The roles of Contactin-2 (CNTN2) and ferroptosis in heart failure and cardiac remodeling remain incompletely understood. CNTN2 was significantly upregulated in hypertrophic cardiomyopathy patients and heart failure mice. In cardiomyocyte specific CNTN2 conditional knockout (CNTN2 cKO) mice, transverse aortic constriction (TAC) induced markedly exacerbated heart failure, cardiac remodeling and ferroptosis compared to control mice. Ferroptosis inhibition substantially attenuated heart failure in CNTN2 cKO mice subjected to TAC, indicating that enhanced ferroptosis contributes to the detrimental effects of CNTN2 deficiency. RNA sequencing identified NUPR1, a ferroptosis repressor, as a downstream molecule of CNTN2. Mechanistically, CNTN2 activated the Lyn/eIF2α/ATF4 pathway to regulate NUPR1. CNTN2 overexpression attenuated Angiotensin II-induced cardiomyocyte ferroptosis and pathological remodeling, whereas these protective effects were abolished by Lyn or NUPR1 inhibitors. We further revealed CNTN2 and Lyn interacted with each other, and that CNTN2 interacted with Lyn through its 1-328aa domain. In vivo NUPR1 overexpression via AAV9 significantly mitigated TAC-induced heart failure and cardiac remodeling in CNTN2 cKO mice. Our study demonstrates that CNTN2 protects against pressure overload induced heart failure and cardiac remodeling by regulating ferroptosis through the Lyn/eIF2α/ATF4/NUPR1 pathway, suggesting CNTN2 as a potential therapeutic target.

BACKGROUND:Left atrial appendage (LAA) patency after catheter ablation and LAA closure (LAAC) is a key determinant of postprocedure stroke. The effect of LAA electrical isolation (LAAEI) on LAA patency after LAAC in patients with atrial fibrillation (AF) remains unknown. Our objective was to explore the effect of LAAEI on LAA patency after catheter ablation and LAAC in patients with AF. METHODS:Patients with persistent AF who underwent combined catheter ablation and LAAC were enrolled. LAA patency was detected by cardiac computed tomographic angiography at 3 months after the procedure. RESULTS:A total of 686 patients with persistent AF with 3-month device surveillance by cardiac computed tomographic angiography were included initially. Propensity score matching analysis was conducted on the basis of clinical features, echocardiographic parameters and procedural characteristics, and 469 patients were matched, including 363 with non-LAAEI and 106 with LAAEI. Patients in matched groups showed 53.7% complete closure (no patency). Of the 46.3% of cases with LAA patency, visible peridevice leak was seen in 72.8%, and 27.2% had patency without visible peridevice leak. Compared with the non-LAAEI group, the LAAEI group had a lower rate of LAA patency (49.6% versus 34.9%; adjusted odds ratio, 0.69 [95% CI, 0.48-0.98]; P=0.040). LAAEI was protective against LAA patency with an odds ratio of 0.68, and the other predictor of contrast leak into the LAA was maximum diameter of LAA orifice (odds ratio, 1.04 [95% CI, 1.00-1.08]; P=0.046). CONCLUSIONS:LAAEI is an effective strategy for reducing LAA patency in patients with persistent AF undergoing combined catheter ablation and LAAC. REGISTRATION:URL: clinicaltrials.gov; Unique Identifier: NCT03788941.

INTRODUCTION:Plasma proteins reflect the combined influence of both internal and external factors, making proteomics-based aging clocks a promising approach for quantifying the aging process. OBJECTIVE:This study aims to develop and validate a novel proteomics-based aging clock by integrating plasma proteomics with composite biomarkers. METHODS:We used a prospective cohort of 37,433 participants (median follow-up: 164.73 months) from the UK Biobank (UKB) with Olink Explore data. We calculated biological age (PhenoAge) and used the Boruta-SHAP (SHapley Additive exPlanations) algorithm to select PhenoAge-related proteins. Based on these proteins, six machine learning models were trained to develop a proteomics-based PhenoAge (ProtPhenoAge). We selected the best model as ProtPhenoAge based on the predictive capabilities of each model for PhenoAge and all-cause mortality. Phenome-wide association study (PheWAS) and Mendelian randomization (MR) explored associations between ProtPhenoAge Acceleration (ProtPhenoAgeAccel) and phenotypes. Genome-wide association study (GWAS) and colocalization analysis identified aging-associated loci. RESULTS:A total of 185 PhenoAge-related plasma proteins were used to develop ProtPhenoAge. The ProtPhenoAge model, using extreme gradient boosting (XGBoost), showed strong correlation with PhenoAge (r = 0.96, R2 = 0.92) and performed well in predicting all-cause mortality [area under the curve (AUC) = 0.76], outperforming previous aging clocks: chronological age (CA), PhenoAge and ProtAge. ProtPhenoAgeAccel was significantly associated with 313 disease phenotypes, covering a broad range of aging-related phenotypes. Compared with previous clocks, it identified more age-independent but aging-related phenotypes. In the GWAS, we identified 10 aging-associated loci. Among them, rs1045929 (P = 2.61 × 10-9) and rs429358 (P = 7.96 × 10-12) are respectively related to epigenetic aging and the well-recognized aging gene APOE. CONCLUSION:Based on genomic and phenomic evidences, ProtPhenoAge was regarded to better quantifies the aging process by overcoming the limitations of previous clocks, which failed to detect time-independent aging features. These findings suggested that ProtPhenoAge is a reliable tool to assess aging and supporting aging research.

Introduction Lymphovascular invasion (LVI), an aggressive pathological manifestation of breast cancer, is closely associated with increased risk of distant metastasis and poor prognosis. This study proposes a novel modeling strategy that integrates MRI-derived microvascular atlas parameters with the TwinsSVT deep learning architecture to enable noninvasive prediction of LVI status in breast cancer patients and to explore its biological interpretability.Materials and Methods A total of 436 breast cancer patients from two medical centers, all pathologically confirmed postoperatively, were retrospectively enrolled. All patients underwent high-resolution multi-b-value diffusion-weighted imaging (DWI) prior to surgery. From the MRI data, four types of microvascular simulation parameter maps were reconstructed within tumor regions: apparent diffusion coefficient (ADC), mean flow velocity (v_m), velocity dispersion (v_s), and angiographic branching index (ANB), aiming to characterize intratumoral microcirculation and vascular structural complexity. These functional parametric maps were individually input into separate encoder branches of the TwinsSVT model to extract multi-scale spatial features. A multi-layer Transformer fusion module was then employed to capture structural interactions across modalities, thereby constructing a multi-parametric fusion model. Model performance was evaluated using metrics including area under the curve (AUC) and F1 score.Results Compared with single-parameter models, the multi-parametric fusion model demonstrated significantly improved predictive performance, with AUCs of 0.881 (95% CI: 0.781-0.982) and 0.859 (95% CI: 0.764-0.953) in internal and external validation cohorts, respectively. Grad-CAM visualizations revealed that the model predominantly focused on tumor margins and regions of high vascular density, suggesting a strong correlation between the model's attention and actual pathological structures.Conclusion The deep learning model constructed based on MRI-derived microvascular simulation atlases enables noninvasive preoperative prediction of LVI status in breast cancer patients. By effectively capturing structural information and offering biological interpretability, the model holds promise as a robust imaging-based tool for precision subtyping and clinical decision support.

BACKGROUND:Mechanosensitive nuclear signaling contributes to myocardial ischemia-reperfusion injury, but the substrates and mechanisms of NUAK1 (AMPK-related kinase 5) remain unclear. We investigated whether NUAK1 regulates SYNE1 (Nesprin-1)/linker of nucleoskeleton and cytoskeleton-dependent nuclear gating of YAP1 (Yes-associated protein 1) during hypoxia/reoxygenation and ischemia-reperfusion injury. METHODS:We integrated quantitative phosphoproteomics, biochemical assays, phosphosite-mutant analyses, subcellular fractionation, atomic force microscopy, and genetic or pharmacological NUAK1 inhibition in neonatal mouse ventricular myocytes and mouse models of ischemia-reperfusion injury to define the NUAK1-SYNE1-YAP1 axis. RESULTS:Quantitative phosphoproteomics identified a conserved NUAK1-dependent phosphorylation site in Nesprin1-α2/SYNE1 (S434; S8284 in nesprin-1 giant), and biochemical assays supported direct SYNE1 phosphorylation by NUAK1. NUAK1 inhibition reduced apoptotic signaling and SYNE1 stability, enhanced YAP1 nuclear localization, and altered nuclear YAP1 dynamics. SYNE1 phosphosite-mutant and fractionation analyses indicated that NUAK1-SYNE1 restrains stress-induced YAP1 nuclear accumulation. Atomic force microscopy linked this pathway to nuclear mechanical remodeling. In mouse models of ischemia-reperfusion injury, NUAK1 inhibition reduced acute myocardial damage and improved remodeling indices. CONCLUSIONS:NUAK1-dependent SYNE1 phosphorylation shapes nuclear mechanosignaling during ischemic stress. NUAK1 downregulation promotes cardiomyocyte YAP1 nuclear activity and attenuates injury responses.

BACKGROUND:Membrane lipids play a crucial role in brain function and cell signalling, and they serve as key biological substrates in inflammatory responses, thrombosis, and energy metabolism. Multiple clinical and molecular evidences suggest that membrane lipids are probably involved in the pathogenesis of ischemic stroke (IS). However, current knowledge about the membrane lipid landscape and its involvement in IS pathophysiology is limited. METHODS:We performed untargeted lipidomic analysis on erythrocyte membranes from 56 IS patients and 55 healthy controls. Integrated with gene expression and weighted gene co-expression network analysis, we identified dysregulated lipid signalling pathways and their contributions to IS pathophysiology. RESULTS:A total of 1392 erythrocyte membrane lipids were detected and quantified. Our results revealed significant impairment of membrane lipid homeostasis in IS patients, characterized by a marked reduction in glycerophospholipids (GPLs) and lysophospholipids (LPLs). Further analysis indicated that the impaired lipids were primarily concentrated in three disturbed signalling pathways, including the phospholipase A2-mediated GPL-LPL pathway, the phospholipase C-mediated inositol 1,4,5-trisphosphate/diglyceride pathway, and the sphingosine-1-phosphate (S1P)-S1P receptors pathway. Gene expression results indicated that these pathways were inhibited during the subacute phase of IS. Furthermore, these lipid signalling pathways form a highly interconnected network that collaboratively contributes to inflammation and thrombosis in IS, thereby influencing the progression and prognosis of the disease. CONCLUSION:Our findings reveal impaired erythrocyte membrane lipid homeostasis in IS, which implicates inflammatory processes and thrombosis in IS. This research offers new insights into the role of membrane lipids in IS pathogenesis, potentially informing future monitoring and therapeutic strategies.

CPNE5, a member of the Copine family, is characterized by its membrane-binding properties and functions as a regulatory modulator of intracellular signaling through the spatial redistribution of interacting protein partners. Emerging evidence has demonstrated that CPNE3 exerts cardioprotective effects via anti-apoptotic activity in myocardial ischemia-reperfusion injury models. However, the functional role of CPNE5 in cardiac pathology remains unclear. In this study, the cardiac-specific overexpression of CPNE5 in mice improved cardiac function, reduced cellular apoptosis, and attenuated cardiac fibrosis in both transverse aortic constriction and ischemia-reperfusion models. Conversely, CPNE5 knockout mice exhibited opposite pathological phenotypes. Mechanistic studies revealed that CPNE5 retains FAS within the endoplasmic reticulum and promotes its degradation through the ER-phagy pathway. This process involves CPNE5's interaction with the autophagy marker LC3 and CALCOCO1, a key receptor in the ER-lysosome-associated degradation (ERLAD) pathway. Collectively, these findings indicate that CPNE5 overexpression protects cardiomyocytes against FASL-induced apoptosis under stress and ischemic conditions.

Myocardial inflammation plays a critical role in the progression of injury following myocardial infarction (MI), yet the transcriptional mechanisms regulating cardiomyocyte inflammation to mitigate post-ischemic injury remain poorly understood. This study elucidated the role of Estrogen-Related Receptor Gamma (ERRγ) in modulating the inflammatory response post-MI, demonstrating that ERRγ expression was downregulated in ischemic tissue and hypoxic neonatal mouse ventricular myocytes (NMVMs). Cardiomyocyte-specific overexpression of ERRγ reduced infarct size, improved cardiac function, and suppressed excessive myocardial inflammation and pyroptosis by binding to the GBP5 promoter, thereby inhibiting GBP5 transcription and reducing NLRP3 inflammasome assembly. The protective effects of ERRγ overexpression were reversed by overexpressing GBP5, and the ERRγ agonist DY131 also improved cardiac function after MI. These findings suggest that ERRγ activation reduces myocardial ischemic injury by regulating cardiomyocyte inflammation and pyroptosis, highlighting ERRγ as a potential novel therapeutic target for attenuating post-MI injury.

Effective and precise treatment of breast cancer, particularly with bone metastasis, remains a significant challenge. Here, a dual-gating strategy combining locally delivered borate glass (BG) and ultrasound (US) is developed for the precise and effective inhibition of breast cancer by targeting transient receptor potential vanilloid 2 (TRPV2). The results demonstrate that after local delivery of BG to the solid tumor, US effectively triggers calcium overload by activating the overexpressed TRPV2 channels, leading to mitochondrial autophagy and apoptosis in breast cancer cells, thereby inhibiting tumor growth with high precision. These effects are validated in subcutaneous, orthotopic, and TRPV2-overexpressing breast cancer mouse models. In the bone metastasis model, BG combined with US treatment simultaneously suppresses tumor growth and promotes bone regeneration. Overall, this dual-gating strategy offers a safe and efficient approach for the precise treatment of cancers with high TRPV2 expression and provides new insights into the design and clinical translation of calcium-overload-based cancer therapies.

In the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), the emergence of acquired resistance remains a significant challenge. Elucidating the underlying mechanisms of resistance is crucial for developing novel strategies to overcome or delay therapeutic escape. To this end, this study aimed to identify key drivers of EGFR-TKIs resistance and explore actionable targets for intervention. We investigated resistance mechanisms by integrating CRISPR/Cas9-based genome-wide screening with tandem mass tag (TMT) proteomic analysis, and virtually screened bioactive small molecule libraries to identify compounds capable of restoring EGFR-TKIs sensitivity. The multi-omics approach revealed that CCT2 is a critical mediator of resistance to third-generation EGFR-TKIs in lung cancer, with higher expression of CCT2 observed in resistant cells compared to sensitive cells. Mechanistically, CCT2 recruits tripartite motif-containing protein 28 (TRIM28) to catalyze SUMO2 modification of thioredoxin-related transmembrane protein 1 (TMX1), inhibiting its ubiquitination and enhancing protein stability. This post-translational modification (PTM) promotes TMX1-dependent reactive oxygen species (ROS) clearance, thereby conferring resistance. Importantly, pharmacological inhibition with the compound HY-10127, identified through virtual screening, effectively restored EGFR-TKIs sensitivity in resistant cell lines and delayed the development of resistance in xenograft models. The findings establish the CCT2/TRIM28/TMX1/ROS axis as a novel resistance mechanism in EGFR-mutated lung cancer, and targeting this pathway with HY-10127 represents a promising strategy to overcome resistance to third-generation EGFR-TKIs, providing preclinical rationale for clinical translation. These discoveries advance our understanding of molecular resistance mechanisms and offer potential therapeutic targets for improving lung cancer prognosis.

Atrial fibrosis is the hallmark of structural remodeling in the pathogenesis of atrial fibrillation (AF). Meanwhile, AF causes a hypercoagulable state, and then provokes pro-fibrotic response. To discover a potential effective AF treatment targeting both coagulation and atrial fibrosis, this study investigated the structure–activity relationship of propylene glycol alginate sodium sulfate (PSS) derivatives with heparin-like activity on TGF-β1-induced atrial fibrosis. We found that PSS derivatives had significantly inhibitory effects on proliferation, migration, phenotypic transformation, and secretion/deposition of extracellular matrix of atrial fibroblasts. Among them, PGGS showed the optimal anti-atrial fibrotic activity by suppressing TGF-β1-induced activation of Smad2/3 signaling pathway. Furthermore, the study in vivo indicated that PGGS treatment displayed a reduced atrial fibrosis and AF inducibility, and attenuated the hypercoagulable state by decreasing D-dimer level and thrombin (FIIa) activity in MHC-TGF-β1 cys33ser transgenic mice, which had increased fibrosis in atrium but not in the ventricles. Our results demonstrated that PSS derivatives, especially PGGS, were potential anti-atrial fibrosis and anti-coagulant agents for AF prevention. Our study is beneficial in extending the current understandings of the function of PSS on atrial fibrosis and vulnerability to AF.

Atrial fibrillation (AF) is the most prevalent sustained cardiac arrhythmia, and recent epidemiological studies suggested type 2 diabetes mellitus (T2DM) is an independent risk factor for the development of AF. Zinc finger and BTB (broad-complex, tram-track and bric-a-brac) domain containing 16 (Zbtb16) serve as transcriptional factors to regulate many biological processes. However, the potential effects of Zbtb16 in AF under T2DM condition remain unclear. Here, we reported that db/db mice displayed higher AF vulnerability and Zbtb16 was identified as the most significantly enriched gene by RNA sequencing (RNA-seq) analysis in atrium. In addition, thioredoxin interacting protein (Txnip) was distinguished as the key downstream gene of Zbtb16 by Cleavage Under Targets and Tagmentation (CUT&Tag) assay. Mechanistically, increased Txnip combined with thioredoxin 2 (Trx2) in mitochondrion induced excess reactive oxygen species (ROS) release, calcium/calmodulin-dependent protein kinase II (CaMKII) overactivation, and spontaneous Ca2+ waves (SCWs) occurrence, which could be inhibited through atrial-specific knockdown (KD) of Zbtb16 or Txnip by adeno-associated virus 9 (AAV9) or Mito-TEMPO treatment. High glucose (HG)-treated HL-1 cells were used to mimic the setting of diabetic in vitro. Zbtb16-Txnip-Trx2 signaling-induced excess ROS release and CaMKII activation were also verified in HL-1 cells under HG condition. Furthermore, atrial-specific Zbtb16 or Txnip-KD reduced incidence and duration of AF in db/db mice. Altogether, we demonstrated that interrupting Zbtb16-Txnip-Trx2 signaling in atrium could decrease AF susceptibility via reducing ROS release and CaMKII activation in the setting of T2DM.

Objectives We aimed to develop a multi-modality model to predict axillary lymph node (ALN) metastasis by combining clinical predictors with radiomic features from magnetic resonance imaging (MRI) and mammography (MMG) in breast cancer. This model might potentially eliminate unnecessary axillary surgery in cases without ALN metastasis, thereby minimizing surgery-related complications. Methods We retrospectively enrolled 485 breast cancer patients from two hospitals and extracted radiomics features from tumor and lymph node regions on MRI and MMG images. After feature selection, three random forest models were built using the retained features, respectively. Significant clinical factors were integrated with these radiomics models to construct a multi-modality model. The multi-modality model was compared to radiologists’ diagnoses on axillary ultrasound and MRI. It was also used to assist radiologists in making a secondary diagnosis on MRI. Results The multi-modality model showed superior performance with AUCs of 0.964 in the training cohort, 0.916 in the internal validation cohort, and 0.892 in the external validation cohort. It surpassed single-modality models and radiologists’ ALN diagnosis on MRI and axillary ultrasound in all validation cohorts. Additionally, the multi-modality model improved radiologists’ MRI-based ALN diagnostic ability, increasing the average accuracy from 70.70 to 78.16% for radiologist A and from 75.42 to 81.38% for radiologist B. Conclusion The multi-modality model can predict ALN metastasis of breast cancer accurately. Moreover, the artificial intelligence (AI) model also assisted the radiologists to improve their diagnostic ability on MRI. Clinical relevance statement The multi-modality model based on both MRI and mammography images allows preoperative prediction of axillary lymph node metastasis in breast cancer patients. With the assistance of the model, the diagnostic efficacy of radiologists can be further improved. Key Points • We developed a novel multi-modality model that combines MRI and mammography radiomics with clinical factors to accurately predict axillary lymph node (ALN) metastasis, which has not been previously reported. • Our multi-modality model outperformed both the radiologists’ ALN diagnosis based on MRI and axillary ultrasound, as well as single-modality radiomics models based on MRI or mammography. • The multi-modality model can serve as a potential decision support tool to improve the radiologists’ ALN diagnosis on MRI.

Pulmonary hypertension (PH) is a severe clinical syndrome with pulmonary vascular remodeling and poor long-term prognosis. Neurotensin receptor 1 (Ntsr1), serve as one of the G protein-coupled receptors (GPCRs), implicates in various biological processes, but the potential effects of Ntsr1 in PH development are unclear. The Sugen/Hypoxia (SuHx) or monocrotaline (MCT) induced rat PH model was used in our study and the PH rats showed aggravated pulmonary artery remodeling and increased right ventricular systolic pressure (RVSP). Our results revealed that Ntsr1 induced endoplasmic reticulum (ER) stress response via ATF6 activation contributed to the development of PH. Moreover, RNA-sequencing (RNA-seq) and phosphoproteomics were performed and the Ntsr1-JAK2-STAT3-thrombospondin 1 (Thbs1)-ATF6 signaling was distinguished as the key pathway. In vitro, pulmonary artery smooth muscle cells (PASMCs) under hypoxia condition showed enhanced proliferation and migration properties, which could be inhibited by Ntsr1 knockdown, JAK2 inhibitor (Fedratinib) treatment, STAT3 inhibitior (Stattic) treatment, Thbs1 knockdown or ATF6 knockdown. In addition, adeno-associated virus 1 (AAV1) were used to knockdown the expression of Ntsr1, Thbs1 or ATF6 in rats and reversed the phenotype of PH. In summary, our results reveal that Ntsr1-JAK2-STAT3-Thbs1 pathway can induce enhanced ER stress via ATF6 activation and increased PASMC proliferation and migration capacities, which can be mechanism of the pulmonary artery remodeling and PH. Targeting Ntsr1 might be a novel therapeutic strategy to ameliorate PH.

Background and objectivesHepatectomy is the preferred treatment for patients with liver tumors. Post-hepatectomy liver failure (PHLF) remains one of the most fatal postoperative complications. We aim to explore the risk factors of PHLF and create a nomogram for early prediction of PHLF.MethodsWe retrospectively analyzed patients undergoing hepatectomy at the Affiliated Huaian No. 1 People’s Hospital of Nanjing Medical University between 2015 and 2022, and the patients were divided into training and internal validation cohorts at an 8:2 ratio randomly. The patients undergoing liver resection from the Affiliated Huaian Hospital of Xuzhou Medical University worked as external validation. Then, a nomogram was developed which was based on multivariate analyses to calculate the risk of PHLF. The area under the ROC curve (AUROC) and Hosmer -Lemeshow test was used to evaluate the prediction effect of the model.ResultsA total of 421 eligible patients were included in our study. Four preoperative variables were identified after multivariate analysis as follows, ASA (American Society of Anesthesiologists) score, Child-Pugh score, SMI (Skeletal muscle index), and MELD (Model for end-stage liver disease) score as independent predictors of PHLF. The area under the ROC curve of the predictive model in the training, internal, and external validation cohorts were 0.89, 0.82, and 0.89. Hosmer -Lemeshow P values in the training, internal, and external validation cohorts were 0.91, 0.22, and 0.15. The Calibration curve confirmed that our nomogram prediction results were in accurate agreement with the actual occurrence of PHLF.ConclusionWe construct a nomogram to predict the grade B/C PHLF of ISGLS (International Study Group of Liver Surgery) in patients who underwent hepatic resection based on risk factors. This tool can provide a visual and accurate preoperative prediction of the grade B/C PHLF and guide the next step of clinical decision-making.

To the Editor: Atrial fibrillation (AF) is a well-recognized cause of left atrial (LA) structural remodeling.[1] LA sphericity (LASP), a new shape-based remodeling parameter, is an independent predictor for AF ablation outcome.[2,3] Improvement of LA structure was defined as LA reverse remodeling (RR). This study compared the impact of combined left atrial appendage (LAA) closure (LAAC) and catheter ablation (CA) of AF with a single procedure on LA volume (LAV) and LASP. We retrospectively enrolled consecutive patients with non-valvular AF between January 2017 and December 2020. The study protocol was approved by the Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (No. NCT03788941). All patient provided inform consent. The inclusion criteria were the following: (1) age ≥18 years; (2) CHA2DS2-VASc (congestive heart failure, hypertension, age ≥75 [doubled], diabetes, stroke [doubled], vascular disease, age 65–74 years, and sex category) Score ≥2; (3) symptomatic AF refractory to antiarrhythmic drugs with CA indication; and (4) LAAC with at least one of the following indications: HAS-BLED (score hypertension, abnormal renal/liver function, stroke, bleeding history or predisposition, labile international normalized ratio, elderly, drugs/alcohol concomitantly) ≥3; thromboembolism (TE) even with oral anticoagulants (OAC); and contraindications or unwillingness to receive long-term OAC. We excluded patients with severe valvular heart disease, hyperthyroidism, or incomplete data. Patients were divided into CA-only, LAAC-only, and combined groups. The combined group was 1:1 matched with the other two groups by propensity score matching (PSM), respectively (combined vs. CA-only and combined vs. LAAC-only). LAA thrombus was ruled out by preoperative transesophageal echocardiography (TEE). CARTO (Biosense Webster, Diamond Bar, CA, USA) or EnSite (St. Jude Medical, St. Paul, MN, USA) electroanatomical mapping systems were used for LA reconstruction and ablation. For patients with paroxysmal AF, pulmonary vein (PV) isolation was performed. Additional linear ablations were subsequently performed for those with persistent AF. Sinus rhythm (SR) was restored by ablation or electric cardioversion. In the combined group, Watchman device (Boston Scientific Corporation, Natick, MA, USA) implantation was performed after CA. Immediate intraprocedural TEE and/or angiography were performed to verify appropriate device implantation. During follow-up visits performed at 3 months and 6 months postoperatively and every 6 months thereafter, electrocardiography (ECG) and Holter tests were performed to detect AF recurrence. TEE and coronary computed tomography angiography (CCTA) were performed for detecting peri-device leaks (PDL) and device-related thrombus. The OAC strategy for patients who received LAAC was adjusted according to the satisfactory seal results. LAV and LAA volume (LAAV) were measured by Mimics Medical 17.0 (Materialise NV, Leuven, Belgium), a software tool for visualizing and segmenting medical images and rendering 3D objects, [Figures 1A and 1B]. Separation of the LA from the left ventricle, PVs, and LAA was bounded by the mitral valve annulus, PV ostia, and LAA orifice, respectively. LAV and LAAV were automatically calculated using the Mimics software. Original CCTA images were reconstructed for chamber measurements using Extended Brilliance Workspace version 4.5 (Philips Healthcare Cleveland, OH, USA). From three diameters of the LA, including the largest transverse and anterior-posterior diameters measured on axial planes and the largest craniocaudal diameter on the sagittal plane, the maximum was selected for calculating LASP [Figures 1C and 1D].Figure 1: Three-dimensional images of the LA (yellow) and LAA (blue-green) (A) and separated LA (B), as well as the measurement of largest LA transverse, anterior-posterior diameters on the axial plane (C) and craniocaudal diameter on the sagittal plane (D). The volume of the given LA is 77.13 cm3 and 6.95 cm is chosen as the maximum diameter. The diameter of the sphere with the same volume of 77.13 cm3 is 5.28 cm. From these values, the LASP is calculated as 75.99% (5.28/6.95 = 0.7599). LA: Left atrial; LAA: Left atrial appendage; LASP: LA sphericity.Statistical analysis was performed using SPSS 26.0 (IBM Software, Armonk, NY, USA). Two-sample t-test, paired t-test, or Mann–Whitney U test was used to compare continuous variables, and the Pearson chi-squared test or Fisher's exact test was used to compare categorical data as appropriate. Survival data were analyzed using the Kaplan–Meier method with a log-rank test. Multiple linear/logistic regression analyses were performed to identity the clinical factors associated with outcomes. A P <0.05 was considered statistically significant. There were 252, 157, and 63 patients meeting the criteria in the combined, CA-only, and LAAC-only groups, respectively. CA-only and LAAC-only groups showed significant differences in several clinical characteristics with the combined group, respectively. After PSM, 141 and 52 pairs were successfully matched in the combined group vs. CA-only group and combined vs. LAAC-only comparisons, respectively. All characteristics were comparable at baseline, except for the HAS-BLED score in the combined group vs. CA-only group (3.1 ± 1.2 vs. 1.6 ± 1.0, P <0.001). During the procedure, SR was restored in all patients who underwent CA. Watchman devices (Boston Scientific Corporation) were successfully implanted with a 100% satisfactory seal in patients who received LAAC. No major bleeding occurred during hospitalization, and all patients were switched to OAC before discharge. One year after the intervention, SR was maintained in 78.2% (197/252) and 75.8% (119/157) of patients in the combined and CA-only groups, respectively. There were four patients with TE, one with MB in both the combined and CA-only groups, one patient with TE, and one with MB in the LAAC-only group. No device-related thrombus or major PDL was detected on the follow-up imaging examination. In the combined and CA-only groups, LAV significantly decreased from 128.38 ± 41.37 mL to 111.79 ± 35.17 mL (P <0.001) and 114.80 ± 43.65 mL to 99.39 ± 38.88 mL (P <0.001) after therapy, respectively. A significant LAV increase was noticed in the LAAC-only group (175.98 ± 49.61 mL vs. 185.96 ± 55.96 mL; P <0.001). LASP significantly increased after treatment in both the combined ([79.60 ± 5.25]% vs. [80.90 ± 5.74]%, P <0.001) and CA-only groups ([80.43 ± 5.92]% vs. [82.89 ± 5.56]%, P <0.001), but not in the LAAC-only group ([81.60 ± 5.37]% vs. [82.33 ± 5.30]%, P = 0.221). After PSM, LAV changes (△-volume) were similar in the combined and CA-only comparison (-13.52 ± 22.72 mL vs. -16.63 ± 24.59 mL; P = 0.250), but differed significantly between the combined and LAAC-only comparison (-21.68 ± 22.22 mL vs. 9.11 ± 19.69 mL; P <0.001). Post-procedural LASP changes (△-sphericity) differed significantly between the combined and CA-only groups ([1.42 ± 4.40]% vs. [2.54 ± 4.72]%, P = 0.028), but were similar between the combined and LAAC-only groups ([1.46 ± 4.10]% vs. [0.68 ± 5.00]%, P = 0.346). Combined application of LAAC and CA was positively correlated with spherical RR (odds ratio [OR], 1.66; 95% confidence interval [CI], 1.009–2.716; P = 0.045) when taking CA as a reference. Compared with patients (92, 36.5%) exhibiting both volumetric and spherical RR, those patients (44, 17.5%) with neither volumetric nor spherical RR had a higher major adverse cardiovascular event (MACE) rate (P <0.05). Multiple logistic analysis showed that in the combined group, RR were independently associated with the TE events (OR, 0.067; 95% CI, 0.007–0.658; P <0.020), but the AF recurrence (HR, 1.570; 95% CI, 0.162–15.188; P = 0.697), age (HR, 1.082; 95% CI, 0.921–1.271; P = 0.341), or other characteristics were not associated with TE events. Preoperative LASP (B = 0.335; P <0.001), △-volume (B = 0.055; P <0.001), and combined LAAC (B = 2.373; P <0.001) interactively affected the extent of spherical RR after ablation and explained 49.0% of its variation (adjusted R2 = 0.490). The multiple linear regression equation could be written as: Y= -30.259+ 0.335 × preoperative LASP + 0.055 × △–volume+ 2.373 × combining LAAC where Y = extent of LA spherical RR (%);preoperative LASP = LASP (%); △–volume = post-ablation LAV changes (mL);and combining LAAC = 1, otherwise = 0 in the equation. We found that the addition of LAAC to the CA procedure did not affect the improvement of LAV after CA but could alleviate spheroidization from the ablation scar. Compared to LAAC alone, combined LAAC and CA resulted in a significant reduction in LAV. LA structural changes after CA-only could be explained by scar-induced retraction. Bisbal et al[1] believed that post-ablation RR is caused by scarring and myocardial structural recovery. Real RR was presented as reductions in both volume and sphericity, and the scar contraction caused by ablation played a more important role in volumetric RR only. The reduced volume after ablation might mainly locate at the PVs ridge junction to the LA, leading to a more symmetric structure of the LA. Gottlieb et al[4] discovered that the diameter of the ablated PV decreased similarly in patients with AF and healthy sheep, further supporting this hypothesis. We noticed that the probability of spherical RR after CA was 0.66 times higher than the combination of LAAC and CA. The Watchman device limited the volume reduction in the LAA ostium, which increased the irregularity of the LA. The following three structural changes in the LA may occur after different treatments: reduction in both volume and sphericity caused by real RR; volume reduction and sphericity increase caused by ablation scar; and increase in both volume and sphericity as a result of remodeling progression. Both combined therapy and LAAC alone resulted in increased sphericity through different mechanisms, leading to no significant difference in △-sphericity. And, real RR was associated with better cardiovascular outcomes after combined procedures. The symmetric structure generated less vortical flow and more areas with slow flow and stasis, facilitating the formation of thrombi, which supports the value of the LASP for non-LAA thrombogenesis risk assessment.[5] Combining LAAC and CA for AF eliminates LAA thrombosis directly and reduces the probability of cardioembolic stroke from non-LAA thrombosis by easing spheroidization caused by ablation scar. The study has some limitation. First, this was a single-center retrospective study with a cohort consisting of three different non-randomized groups and the bias may exist. We attempted to minimize the patient selection bias using PSM. Second, the number of patients and the follow-up time in this study were limited, and larger and longer studies are required to confirm these conclusions. Third, LAV and LASP measurements may differ betweem pre- and post-LAAC (sometimes the device protrudes into the LA), and the reduction of LAV and LASP may be overestimated. Compared with a single procedure, combined CA and LAAC resulted in LAV reduction with less spheroidization. The combination of LAAC, larger preoperative LASP, and more LAV reduction post-procedure were associated with more notable LASP reduction after ablation. Future cohort studies with larger sample size are needed to validate these results. Funding This work was supported by grants from the State Key Program of National Natural Science Foundation of China (No. 82130009), the National Science Foundation of China (No. 82070515), and the Clinical Research Plan (No. SHDC2020CR2026B). Conflicts of interest None.

Abnormal function of endothelial cells (ECs) is an important reason for vascular endothelial remodeling and atherosclerotic plaque formation in patients with atherosclerosis (AS). Here, we report for the first time that the vascular ECs with apoptosis resistance phenotype (ARECs) exist in peripheral blood of AS patients. Our research data showed that the switch of regulation modes between HIF-1α and Bax operated by lncRNA-ASLNC18810 is the direct cause for the formation of ARECs. When ASLNC18810 is low or missing, HIF-1α indirectly negatively regulates the Bax in post-transcription through HIF-1α/miR-559/Bax pathway which makes ECs acquire apoptosis resistance and form ARECs. The functional experiments results showed that ASLNC18810 could effectively eliminate the anti-apoptotic properties of ARECs by blocking the HIF-1α/miR559/Bax pathway and maintaining HIF-1α/Bax pathway. In a word, our study shows that ASLNC18810 has full potential to become a biological target for the prevention and treatment of atherosclerotic plaques by regulating ARECs. ASLNC18810 was significantly upregulated in ECs compared to ARECs. With high level of ASLNC18810 in ECs, ASLNC18810 binds to miR-559 as a miRNA sponge and suppresses the inhibition effect of miR-559 on Bax protein, this direct positive transcriptional regulation between HIF-1α and Bax endows the apoptotic property in ECs induced by Ox-LDL. However, with low expression of ASLNC18810 in ARECs, the post-transcriptional regulation of Bax by miR-559 dominates and the indirect negative regulation between HIF-1α and Bax endows the anti-apoptotic property of ARECs. To sum up, low ASLNC18810 expression-mediated switching of HIF-1α/Bax pathway to HIF-1α/miR-559/Bax pathway is the internal reason for ECs to obtain apoptosis resistance and the formation of ARECs under the ox-LDL induction.

Background:The progression and recurrence of pterygium mainly occur due to the abnormal proliferation and migration of stromal pterygium fibroblasts. This research explores the aberrant expression of small nucleolar RNA U3 (U3 snoRNA) in pterygium and elucidates the molecular mechanisms of U3 snoRNA in pterygium development.Methods:Primary human conjunctival fibroblasts (HCFs) and human pterygium fibroblasts (HPFs) were separated and cultured from fresh conjunctiva grafts and pterygium tissues. The PLKO.1 lentiviral system and CRISPR/Cas9 recombinant construct were, respectively, used to overexpress and silence U3 snoRNA in HPFs and HCFs for further specific phenotype analysis. RNA-seq and TMT-labeled quantitative protein mass spectrometry were utilized to evaluate the effect of U3 snoRNA on mRNA transcripts and protein synthesis.Results:Reduced U3 snoRNA in pterygium promotes HCF or HPF cells' proliferation, migration, and cell cycle but has no significant effect on apoptosis. U3 snoRNA modulates 18S rRNA synthesis through shearing precursor ribosomal RNA 47S rRNA at the 5' external transcribed spacer (5' ETS). Moreover, the altered U3 snoRNA causes mRNA and protein differential expression in HCF or HPF cells.Conclusions:The atypical U3 snoRNA regulates the translation of specific proteins to exert a suppressive function in pterygium through modulating the 18S rRNA synthesis. Here, we uncover a novel insight into U3 snoRNA biology in the development of pterygium.

The activation of activin receptor-like kinase 4 (ALK4) signaling plays a pivotal role in the pressure-overloaded heart, and haplodeficiency of ALK4 can alleviate cardiac fibrosis secondary to myocardial infarction and preserve cardiac function through partially inactivating the Smad3/4 pathway. However, whether transforming growth factor (TGF) β signaling is involved in the beneficial effects of ALK4 knockdown on the ischemic heart is still unclear. This study was undertaken to investigate the change in the TGFβ signaling after ALK4 knockdown in vivo and in vitro. Forty C57BL/6J mice were randomized into ALK4+/- ischemia/reperfusion (I/R) group (ALK4+/-+I/R, n = 10 ), ALK4+/- sham group (ALK4+/-+sham, n = 10 ), wild-type sham group (WT+sham, n = 10 ), and WT I/R group (WT+I/R, n = 10 ). Heart histology and the levels of cytokines related to antioxidant and inflammation, as well as protein and mRNA expressions of molecules associated with TGFβ pathway, were examined in different groups. Our results showed that the reduction of ALK4 expression ameliorated myocardial I/R injury through inhibiting TGFβ signaling pathway. Our findings indicate that ALK4 may become a novel target for the therapy of myocardial I/R injury.