between BCI-literate and -illiterate groups was constant regardless of the metrics and datasets used. Although we observed that BCI-literacy had stronger connectivity, no other significant constructional differences were found.Significance.Based upon our findings, we predicted MI-BCI performance for the entire dataset. We discovered that combining several graph features could improve the prediction's accuracy.

Prediction of Acute Hepatotoxicity With Human Pluripotent Stem Cell-Derived Hepatic Organoids.

Recent development of hepatic organoids (HOs) derived from human pluripotent stem cells (hPSCs) provides an alternative in vitro model that can mimic the human liver detoxification pathway for drug safety assessment. By recapitulating the high level of maturity and drug-metabolizing capacity of the liver in a three-dimensional organoid culture, HOs may allow researchers to assess drug toxicity and metabolism more accurately than animal models or hepatocellular carcinoma cells. Although this promising potential has contributed to the development of various protocols, only a few protocols are available to generate functional HOs with guaranteed CYP450 enzymatic activity, the key feature driving toxic responses during drug metabolism. Based on previously published protocols, we describe an optimized culture method that can substantially increase the expression and activity of CYP450s, in particular CYP3A4, CYP2C9, and CYP2C19, in HOs. To generate mass-produced and highly reproducible HOs required as models for toxicity evaluation, we first generated hepatic endodermal organoids (HEOs) from hPSCs capable of in vitro proliferation and cryopreservation. The stepwise protocol includes generating HEOs as well as efficient methods to enhance CYP450 expression and activity in terminally differentiated HOs. Furthermore, we present a simple protocol for the assessment of HO cytotoxicity, one of the hallmarks of drug-induced acute hepatotoxicity. The protocols are relatively straightforward and can be successfully used by laboratories with basic experience in culturing hPSCs. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of hepatic endodermal organoids from human pluripotent stem cells Basic Protocol 2: Expansion and cryopreservation of hepatic endodermal organoids Basic Protocol 3: Differentiation of hepatic organoids from hepatic endodermal organoids Basic Protocol 4: Evaluation of hepatotoxicity using hepatic organoids Support Protocol: Human pluripotent stem cell culture.

hiPSC-derived macrophages improve drug sensitivity and selectivity in a macrophage-incorporating organoid culture model.

Accurate simulation of different cell type interactions is crucial for physiological and precisein vitrodrug testing. Human tissue-resident macrophages are critical for modulating disease conditions and drug-induced injuries in various tissues; however, their limited availability has hindered their use inin vitromodeling. Therefore, this study aimed to create macrophage-containing organoid co-culture models by directly incorporating human-induced pluripotent stem cell (hiPSC)-derived pre-macrophages into organoid and scaffold cell models. The fully differentiated cells in these organoids exhibited functional characteristics of tissue-resident macrophages with enriched pan-macrophage markers and the potential for M1/M2 subtype specialization upon cytokine stimulation. In a hepatic organoid model, the integrated macrophages replicated typical intrinsic properties, including cytokine release, polarization, and phagocytosis, and the co-culture model was more responsive to drug-induced liver injury than a macrophage-free model. Furthermore, alveolar organoid models containing these hiPSC-derived macrophages also showed increased drug and chemical sensitivity to pulmonary toxicants. Moreover, 3D adipocyte scaffold models incorporating macrophages effectively simulated in vivo insulin resistance observed in adipose tissue and showed improved insulin sensitivity on exposure to anti-diabetic drugs. Overall, the findings demonstrated that incorporating hiPSC-derived macrophages into organoid culture models resulted in more physiological and sensitivein vitrodrug evaluation and screening systems.

Artificial intelligence estimated electrocardiographic age as a recurrence predictor after atrial fibrillation catheter ablation.

The application of artificial intelligence (AI) algorithms to 12-lead electrocardiogram (ECG) provides promising age prediction models. We explored whether the gap between the pre-procedural AI-ECG age and chronological age can predict atrial fibrillation (AF) recurrence after catheter ablation. We validated a pre-trained residual network-based model for age prediction on four multinational datasets. Then we estimated AI-ECG age using a pre-procedural sinus rhythm ECG among individuals on anti-arrhythmic drugs who underwent de-novo AF catheter ablation from two independent AF ablation cohorts. We categorized the AI-ECG age gap based on the mean absolute error of the AI-ECG age gap obtained from four model validation datasets; aged-ECG (≥10 years) and normal ECG age (<10 years) groups. In the two AF ablation cohorts, aged-ECG was associated with a significantly increased risk of AF recurrence compared to the normal ECG age group. These associations were independent of chronological age or left atrial diameter. In summary, a pre-procedural AI-ECG age has a prognostic value for AF recurrence after catheter ablation.

European and US Guideline-Based Statin Eligibility, Genetically Predicted Coronary Artery Disease, and the Risk of Major Coronary Events.

BACKGROUND: A study was designed to investigate whether the coronary artery disease polygenic risk score (CAD-PRS) may guide lipid-lowering treatment initiation as well as deferral in primary prevention beyond established clinical risk scores. METHODS AND RESULTS: Participants were 311 799 individuals from the UK Biobank free of atherosclerotic cardiovascular disease, diabetes, chronic kidney disease, and lipid-lowering treatment at baseline. Participants were categorized as statin indicated, statin indication unclear, or statin not indicated as defined by the European and US guidelines on statin use. For a median of 11.9 (11.2-12.6) years, 8196 major coronary events developed. CAD-PRS added to European-Systematic Coronary Risk Evaluation 2 (European-SCORE2) and US-Pooled Cohort Equation (US-PCE) identified 18% and 12% of statin-indication-unclear individuals whose risk of major coronary events were the same as or higher than the average risk of statin-indicated individuals and 16% and 12% of statin-indicated individuals whose major coronary event risks were the same as or lower than the average risk of statin-indication-unclear individuals. For major coronary and atherosclerotic cardiovascular disease events, CAD-PRS improved C-statistics greater among statin-indicated or statin-indication-unclear than statin-not-indicated individuals. For atherosclerotic cardiovascular disease events, CAD-PRS added to the European evaluation and US equation resulted in a net reclassification improvement of 13.6% (95% CI, 11.8-15.5) and 14.7% (95% CI, 13.1-16.3) among statin-indicated, 10.8% (95% CI, 9.6-12.0) and 15.3% (95% CI, 13.2-17.5) among statin-indication-unclear, and 0.9% (95% CI, 0.6-1.3) and 3.6% (95% CI, 3.0-4.2) among statin-not-indicated individuals. CONCLUSIONS: CAD-PRS may guide statin initiation as well as deferral among statin-indication-unclear or statin-indicated individuals as defined by the European and US guidelines. CAD-PRS had little clinical utility among statin-not-indicated individuals.

Generation of a PDGFRB-mCherry knock-in reporter human induced pluripotent stem cell line (KITi001-A-1), using CRISPR/Cas9 nuclease.

PDGFRB encodes platelet-derived growth factor receptor beta (PDGFR-ß), a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor family. It is required for the normal development of the vascular and nervous systems and rearrangement of the actin cytoskeleton. PDGFR-ß plays an essential role in early liver diseases, including liver fibrosis. Here, we generated a human induced pluripotent stem cell (iPSC) line, KITi001-A-1, using CRISPR/Cas9. This reporter iPSC line and its derivatives are useful for tracing PDGFR-ß-expressing cells and for screening for liver fibrosis-inducing compounds.

Establishment of a human embryonic stem cell line, WAe009-A-99, with constitutive expression of the dCas9-p300 fusion protein.

CRISPR/Cas9-based transcriptional regulation systems can induce the site-specific activation or repression of endogenous genes. p300 is a transcriptional co-activator that functions as a histone acetyltransferase that regulates gene transcription via chromatin remodeling. Here, we generated a human embryonic stem cell line stably expressing catalytically dead Cas9 (dCas9) fused to the catalytic core domain of human p300 via lentiviral transduction. This cell line can be used for locus-specific histone acetylation in combination with guide RNAs, and is a valuable tool for gene regulation in stem cell research.

Comparison of pulmonary vein isolation using cryoballoon, high-power short-duration, and conventional radiofrequency ablation for atrial fibrillation: a propensity score-weighted study.

Background: The comparative efficacy, saftey, and heart rate variability (HRV) parameters after pulmonary vein isolation using cryoballoon (Cryo-PVI), high-power short-duration (HPSD-PVI), and conventional radiofrequency ablation (conventional-PVI) for atrial fibrillation (AF) is unclear. Materials and methods: In this propensity score-weighted, retrospective analysis of a single-center cohort, we analyzed 3,395 patients (26.2% female, 74.5% paroxysmal AF) who underwent AF catheter ablation without an empirical left atrial ablation. Procedural factors, recurrence rates, complication rates, and the post-procedural HRV parameters were compared across the Cryo-PVI (n = 625), HPSD-PVI (n = 748), and conventional-PVI (n = 2,022) groups. Results: Despite the shortest procedural time in the Cryo-PVI group (74 min for Cryo-PVI vs. 104 min for HPSD-PVI vs. 153 min for conventional-PVI, p < 0.001), the major complication (p = 0.906) and clinical recurrence rates were similar across the three ablation groups (weighted log-rank, p = 0.824). However, the Cryo-PVI group was associated with a significantly lower risk of recurrent AF in patients with paroxysmal AF [weighted hazard ratio (WHR) 0.57, 95% confidence interval (CI) 0.37-0.86], whereas it was associated with a higher risk of recurrent AF in patients with persistent AF (WHR 1.41, 95% CI 1.06-1.89, p for interaction of <0.001) compared with the conventional-PVI group. In the subgroup analysis for the HRV, the Cryo-PVI group had the highest low-frequency-to-high-frequency ratio at 1-year post-procedure, whereas the HPSD-PVI group had the lowest low-frequency-to-high-frequency ratio at 1-year post-procedure (p < 0.001). Conclusions: The Cryo-PVI group had better rhythm outcomes in patients with paroxysmal AF but worse rhythm outcomes in patients with persistent AF and a higher long-term post-procedural sympathetic nervous activity and sympatho-vagal balance compared with the conventional-PVI group.

Association Between the Combined Effects of Physical Activity Intensity and Particulate Matter and All-Cause Mortality in Older Adults.

OBJECTIVE: To investigate the association between the combined effects of physical activity (PA) intensity and particulate matter ≤10 µm in diameter (PM10) and mortality in older adults. METHODS: This nationwide cohort study included older adults without chronic heart or lung disease who engaged in regular PA. Physical activity was assessed by a standardized, self-reported questionnaire that asked the usual frequency of PA sessions with low (LPA), moderate (MPA), or vigorous intensity (VPA). Each participant's annual average cumulative PM10 was categorized as low to moderate and high PM10 on the basis of a cutoff value of 90th percentile. RESULTS: A total of 81,326 participants (median follow-up, 45 months) were included. For participants engaged in MPA or VPA sessions, every 10% increase in the proportion of VPA to total PA sessions resulted in a 4.9% (95% CI, 1.0% to 9.0%; P=.014) increased and 2.8% (95% CI, -5.0% to -0.5%; P=.018) decreased risk of mortality for those exposed to high and low to moderate PM10, respectively (Pinteraction, <.001). For participants engaged only in LPA or MPA sessions, every 10% increase in the proportion of MPA to total PA sessions resulted in a 4.8% (95% CI, -8.9% to -0.4%; P=.031) and 2.3% (95% CI, -4.2% to -0.3%; P=.023) decreased risk of mortality for those exposed to high and low to moderate PM10, respectively (Pinteraction, .096). CONCLUSION: We found that for the same level of total PA, MPA was associated with delayed mortality whereas VPA was associated with hastened mortality of older adults in high levels of PM10.

Generation of multilineage liver organoids with luminal vasculature and bile ducts from human pluripotent stem cells via modulation of Notch signaling.

BACKGROUND: The generation of liver organoids recapitulating parenchymal and non-parenchymal cell interplay is essential for the precise in vitro modeling of liver diseases. Although different types of multilineage liver organoids (mLOs) have been generated from human pluripotent stem cells (hPSCs), the assembly and concurrent differentiation of multiple cell types in individual mLOs remain a major challenge. Particularly, most studies focused on the vascularization of mLOs in host tissue after transplantation in vivo. However, relatively little information is available on the in vitro formation of luminal vasculature in mLOs themselves. METHODS: The mLOs with luminal blood vessels and bile ducts were generated by assembling hepatic endoderm, hepatic stellate cell-like cells (HscLCs), and endothelial cells derived entirely from hPSCs using 96-well ultra-low attachment plates. We analyzed the effect of HscLC incorporation and Notch signaling modulation on the formation of both bile ducts and vasculature in mLOs using immunofluorescence staining, qRT-PCR, ELISA, and live-perfusion imaging. The potential use of the mLOs in fibrosis modeling was evaluated by histological and gene expression analyses after treatment with pro-fibrotic cytokines. RESULTS: We found that hPSC-derived HscLCs are crucial for generating functional microvasculature in mLOs. HscLC incorporation and subsequent vascularization substantially reduced apoptotic cell death and promoted the survival and growth of mLOs with microvessels. In particular, precise modulation of Notch signaling during a specific time window in organoid differentiation was critical for generating both bile ducts and vasculature. Live-cell imaging, a series of confocal scans, and electron microscopy demonstrated that blood vessels were well distributed inside mLOs and had perfusable lumens in vitro. In addition, exposure of mLOs to pro-fibrotic cytokines induced early fibrosis-associated events, including upregulation of genes associated with fibrotic induction and endothelial cell activation (i.e., collagen I, α-SMA, and ICAM) together with destruction of tissue architecture and organoid shrinkage. CONCLUSION: Our results demonstrate that mLOs can reproduce parenchymal and non-parenchymal cell interactions and suggest that their application can advance the precise modeling of liver diseases in vitro.

Oral Anticoagulation Therapy in Atrial Fibrillation Patients with Advanced Chronic Kidney Disease: CODE-AF Registry.

PURPOSE: Advanced chronic kidney disease (CKD), including end-stage renal disease (ESRD) on dialysis, increases thromboembolic risk among patients with atrial fibrillation (AF). This study examined the comparative safety and efficacy of direct-acting oral anticoagulant (DOAC) compared to warfarin or no oral anticoagulant (OAC) in AF patients with advanced CKD or ESRD on dialysis. MATERIALS AND METHODS: Using data from the COmparison study of Drugs for symptom control and complication prEvention of AF (CODE-AF) registry, 260 non-valvular AF patients with advanced CKD (defined as estimated glomerular filtration rate <30 mL/min per 1.73/m²) or ESRD on dialysis were enrolled from June 2016 to July 2020. The study population was categorized into DOAC, warfarin, and no OAC groups; and differences in major or clinically relevant non-major (CRNM) bleeding, stroke/systemic embolism (SE), myocardial infarction/critical limb ischemia (CLI), and death were assessed. RESULTS: During a median 24 months of follow-up, major or CRNM bleeding risk was significantly reduced in the DOAC group compared to the warfarin group [hazard ratio (HR) 0.11, 95% confidence interval (CI) 0.01 to 0.93, p=0.043]. In addition, the risk of composite adverse clinical outcomes (major or CRNM bleeding, stroke/SE, myocardial infarction/CLI, and death) was significantly reduced in the DOAC group compared to the no OAC group (HR 0.16, 95% CI 0.03 to 0.91, p=0.039). CONCLUSION: Among AF patients with advanced CKD or ESRD on dialysis, DOAC was associated with a lower risk of major or CRNM bleeding compared to warfarin and a lower risk of composite adverse clinical outcomes compared to no OAC. ClinicalTrials.gov (NCT02786095).

Gene expression analysis of toxicological pathways in TM3 leydig cell lines treated with Ethane dimethanesulfonate.

Ethane dimethanesulfonate (EDS), a well-known alkylating agent, selectively destroys Leydig cells. To clarify the molecular pathways underlying EDS action on Leydig cells, we analyzed gene expression profiles of an EDS-treated TM3 Leydig cell line. In this study, we analyzed the representative canonical pathways and toxicity pathways/gene lists using the Ingenuity Pathways Analysis program. In TM3 cells, 677 and 6756 genes were identified as being up- or downregulated after 3 and 24 h EDS treatments, respectively, (>1.3-fold changes, p < 0.05). Toxicological pathway analysis revealed that expression of genes related to Nrf2-mediated oxidative stress response showed remarkable changes in early or later stage of EDS-treated TM3 cells. Several genes related to steroidogenesis and apoptosis were also differentially expressed at 24 h in EDS-treated TM3 cells. Overall, toxicological pathway analysis using gene expression profiling showed that oxidative stress might be an important factor in cell death in TM3 cells affected by EDS treatment.