Citations

AbstractBackgroundFerroptosis is an important iron‐dependent form of cell death in hepatocellular carcinoma (HCC). Sorafenib, a potent ferroptosis inducer, is used to treat advanced HCC but its efficacy is limited by the development of drug resistance.MethodsThe effects of DUXAP8 expression on HCC progression were evaluated by TCGA database, Kaplan‐Meier analysis, and in situ hybridization analysis. Sorafenib resistant HCC cell lines were modeled in vitro to study the regulation of DUXAP8 on ferroptosis in HCC induced by sorafenib. We used RNA pull‐down, immunofluorescence assays, acyl‐biotinyl exchange assay and mass spectrometry analysis to assess the molecular mechanism of ferroptosis regulation by DUXAP8. Syngeneic subcutaneous and orthotopic CDX models were used to assess whether DUXAP8 inhibition improves HCC in vivo.ResultsLncRNA DUXAP8, which is highly expressed in liver cancer and associated with poor prognosis, contributes to sorafenib resistance through suppression of ferroptosis. In vitro tests revealed that DUXAP8 reduced the sensitivity of HCC to sorafenib‐induced ferroptosis by acting on SLC7A11, a subunit of the amino acid antiporter system xc‐. DUXAP8 facilitates SLC7A11 palmitoylation and impedes its lysosomal degradation, thereby enhancing SLC7A11 action and suppressing ferroptosis. RNA pull‐down and immunofluorescence assays confirmed that DUXAP8 decreased membrane translocation and promoted sorting of de‐palmitoylated SLC7A11 to lysosomes by binding of DUXAP8 to SLC7A11. In addition, mass spectrometric analysis found that the Cys414 residue of SLC7A11 might be the predominant mutant site responsible for molecular masking of SLC7A11 lysosomal sorting. Further, the antitumor effect of DUXAP8 knockdown was verified in orthotopic and subcutaneous CDX models.ConclusionsOur findings suggest that a novel translational strategy combining sorafenib with DUXAP8 silencing to overcome drug resistance may improve treatment efficacy in patients with advanced HCC.

Platinum-containing stents are commonly used in humans with hypercholesterolemia, whereas preclinical stent evaluation has commonly been performed in healthy animal models, providing inadequate information about stent performance under hypercholesterolemic conditions. In this investigation, we used an ApoE−/− mouse model to test the impact of hypercholesterolemia on neointima formation on platinum-containing implants. We implanted 125 μm diameter platinum wires into the abdominal aortas of ApoE−/− and ApoE+/+ mice for 6 months, followed by histological and immunofluorescence examination of neointimal size and composition. It was found that ApoE−/− mice developed neointimas with four times larger area and ten times greater thickness than ApoE+/+ counterparts. Neointimas developed in the ApoE−/− mice also contained higher amounts of lipids quantified as having 370 times more coverage compared to ApoE+/+, a 3-fold increase in SMCs, and a 22-fold increase in macrophages. A confluent endothelium had regenerated in both mouse strains. The ApoE−/− mice experienced luminal reductions more closely resembling clinically relevant restenosis in humans. Overall, the response to platinum arterial implants was highly dependent upon the atherogenic environment.

AbstractBackground: Liver transplant (LT) recipients with untreated hepatitis C (HCV) are at risk for cirrhosis graft failure. The advent of direct acting antiviral agents (DAA) has improved outcomes in HCV.Aims: We aim to examine liver transplant outcomes and allograft fibrosis development/progression after sustained virologic response (SVR).Methods: We performed a retrospective cohort study of 226 consecutive liver transplant recipients with HCV from 2007 to 2018. The cohort was split into transplants pre (Group A) and post (Group B) 2014 to reflect the introduction of DAAs. Fibrosis was monitored with liver biopsy and non‐invasive imaging.Results: Group B had significantly improved HCV treatment rates and earlier SVR compared to Group A, with a cumulative incidence rate of SVR at 2 years of 86.7% versus 15.4% (HR = .11, p < .001). Prior to achieving SVR, Group A demonstrated worsening of fibrosis stage per year (+.21, p < .001) whereas Group B showed minimal change on protocol annual biopsy (−.02, p = .80). After SVR, most patients were followed non‐invasively and demonstrated stable or improved fibrosis stage over time. Patients undergoing transient elastography showed regression in fibrosis stage per year (−.19, p < .001).Conclusion: HCV patients undergoing LT after 2014 had higher rates of SVR and improved clinically relevant transplant outcomes, namely less graft loss and death relating to HCV. Fibrosis progression halted or improved after SVR in both cohorts, suggesting that LT recipients with SVR do not require fibrosis monitoring even with established fibrosis prior to SVR.

AbstractBackgroundGene expression changes have been identified in post‐mortem brain tissue of AD cases when compared with controls, implicating various genes and pathways. Such findings provide important insights into molecular dysregulation associated with this disease and nominate therapeutic targets. Here we investigate brain gene and co‐expression network changes in a cohort of AD cases to identify associations with degree of AD neuropathology including Braak stage (Tau), Thal phase (Amyloid beta) and brain tissue levels of five AD‐related proteins (apoE, Aβ40, Aβ42, tau, and p‐tau).MethodGene expression measures were collected from temporal cortex (TCX) tissue of 477 AD cases, available through the Mayo Clinic Brain Bank, using RNA sequencing (Illumina TruSeq mRNA) performed on the HiSeq4000. Following quality control 456 cases remained for analysis. Braak and Thal were provided by an experienced neuropathologist and TCX levels of five AD‐related proteins from three fractions (buffer‐soluble, detergent‐soluble, and insoluble) were measured by ELISA (Liu et al. 2020). Weighted gene coexpression network analysis (WGCNA) R package was used to build co‐expression networks. Genes and networks were tested for association with neuropathology and AD‐related protein levels, adjusting for relevant covariates.ResultA total of 57,353 genes were profiled, of which 19,044 were reliably expressed above background levels and formed 20 co‐expression modules. 36 genes were associated with Braak stage, 10 with Thal phase and up to 9,000 with the AD‐related protein fractions. Brain levels of pTau had the greatest number of associations, with an overrepresentation of genes involved in synaptic signalling and immune response gene ontology biological processes. 16 of the coexpression modules associated with at least one trait after Bonferroni correction for number of tests.ConclusionWe have identified a number of genes and networks associated with different aspects of AD neuropathology and associated proteins, further lending support to the involvement of synaptic and immune biology in AD. However it is not clear whether observed changes cause disease or are a consequence of the neurodegenerative process. Comparison of these findings across different proteins and fractions is expected to reveal common and distinct vulnerabilities associated with amyloid and tau, and different stages of protein aggregation.

Abstract The recycling of membrane proteins from endosomes to the cell surface is vital for cell signaling and survival. Retriever, a trimeric complex of VPS35L, VPS26C and VPS29, together with the CCC complex comprising CCDC22, CCDC93, and COMMD proteins, plays a crucial role in this process. The precise mechanisms underlying Retriever assembly and its interaction with CCC have remained elusive. Here, we present the first high-resolution structure of Retriever determined using cryogenic electron microscopy. The structure reveals a unique assembly mechanism, distinguishing it from its remotely related paralog, Retromer. By combining AlphaFold predictions and biochemical, cellular, and proteomic analyses, we further elucidate the structural organization of the entire Retriever-CCC complex and uncover how cancer-associated mutations disrupt complex formation and impair membrane protein homeostasis. These findings provide a fundamental framework for understanding the biological and pathological implications associated with Retriever-CCC-mediated endosomal recycling.

Introduction: Percutaneous left atrial appendage occlusion (LAAO) has been rapidly evolving since FDA’s approval in 2015, and has become more of a same-day-discharge procedure. Cancer patients who underwent LAAO are known to have more inpatient complications and longer hospital stay. Readmission data, however, were rarely studied in this patient population. Hypothesis: Readmission burden is relatively high in cancer patients who received LAAO. Methods: Data were derived from the National Readmission Database from 2016 to 2019. Patients with primary diagnosis of atrial fibrillation or flutter admitted for percutaneous LAAO (ICD-10 code 02L73DK) were grouped by cancer as a secondary diagnosis. We assessed inpatient mortality, length of stay, total charges and procedure complications from index hospitalization. We also compared readmission rate and most common readmission reasons. Stata SE 17.0 was used for data analysis. Results: There were 49882 index hospitalizations for LAAO, among which 1545 (3.1%) patients had a secondary diagnosis of cancer. From index hospitalizations, Patients with cancer had higher inpatient mortality (0.65% vs 0.14%, p=0.007), longer length of stay (1.54±0.08 vs 1.31±0.01, p<0.001) and higher total charges (120.25±3.44 vs 117.29±2.40, p<0.001). There was no statistical significance of 30-day readmission rate between patients with cancer and patients without cancer (10.0% vs 9.1%, p=0.34). The most common readmission reason in cancer patients was gastrointestinal bleeding. Conclusions: Readmission utilization is comparable between patients with cancer and patients without cancer who underwent LAAO.

Introduction: Hypoplastic left heart syndrome (HLHS) is a severe form of single ventricle congenital heart disease characterized by the underdeveloped left ventricle. Early serial postmortem examinations revealed a high rate of coronary artery abnormalities in HLHS fetal hearts (e.g., thickened wall and kinking arteries). However, the intrinsic defect in HLHS coronary vessels and its genetic basis remain unclear. Methods: We profiled human fetal heart with an underdeveloped left ventricle (ULV) and induced pluripotent stem cells derived endothelial cells (iPSC-ECs) from HLHS patients at single-cell resolution. CD144 + / NPR3 - vascular ECs were selected and classified as venous, arterial, and late arterial populations. To study the arterial EC phenotypes, we generated iPSC-arterial ECs (AECs, CDH5 + CXCR4 + NT5E -/low ) derived from 3 HLHS patients and 3 age-matched controls, and evaluated their functionalities including cell cycle regulation, angiogenesis, and inflammatory response. Results: Revealed by single cell RNA-seq and subsequent gene ontology analysis, ULV late arterial EC population showed significant defects in EC development, proliferation, angiogenesis, and Notch signaling compared to the control. Consistently, HLHS iPSCs exhibited impaired AEC differentiation judged by the reduced CXCR4 + NT5E -/low AEC progenitors. Mature HLHS iPSC-AECs showed reduced angiogenesis and enhanced G0/G1 cell cycle arrest with downregulated cell cycle-related genes (e.g., Ki67, CCND1/2 ). Healthy human aortic smooth muscle cells exhibited abnormal proliferation and synthetic phenotypes when co-cultured with HLHS iPSC-AECs. Additionally, NOTCH pathway genes (e.g., DLL4, HEY1, GJA5 ) were suppressed in both ULV AECs and HLHS iPSC-AECs. HLHS de novo variant KMT2D directly regulated the transcription of NOTCH targeted genes involved in arterial development and proliferation via H3K4me2. Intriguingly, the treatment of NOTCH ligands (Jag1, Dll1) significantly improved the proliferation of HLHS AECs. Conclusions: Our study revealed that HLHS coronary AECs were dysfunctional in angiogenesis, proliferation, and EC-SMC interaction. KMT2D-NOTCH signaling may contribute to the impaired development and proliferation of HLHS AECs.

Introduction: Over 100 FDA-approved medications, electrolyte perturbations, and many disease states can prolong the QTc beyond its 99 th percentile value resulting in acquired QT prolongation. In contrast, approximately 1 in 2000 people have congenital long QT syndrome (LQTS) hallmarked by pathological QT prolongation secondary to genetic defects in the heart. Hypothesis: An artificial intelligence (AI) deep neural network (DNN) can distinguish patients with LQTS from those with acquired QT prolongation. Methods: The study cohort included all patients with LQTS evaluated in the Windland Smith Rice Genetic Heart Rhythm Clinic and controls from Mayo Clinic’s ECG data vault comprising over 2.5 million patients. For the AI-DNN model, every patient/control with ≥ 1 ECG above age- and sex- specific 99 th percentile values for QTc [> 460 ms for all patients (male/female) < 13 years of age, or > 470 ms for men and > 480 ms for women above this age] was included. An AI-DNN involving a multi-layer c onvolutional n eural n etwork (CNN) was developed to classify patients. LQTS patients were age- and sex- matched to controls at 1:5 ratio. Results: Among the 1,599 patients with genetically confirmed LQTS, 808 had ≥ 1 ECG with QTc above the threshold (2,987 ECGs) compared to 361,069/2.5M controls (14% of Mayo Clinic patients getting an ECG, ‘presumed negative’; 989,313 ECGs). Following age- and sex- matching and splitting, 3,309 (training), 411 (validation) and 887 (testing) control ECGs were used. This model distinguished LQTS from those with acquired QT prolongation with an AUC of 0.896 (accuracy 85%, sensitivity 77%, specificity 88%, PPV 0.58, NPV 0.94). After exclusion of patients with a wide QRS (>150 ms) or pacemaker, the model remained successful in distinguishing the two groups (AUC 0.85, accuracy 78%, sensitivity 78%, specificity 87%, PPV 59%, NPV 94%). Conclusions: For patients with a QTc exceeding its 99 th percentile values, this novel AI-DNN functions as a LQTS mutation detector being able to identify patients with abnormal QT prolongation secondary to a LQTS-causative mutation rather than acquired QT prolongation with a >50% positive predictive value. This algorithm may facilitate screening for this potentially lethal, yet highly treatable, genetic heart disease.

Introduction: Genome-wide association studies have identified common genetic variants associated with hypertrophic (HCM) and dilated cardiomyopathy (DCM) and left ventricular (LV) characteristics. However, the association of these variants with myocardial fibrosis (MF), an important pathophysiological mediator of cardiomyopathy, is unknown. Hypothesis: We used the Multi-Ethnic Study of Atherosclerosis (MESA), to evaluate the association of these loci with interstitial MF measured by cardiac MRI (CMR). Methods: Unrelated MESA participants with T1-mapping CMR and no history of MI or heart failure were included (n=1,235). We used the extracellular volume (ECV) fraction as a well-established surrogate of interstitial MF. Genotyping was performed by Affymetrix Human SNP array 6.0, and imputation was performed using the TOPMED reference panel. Log ECV(%) was regressed on 50 candidate SNPs, in each ethnicity/race group, adjusting for age, sex, and population stratification principal component. The race-specific results were pooled by fixed-effect meta-analyses using METASOFT. Results: The SMARCB1 rs2186370 intronic variant, previously identified as a risk variant for DCM and HCM, was associated with ECV (p=0.0004). The rs2186370 A allele (frequency: 0.18 in Whites, and 0.50 in African Americans) was associated with a 1.6% increase in ECV. The rs2070458 A allele (another SMARCB1 intronic variant), in complete linkage disequilibrium with rs2186370, was known previously to be associated with increased LV wall thickness, mass, and concentricity. The direction of effect was similar in all four ancestry groups, but the effect was strongest in African Americans. These variants are strong expression quantitative loci (eQTLs) in human LV tissue and are associated with genotype-dependent expression of SMARCB1 ( p =7.3х10 -22 ) and MMP11 ( p =2.5х10 -24 ). Conclusions: In conclusion, variants in two linked SMARCB1 loci previously associated with HCM, DCM and LV traits, are associated with CMR-based interstitial MF. These loci are eQTL not only for SMARCB1 but also for MMP11 an important mediator of MF . These findings underscore the important link between MF and CM, and potentially provide novel mechanistic targets in understanding the development of CM.

Introduction: It has been previously demonstrated that cardiomyopathy predisposing common genetic variation in SMARCB1 is associated with interstitial myocardial fibrosis (MF) as determined by cardiac MRI (CMR) linking the development of cardiomyopathy with MF. SMARCB1 is known to be a tumor suppressor, but it’s role in the pathophysiology of MF and fibroblast physiology has not been functionally validated. Hypothesis: SMARCB1 plays an important role in regulating human cardiac fibroblast (HCF) differentiation/activation and collagen deposition, which are the critical steps in the development of MF. Methods: Cultured HCFs were treated with TGF-β1 (10ng/ml) for 48hrs to transdifferentiate into myofibroblasts, which are characterized by increased expression of α-smooth muscle actin (α-SMA, ACTA2) and collagen I (COL1A1). HCFs were transfected by siRNA using Lipofectamine™ RNAiMAX Transfection Reagent to knock-down (KD) the expression of SMARCB1 . Total RNA and protein were extracted from cell lysate followed by qPCR and Western blot (WB) to quantify the expression level of SMARCB1, ACTA2 and COL1A1. Triplicate experiments were performed using biologically independent samples. For statistical analysis, a paired two-tail t-test was used. Results: HCFs treatment with TGF-β1, resulted in a significant decrease in expression of SMARCB1 (p=0.031) with accompanying significant increase in expression of ACTA2 (p=0.025) and COL1A1 (p=0.0015) as compared to control by qPCR and WB. SMARCB1 KD resulted in a further significant increase in expression of ACTA2 (p=0.018) and COL1A1 (p=0.028) as compared to non-target siRNA control group at both the transcript and protein level. Conclusion: We demonstrate for the first time that a gene associated with the development of cardiomyopathy SMARCB1 mediates TGF-β1 induced cardiac fibrosis. These findings suggest SMARCB1 as a potential therapeutic target to attenuate MF in cardiomyopathy.