bluuebunny/crossref_metadata_2025 huggingface dataset:
A Maltese landscape of cardiac genetics
Authors: Abela M , Schembri J , Scerri J , Debattista J , Felice T , Caruana M , Sammut M A , Said E , Scerri C
Published: November 2023

Introduction

Advances in human genetics have improved our understanding of various inherited cardiovascular conditions (ICC). Genetic testing is now an important pillar in the management of patients suffering from cardiomyopathies, channelopathies and aortic and other vascular disorders. Existing population databases may not cater for the local gene pool. The local genetic yield of ICCs has never been explored.

Objective

The aim of this study was to evaluate the population referred for genetic testing and to assess the genetic yield according to the referring phenotype.

Methodology

Probands referred for genetic testing between 2018-2022 were included. Data was retrospectively reviewed in close liaison with the molecular genetics lab at a university. Variants classified as pathogenic or likely pathogenic by ACMG criteria were labelled as ‘gene positive’ for the purpose of this study.

Results

328 patients were referred for genetic testing (63.7% Male, 51.3±18.7 years). The majority were referred because of a cardiomyopathy phenotype (64.02%) followed by arrhythmic disorders (20.7%), aortic/vascular disorders (12.5%), familial hyperlipidaemia (1.5%) and complex congenital heart disease (0.6%). Two (0.6%) did not have a reason specified.

94 (28.7%) probands were gene positive, 5 patients (1.5%) had two mutations, 41.4% of all mutations were novel. The highest genetic yield was seen in cardiomyopathy patients (34.8%), followed by aortic/vascular disorders (24.4%) and arrhythmic disorders (14.7%).

A separate analysis was also performed for each individual phenotype (Table 1). The dominant genes for each condition: MYH7 (24.4%) and MYBPC3 (22.0%) for hypertrophic cardiomyopathy; TTN (52.9%) and FLNC (23.5%) for dilated cardiomyopathy; DSG2 (45.5%) and FLNC (18.2%) for arrhythmogenic cardiomyopathy; TTN (40.0%) and MYH7 (40.0%) for left ventricular non-compaction cardiomyopathy; TTR (100%) for cardiac amyloid; SCN5A (37.5%) for non-specific cardiomyopathy; KCNH2 (50.0%) and KCNQ1 (50.0%) for long QT syndrome; SCN5A (37.5%) for Brugada Syndrome; FBN1 (40.0%) for skeletal phenotype; FBN1 (33.3%) for aortic phenotype; and LDL-R (100.0%) for familial hyperlipidaemia

Conclusion

The overall ICC genetic yield in Malta is 28.7%. A substantial proportion were novel mutations. The highest genetic yield is in the cardiomyopathy group. The yield for most phenotypes is comparable to other labs. The proportion of gene positive LQTS patients is surprisingly very small, a phenotype with an established high genetic yield in various studies (up to 80%). Clinicians caring for patients with ICCs now have local diagnostic yield data to better counsel patients when referred for genetic testing.
Prognostic value of modified ADMIIRE-HF risk score in patients with implantable cardioverter defibrillator: a prospective comparative study with 6-minute walk distance
Authors: Kawasaki M , Yamada T , Watanabe T , Morita T A K A S H , Kikuchi A , Kondo T , Kawai T , Nishimoto Y , Nakamura J , Fujita T , Chang Y , Taniichi M , Oshita T , Fukunami M
Published: November 2023

Background

ADIMIRE-HF risk score is a novel risk score that combines clinical characteristics and MIBG imaging variables to predict serious arrhythmic risk in patients with chronic heart failure (CHF) and recent studies showed that highest occurrence of severe arrhythmic events (SAE) was seen in patients with an intermediate decrease in MIBG uptake rather than the lowest values. Six-minute walk distance (6MWD) has also been shown to detect high risk patients with CHF. However, there is no information available on the long-term prognostic value of these indices for the prediction of ventricular tachyarrhythmias in patients with implantable cardioverter defibrillator (ICD).

Methods and Results

We prospectively enrolled 231 consecutive outpatients with ICD (age: 65±14 years, male: 81%, NYHA class: 1.7±0.7, LVEF: 47±17%). At entry, cardiac MIBG imaging, echocardiography and 6-minute walk test were performed. Modified ADMIRE-HF risk score was derived from the sum of the point values of the following parameters: LVEF (<25%:5 points), MIBG heart-to-mediastinum ratio on delayed planar image (HMR 1.4-1.9: 12 points) and systolic blood pressure (<120: 3 points, 120-139: 0 point, >140: -3 points). During a follow-up period of 4.4±2.8 years, 72 patients had appropriate ICD therapy (ATx). At multivariate Cox analysis, modified ADMIRE-HF risk score and 6MWD were significantly and independently associated with ATx after adjustment of age, sex, non-sustained ventricular tachycardia and NYHA functional class. ATx was significantly more frequently observed in patients with both higher modified ADMIRE HF risk score (>9: median value) and shorter 6MWD (<300m) and with either, than with none of them (54% vs 34% vs 18%, respectively p<0.0001).

Conclusion

Combination of modified ADMIRE-HF risk score and 6MWD would be strongly associated with an increased risk for ventricular tachyarrhythmias in ICD patients.
Large scale genome-wide association analyses identify novel genetic loci and mechanisms in hypertrophic cardiomyopathy
Authors: Tadros R , Zheng S L , Grace C , Jorda P , Francis C , Jurgens S J , Thomson K L , Harper A R , Wilde A A , Olivotto I , Adler A , Goel A , Ware J S , Bezzina C R , Watkins H
Published: November 2023

Background

Hypertrophic cardiomyopathy (HCM) is an important cause of morbidity and mortality with both monogenic and polygenic components. Prior genome-wide association studies (GWAS) identified few genomic loci and disease genes due to limited sample size.

Purpose

To discover novel genetic loci, genes and mechanisms implicated in HCM using a large scale GWAS and multi-trait analysis of GWAS (MTAG).

Methods and results

We performed the largest HCM GWAS meta-analysis and MTAG to date including 5,900 HCM cases, 68,359 controls, and 36,083 UK Biobank (UKB) participants with cardiac magnetic resonance (CMR) imaging. We estimated the heritability of HCM attributable to common genetic variation (h2SNP) to be 0.25±0.02 using genome-based restricted maximum likelihood (GREML), with higher h2SNP in non-sarcomeric (0.29±0.02) compared to sarcomeric HCM (0.16±0.04). We identified a total of 70 loci (50 novel) associated with HCM (Figure 1), and 62 loci (32 novel) associated with relevant left ventricular (LV) structural or functional traits. Amongst the common variant HCM loci, we identify a novel HCM disease gene, SVIL, which encodes the actin-binding protein supervillin. We performed rare variant burden analysis including 1,845 clinically-diagnosed unrelated HCM cases and 37,481 controls and demonstrated a 10.5-fold (95% CI: 4.1-26.8; P=0.0000002) excess burden of SVIL loss of function (LoF) variants in HCM cases. Two-sample mendelian randomization analyses using LV contractility as exposure and obstructive (oHCM) and non-obstructive HCM (nHCM) as outcomes support a causal role of increased LV contractility in both oHCM and nHCM (Figure 2), suggesting common disease mechanisms and anticipating shared response to therapy.

Conclusion

We identify 50 novel genomic loci associated with HCM. Our data suggest that LoF variants in SVIL are a cause of HCM, and that increased contractility mediate both nHCM and oHCM. Taken together, the findings significantly increase our understanding of the genetic basis and molecular mechanisms of HCM, with potential implications for disease management.Figure 1Figure 2
Porphyromonas gingivalis systemic infection is associated with calcific aortic valve stenosis through IL-1-dependent signaling pathways
Authors: Zhang E , Meng Z , Wang C , Xu C S , Wang M , Natarajan R , Wu Y
Published: November 2023

Background

Calcific aortic valve stenosis (CAVS) is characterized by inflammation mediated valvular calcification. Porphyromonas gingivalis, a keystone pathogen for periodontitis, has been associated with various chronic inflammatory diseases. We assessed the impact of intravenous inoculation with P. gingivalis on the progression of CAVS.

Methods

Wide-type (WT) C57BL/6, ApoE deficient (ApoE-/-), and IL1B deficient (IL-1β-/-) mice were fed with normal chow or high-fat diet (HFD). A series of metagenomic, morphometric, and molecular signaling experiments, including 16S rRNA gene sequencing, RNA sequencing (RNA-Seq), quantitative real-time polymerase chain reaction (qPCR), immunostaining, and western blotting, were conducted. Valvular calcification and aortic stenosis were evaluated by Alizarin red staining, and mice echocardiography, respectively.

Results

By using 16S rRNA gene sequencing and qPCR, we found that P. gingivalis was the 3rd most significantly enriched bacterial species in aortic valves from CAVS patients compared with controls. Morphometric assays showed predominant P. gingivalis infiltration inside of human aortic valves. Intravenous inoculation of P. gingivalis in either WT or ApoE-/- mice resulted in increased bacterial infiltration in aortic valves, irrespective of chow- or HFD. Alizarin red staining and echocardiographic evaluation further revealed that P. gingivalis bacteremia markedly enhanced valvular calcification, leading to hemodynamically significant aortic stenosis in the murine models, which were ameliorated by intraperitoneal metronidazole prophylaxis. RNA-Seq and qPCR validation showed that the gene expression of IL1B, RUNX2, and BMP2 was upregulated in P. gingivalis-stimulated valvular interstitial cells (VIC). Similar changes on IL1B were observed in aortic valves form P. gingivalis-challenged WT and ApoE-/- mice, and in valvular specimens from CAVS patients. qPCR and Alizarin red staining showed that IL1B knockdown decreased P. gingivalis-induced RUNX2 and BMP2 expression, and attenuated calcification in VICs. Consistently, P. gingivalis-induced changes on bacterial infiltration, valvular calcification, and hemodynamically significant aortic stenosis were markedly blunted in IL-1β-/- mice. Furthermore, the level of serum IL-1β and SAA were increased in response to P. gingivalis challenge in ApoE-/- mice, but not in WT or IL-1β-/- mice.

Conclusions

This study demonstrated, for the first time, that P. gingivalis infiltration in aortic valves promoted IL-1β-mediated local inflammation and valvular calcification, thus resulting in the progression of CAVS.Central Illustration: Pg promotes CAVS
A novel mouse model for mineral homeostasis disrupting induced calcific aortic valve stenosis
Authors: Qian N J , Liu X B , Wang J A
Published: November 2023

Background

Calcific aortic valve disease (CAVD) is the most common valve disease that has been occurring with rapidly increasing morbidity. Chronic kidney disease (CKD) which disrupts mineral homeostasis due the impaired renal function, is one of the known factors, largely contributing to risk of CAVD, independent of atherosclerosis. To the best of our knowledge, there are few calcific aortic valve stenosis mouse models simulating conditions under CKD that develop hemodynamically significant stenosis as well as calcification,

Purpose

The purpose was to assess the feasibility to develop a mouse model of CAVD considering risks under CKD.

Methods

For this model, C57/BL6 mice were firstly underwent aortic valve wire injury by spring guidewires inserting into left ventricle via right common carotid artery. Vitamin D was subcutaneous injected on 3 consecutive days from the second day of aortic valve wire injury. Echocardiography was performed to evaluate aortic valve function at 7, 14 and 28 days. Then the mice were sacrificed and aortic valves were collected to make histological analysis.

Results

An obvious increase in aortic velocity was already observed at 7 days after injury by continuous echocardiographic assessment and remained stable from 14 days compared with sham mice. Alizarin red staining showed calcium nodules formation in valve leaflets 14 days after injury and became more conspicuous at 28 days. Aortic valve thickening, increased macrophage infiltration, fibrosis and remarkable osteogenic markers were found in aortic valves at 28 days after injury.

Conclusions

We develop a novel mouse model of CAVD, especially simulating risks under CKD and mineral homeostasis disrupting. This model may be a convenient and valuable tool to investigate the mechanism of CAVD and further explore potential therapeutic strategies.Fig1.Alizarin red staining aortic valvesFig2.Immunostaining of aortic valves
Valvular presence of coagulation factor XI correlates with the severity of aortic stenosis
Authors: Kopytek M , Zabczyk M , Undas A , Natorska J
Published: November 2023

Background

The role of coagulation system as a factor involved in the progression of aortic stenosis (AS) has been proven. Considering recent studies about oral factor XIa (FXIa) inhibitor – asundexian with antithrombotic efficacy, our aim was to investigate whether FXI is present within stenotic leaflets in patients with severe AS and whether its expression correlates with valvular calcification.

Methods

We recruited 20 patients aged 66 ±9 years with severe AS defined as mean transvalvular pressure gradient (PGmean) ≥ 40mmHg, peak transvalvular velocity (Vmax) of ≥4.0 m/s and aortic valve area (AVA) <1 cm2. Stenotic aortic valves were obtained during valve replacement surgery. Aortic leaflets obtained from AS patients and from age-matched apparently healthy autopsy donors (n=3) were used to evaluate valvular expression of FXI, FIX, tissue factor (TF) and bone morphogenetic protein 2 (BMP2) by single and double-labeled immunostaining. The immunopositive valve area was computed as the ratio (%) of positively and negatively stained areas. The Ethical Committee approved the study and all participants provided written informed consent.

Results

AS patients with median PGmean of 54 [46-67] mmHg, Vmax of 4.5 [4.4-5.0] m/s and AVA of 0.8 [0.7-0.9] cm2 were treated with angiotensin converting enzyme inhibitors (80%), beta-blockers (90%), statins (70%) and acetylsalicylic acid (80%). In loco analysis revealed valvular expression of FXI within all studied stenotic valves, but not within control ones (Figure 1 A and B). The FXI-immunopositive valve area constituted 21.4±4.5% of the total leaflet area, while these for FIX and TF were 17.2 ±3.8% and 26.5±5.1%, respectively. The mean BMP2-positive area was 22.9±4.1%. The expression of studied proteins was observed at the aortic side of the stenotic leaflets and presented a condensed pattern of fluorescence. Moreover, the expression of FXI co-expressed with FIX in 76% and TF in 71%, suggesting local activation of FXI (Figure 1 D and E). Moreover, FXI co-expressed with BMP2 in 83% (Figure C), which may indicate that FXI activation occurs at the site of leaflet calcification. Interestingly, valvular amounts of FXI correlated with AS severity measured as PGmean (r=0.49, p=0.03), PGmax (r=0.53, p=0.02), Vmax (r=0.54, p=0.01) and AVA (r=-0.53, p=0.02).

Conclusions

We are the first to show the valvular presence of FXI, co-expressing with FIX and TF, which indicates FXI in loco activation. A strong co-expression of FXI with BMP2 and associations with disease severity highlight the impact of coagulation on valve calcification. It is tempting to speculate that FXIa inhibition could attenuate not only coagulation activation but also valvular calcification and thus retard AS progression. Clinical relevance of these findings requires further studies. This work was supported by the Polish National Science Centre (UMO-2021/41/N/NZ5/03323).Figure 1_ESC_2023
Viral infection causes long term changes of the immunological milieu within the heart
Authors: Sustic M , Cokaric Brdovcak M , Materljan J , Jonjic S
Published: November 2023

Background

The last two decades have brought plentiful insights and new advances in understanding virus-host interactions, and the importance of acute and latent viral infections in the modulation of the structure and function of the mammalian immune system became apparent. Despite abundant evidence demonstrating substential changes after viral infections in the immune cell composition and the local immune response within various organs such as the lungs and the gut, the same phenomena within the heart tissue has remained poorly investigated.

Methods

C57BL6/J 6–12-week-old female mice were infected intravenously with murine cytomegalovirus (MCMV) or left uninfected. Kinetics of viral replication within the heart were determined using a standard plaque assay. Furthermore, immune cell composition within the heart was determined by flow cytometry during acute and latent viral infection.

Results

Active MCMV replication within the heart peaked at day 5 post-infection (p.i.) and the replicating viral particles became undetectable by day 12 in all of the infected animals. The initial immune cell infiltration was led by the increase in the NK cell compartment, followed by a substantial increase in the T cell compartment which peaked around day 7 p.i. Furthermore, there were substantial phenotypic changes in the population of cardiac macrophages which became predominantly MHCIIhigh and Ly6Chigh. Most notable was an enlargement within the CD8 T cell population which increased around 60-fold compared to the steady state conditions. Crucially, the changes in the immune cell milieu were maintained long after cessation of active viral replication as the hearts of latently infected mice contained around 2.5 times more leukocytes (CD45+) than the hearts of non-infected animals, around 18% percent of which were CD8 T cells, compared to 5% observed in the mock-infected group.

Conclusion

Systemic viral infection with a ubiquitous herpesvirus causes long term alterations in the composition and phenotype of the heart immune cells which could have major consequences for myocardial coping with different pathological injuries such as a subsequent microbial infection, ischemic conditions or stress induced by hypertension or volume overload.
Single-cell RNA-sequencing to interrogate TCR-Repertoire and antigen-specificity in human atherosclerosis
Authors: Horstmann H , Anto-Michel N , Kueckelhaus J , Hansen S , Marchini T , Olawale T , Gissler C , Heiland H , Maerz W , Westermann D , Zirlik A , Wolf D
Published: November 2023

Background

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of distinct immune cells and lipids in the arterial wall. Despite recent advances in immunophenotyping, the molecular characteristics of T cells and their T cell receptor (TCR) repertoire in patients with atherosclerosis remain elusive. Studies using bulkRNA-sequencing of the TCR in both mice and humans have suggested a significant proportion of T cells in atherosclerotic plaques arises from a small number of antigen-specific T cells that express a limited TCR repertoire. However, it is unknown whether this restriction of TCR usage is caused by CD8+ or CD4+ T cells.

Methods

Here, we performed paired Single-cell RNA-sequencing (scRNAseq) and TCR-sequencing on atherosclerotic plaques (n=8) and peripheral blood mononuclear cells (PBMCs) from patients with and without coronary artery disease (CAD: n=31, noCAD: n=15). To analyze the paired scRNAseq and TCR sequencing data, we developed a bioinformatic tool that enables the identification of clonally expanded T cells in scRNAseq datasets. This tool allowed to investigate the T cell response in human atherosclerosis at a single-cell level and link clonal expansion with specific gene expression.

Results

Our analysis revealed that CD8+, rather than CD4+ T cells, exhibit an oligoclonal phenotype in the atherosclerotic plaques. By applying bioinformatic pseudotime analysis, we were able to recapitulate the developmental relationships within the T cell compartment. Our results demonstrate that clonally expanded CD8+ T cells show a TEMRA phenotype (Effector Memory–Expressing CD45RA) and originate from naive T cells within the plaque microenvironment. Pathway analyses of enriched CD8+ Plaque TEMRAs revealed increased signalling for cellular activation and migration, response to stress, and inflammation. In addition, clonally expanded CD8+ cells were also detected in PBMCs from patients with CAD and appear to be overrepresented in comparison to patients without CAD.

Conclusion

Our study provides new insights into the T cell response in human atherosclerosis. We demonstrate that CD8+ T cells show an oligoclonal phenotype in the atherosclerotic plaque and are also present in PBMCs from patients suffering from CAD. Furthermore, these cells appear to be dysregulated, suggesting a possible mechanism by which they contribute to disease progression. These findings highlight the power of paired scRNAseq and TCR sequencing which identify dysregulated genes and pathways in clonally expanded T cells, providing new insights into their potential role in atherosclerosis.
Association of ventricular tachycardia morphology induced on electrophysiology study after myocardial infarction: long-term survival and ventricular tachyarrhythmia recurrence
Authors: Deshmukh T , Kovoor J , Byth K , Zaman S , Chow C , Chong J , Thiagalingam A , Kovoor P
Published: November 2023

Background

Electrophysiology study (EPS) presents a valuable risk stratification tool to facilitate the implantation of implantable cardioverter-defibrillators (ICDs) to prevent sudden cardiac death from ventricular tachyarrhythmia early after myocardial infarction.

Purpose

To investigate long-term survival of patients with inducible ventricular tachycardia (VT) at electrophysiology study (EPS) early after ST elevation myocardial infarction (MI) and possible association with site of origin of the induced VT.

Methods

Consecutive patients with LVEF ≤40% on day 3–5 after MI who underwent EPS were prospectively recruited. Positive EPS was defined as sustained monomorphic VT cycle length ≥200ms for >10 seconds, or shorter duration if hemodynamic compromise occurred. The primary outcome was time to death from any cause. The secondary outcome was time to the composite endpoint of first activation of ICD for ventricular tachyarrhythmia or death.

Results

A total of 126 patients with inducible VT on EPS early after MI were included with median follow up 6.7 years. 54% had LBBB and 46% had RBBB morphologies during induced VT. The distributions of age, sex, left ventricle ejection fraction, cardiovascular risk factors, infarct artery and EPS characteristics did not differ significantly by induced VT morphology. The estimated risk of death was independently associated with RBBB morphology of VT induced at EPS (adjusted HR 3.2, 95%CI 1.0-10.0, p=0.044). The estimated risk of the composite end-point of spontaneous ventricular tachyarrhythmia recurrence or death was also independently associated with RBBB morphology of VT induced at EPS (HR 1.8, 95%CI 1.0-3.2, p=0.043).

Conclusion

There is an increased risk of death and spontaneous ventricular tachyarrhythmia recurrence at median follow up of 6.7 years, associated with RBBB morphology of VT (likely origin from left ventricle) induced at EPS early after MI, in those with LVEF <40%. This study is the first to characterise the association between site of origin of VT induced at EPS after myocardial infarction, indicated by RBBB or LBBB morphology, and overall survival as well as activation free survival. The results could help to identify a group of patients that could benefit from closer monitoring post MI and more intense preventative management to improve long-term outcomes.
RNA elongation contributes to gene expression regulation during early endothelial-to-mesenchymal transition
Authors: Kokot K , Hoba J , Erbe S , Andritschke M , Laufs U , Boeckel J N
Published: November 2023

Background

Endothelial dysfunction is characterized by endothelial-to-mesenchymal transition (EndMT), a process in which endothelial cells (EC) transform into mesenchymal cells and lose part of their endothelial identity. EndMT-positive cells are present in vulnerable atherosclerotic plaques. EndMT is triggered by stress stimuli such as inflammation or hypoxia that require an adaptive change in gene expression mediated at the level of RNA elongation. RNA elongation is controlled by the super elongation complex (SEC), which is built around scaffolding proteins of the AF family (AFF1-AFF4). The SEC controls the pausing of RNA Polymerase II (RNAPII). We hypothesized that RNA elongation plays a regulatory role in EndMT.

Methods and Results

Single cell sequencing data revealed that the SEC scaffold proteins AFF1 and AFF4 are ubiquitously expressed in human carotid artery plaques. AFF1 and AFF4 are higher expressed in EC compared to AFF2 and AFF3. In an in vitro EndMT model, AFF1 and AFF4 were upregulated compared to controls (AFF1: 1.9-fold; AFF4: 2.6-fold). SEC inhibition using chemical compounds reduced the protein levels of AFF1 (-67.4%) and AFF4 (-46.5%). EndMT induction was reduced by SEC inhibition, as measured by mesenchymal marker gene expression (SM22: -88.8%; CNN1: -94.0%). The rapid induction of gene expression depends on the release of paused RNAPII present at the promoter site. ChIP-sequencing revealed a reduced occupancy of RNAPII at promoter sites in EndMT as early as 2 h compared to controls, suggesting a rapid pause release of RNAPII during EndMT. Indeed, transcriptome-wide RNA elongation rates were increased by 20.2% as early as 10 min after induction of EndMT, as determined by analysis of newly synthesized 4sU-labelled RNA. SEC inhibition reduced transcriptome-wide RNA elongation rates (-26.8%). These data suggest a regulatory contribution of RNA elongation in early EndMT. Among the genes that were immediately and prominently upregulated after the induction of EndMT was the SPARC mRNA. RNA initiation and elongation of SPARC mRNA was increased 10 min after EndMT induction, which was reduced by SEC inhibition. 72 hours after EndMT induction, inhibition of SEC resulted in a reduction in SPARC mRNA (-72.2%), protein concentration (-97.2%) and reduced SPARC delivery to the supernatant (-43.9%). Knockdown of SPARC enhanced EndMT induction (SM22: 1.6-fold; CNN1: 3.6-fold), which was attenuated by treatment with recombinant SPARC.

Conclusions

We observed increased RNA elongation rates during EndMT associated with decreased pausing of RNA Polymerase II. The RNA elongation mediated specific gene expression such as the upregulation of SPARC. Taken together, our results indicate that RNA elongation plays a regulatory role in EndMT.
Identifying pro-angiogenic lncRNAs to regenerate the damaged myocardium following myocardial infarction
Authors: Belluomo R , Bennet M , Beker A H , Brandes R P , Boon R A
Published: November 2023

Cardiovascular diseases are the most common cause of death worldwide and Myocardial Infarction (MI) is the primary symptom of cardiovascular disease. In MI, Damage-Associated Molecular Patterns (DAMPs) released by dying cells play an important role in driving the inflammatory response following myocardial damage with potentially adverse or pro-regenerative outcomes. Since angiogenesis is a key process to ameliorate the sequelae of MI and the effect of DAMPs on cardiac cells are still widely unknown, hereby we present a novel model to study how DAMPs affect the gene expression of human Cardiac Microvascular Endothelial Cells (CMECs), one of the most abundant cell types in the heart, aiming to identify lncRNA targets to induce regenerative angiogenesis in patients with MI.

To generate a cocktail of DAMPs, cells were isolated from the heart tissue of patients undergoing septal myectomy via collagenase treatment and exposed to repeated cycles of freeze-thawing to induce cell rupture. The release of endogenous molecules (DAMPs), such as DNA, was confirmed by nano drop. CMECs were then stimulated with DAMPs for 24h and RNA sequencing was performed. Finally, to predict potential co-regulation between lncRNAs and angiogenic proteins, a combination of PLAIDOH and NoRCE were used. The RNA sequencing revealed a total of 1294 Differently Expressed Genes (DEGs) in CMECs stimulated with DAMPs, of which 218 were lncRNAs annotated in GENCODE v42. Among the top 50 DEGs were found proteins involved in cell growth, proliferation, and immune response such as FGF5, CEP128, and IL32 and the GSE analysis revealed that proliferation (mitosis), migration, and angiogenesis were among the most enriched terms. The analysis with PLAIDOH and NoRCe revealed ∼25 differentially expressed lncRNAs which correlate with proteins involved in cell cycle and mitotic entry (Pearson 0.6-0.99 and p-value < 0.05). Among these were found HIF1A-AS3, TGFB-OT1, TGFB-AS1. Following the in-silico analysis, results were confirmed by qPCR. qPCR for HIFA1A-AS3 showed a significant reduction of 40% when CMECs were stimulated with DAMPs (p-value: 0.04) confirming the sequencing results. Whether HIFA1A-AS3 is involved in cell proliferation will now be validated with KO experiments.

In conclusion, the administration of DAMPs to CMECs for 24h induced genes involved in proliferation, migration, and angiogenesis, processes that are also highly regulated in MI. By combining PLAIDOH and NoRCE we were able to identify lncRNAs which correlate in expression with protein-coding genes involved in these processes indicating co-regulation. These experiments recapitulate the main endothelial cell processes found in MI via administration of DAMPs and enable us to identify lncRNA targets to promote regenerative angiogenesis. Hence, this brings us one step closer to identifying gene therapy targets capable of inducing regenerative angiogenesis in patients with MI.
The role of endomembrane homeostasis in vascular calcification
Authors: Goettsch C , Hense N , Mir B , Marx N
Published: November 2023

Background

Cardiovascular diseases are a leading cause of mortality, with cardiovascular calcification as a prominent predictor and contributor. Microcalcifications in thin fibrous caps of atherosclerotic plaques create biomechanical instability, leading to plaque vulnerability. Recent work indicated that cellular-derived extracellular vesicles (EVs) are pivotal for microcalcification nucleation. EVs can originate from the endolysosomal system, and FYVE-Type Zinc Finger Containing Phosphoinositide Kinase (PIKfyve), a lipid kinase, plays a key role in the endolysosomal maturation.

Purpose

We hypothesize that alterations in endomembrane homeostasis will modulate EV cargo and, thereby, vascular calcification.

Methods and Results

In calcified arteries and vascular smooth muscle cells (SMCs), we observed increased PIKfyve expression. Phosphatidylinositol 3-phosphate (PI3P) - the substrate of PIKfyve - was decreased in cellular membranes of calcifying SMCs (-40%) and recovered by Apilimod, a small molecule PIKfyve inhibitor. Apilimod prevented matrix mineralization and collagen network formation in calcifying SMCs (Fig. 1A) accompanied by reduced pro-collagen 1A1 secretion (-90%). Apilimod inhibited tissue non-specific alkaline phosphatase (TNAP) – an early marker of SMC calcification on mRNA, protein and activity level. PIKfyve silencing using siRNA showed similar results. Furthermore, Apilimod caused a dose-dependent increase of EV release assessed by nanoparticle tracking analysis (Fig. 1B). Analysis of the EV cargo demonstrated that Apilimod reduces TNAP protein abundance and activity (-60%) - a characteristic cargo that may determine EV calcification potential. Apilimod-induced EVs exhibited lower mineral content and reduced aggregation potential assessed by osteosense-based flow cytometry and turbidity assays. Next, we performed transcriptome and kinome analyses to discover the underlying mechanism of PIKfye-mediated SMC calcification. These data revealed a link to adipocyte-like differentiation and SMC phenotype specifying pathways. Validation supported increased expression of adipogenic transcription factors (CEBPA, PPARG),genes of the cholesterol and fatty acid metabolism pathways (FABP3, CD36) and enhanced fatty acid (C12) uptake (Fig. 1A) by Apilimod; while SMC-specific markers (ACTA2, SM22) and key SMC transcription factors (GATA6, MYOCD) were downregulated. Finally, in Ldlr-deficient mice fed a high-fat, high-cholesterol diet for 15 weeks, Apilimod application for 5 weeks increased PI3P levels (1.6-fold) and decreased vascular calcification (Fig. 1C), but did not alter plaque size or collagen content in atherosclerotic plaques.

Conclusion

Disrupting endolysosomal maturation with Apilimod promotes the release of EVs with reduced calcification potential and induces a phenotypic adaption towards adipocyte-like SMCs, causing reduced SMC calcification. Anti-arteriosclerotic effects of Apilimod in vivo remain to be further investigated.Figure
Influence of standard modifiable risk factors on ventricular tachycardia after myocardial infarction
Authors: Deshmukh T , Kovoor J , Byth K , Chow C , Zaman S , Chong J , Figtree G , Thiagalingam A , Kovoor P
Published: November 2023

Background

Sudden cardiac death due to ventricular tachyarrhythmia is the single greatest cause of death within two years after myocardial infarction (MI), especially early in the first 30 days.

Purpose

We aimed to assess the incidence of inducible ventricular tachycardia (VT) early after MI in those with standard modifiable risk factors (SMuRFs) versus those without.

Methods

Consecutive patients with LVEF ≤40% on day 3–5 after ST elevation myocardial infarction (STEMI) who underwent electrophysiology study were prospectively recruited. Positive EPS was defined as sustained monomorphic VT cycle length ≥200ms for > 10 seconds, or shorter duration if hemodynamic compromise occurred. The primary outcomes were inducibility of VT and all-cause mortality.

Results

A total of 410 patients, of which 126 had inducible VT on EPS, early after myocardial infarction were included. In our multiple regressions model, when smoking history was considered, ex-smokers were identified at increased risk of VT at EPS (adjusted OR 2.0, p=0.033) whilst those who never smoked or were current smokers had comparable risk. It also showed that the presence of any SMuRF apart from current smoker also conferred an increased risk of inducible VT (adjusted OR 1.9, p=0.043). For every 10 years increase in age the risk of death increased by a multiplicative factor of 1.3, for every 10% decrease in LVEF (%) the risk of death increased by a multiplicative factor of 1.9 and in patients with previous IHD, the risk of death was doubled. Neither the number of SMuRFs nor the presence of any SMuRF were associated with mortality.

Conclusion

In our cohort of patients with LVEF<40% early after STEMI who had an EPS to assess for inducible VT, either the presence of any of the three SMuRFs (hypertension, hypercholesterolemia or diabetes mellitus) or previous smoking doubled the chance of having VT induced. These data provide valuable information that could be integrated into clinical care protocols after myocardial infarction to improve long-term patient outcomes and further reinforces the need to modify SMuRFs in patients with ischemic heart disease.
Cholesterol crystals as the main trigger of interleukin-6 production through innate inflammation in human spontaneously ruptured aortic plaques
Authors: Komatsu S , Yutani C , Takahashi S , Takewa M , Iwa N , Noda H , Ohara T , Kodama K
Published: November 2023

Aim

Cholesterol crystals (CCs) may provoke innate inflammation in experimental and animal model, however, whether CCs are bystanders or play central role for plaque rupture is unknown. This study aimed to clarify whether CCs was the main trigger of innate inflammation in human spontaneously ruptured aortic plaques (SRAPs).

Methods

This study included 260 SRAPs collected during non-obstructive general angioscopy (NOGA) from 126 patients with having or suspected CAD. Interleukin (IL)-6 levels in SRAPs were measured. IL-6 level at the Valsalva sinus, and femoral or brachial arteries were measured. IL-6 ratios, IL-6 level in SRAPs and arteries divided by IL-6 level at the Valsalva sinus at the beginning of aorta, were calculated. The number of CCs were counted in sampled blood. The correlation between the count of CCs and IL-6 levels in SRAPs and that between the counts of CCs and IL-6 ratios in SRAPs were analyzed.

Results

IL-6 ratios of the brachial and femoral arteries were 1.06 (95% CI, 0.99–1.20) and 1.11 (95% CI, 1.04–1.20), respectively. IL-6 ratio in SRAPs was 1.10 [1.00, 1.26]. (median [IQR]). CCs were detected in 94 of 260 SRAPs (36%). The count of CCs was 11,590 (95% CI: 2,386–30,113] per 10 mL in CCs positive samples. There was a moderate correlation between the counts of CCs and IL-6 ratios in SRAPs (r=0.496, r<0.0001)(Fig. 1), whereas there was no correlation between the count of CCs and IL-6 levels in SRAPs.(Fig. 2)

Conclusions

By the in-situ study, CC might be the main trigger of IL-6 through innate inflammation in human SRAPs.Fig 1Fig 2
Antibody against apolipoprotein-A1, Metabolic-Associated Fatty Liver Disease and cardiovascular risk: a translational study
Authors: Pagano S , Bakker S J L , Juillard C , Vossio S , Moreau D , Brandt K J , Mach F , Dullaart R P F , Vuilleumier N
Published: November 2023

Background and aims

Metabolic-Associated Fatty Liver Disease (MAFLD) represents a common cause of liver disease leading to increased cardiovascular disease (CVD) morbidity and mortality. Autoantibodies against apolipoprotein A-1 (AAA-1) are a possible novel CVD risk factor promoting inflammation and disrupting intracellular lipid homeostasis, two prominent pathogenic features of MAFLD. We aimed to evaluate the possible role of AAA-1 in MAFLD and their association with CVD risk.

Methods

HepaRG cells and liver sections from ApoE-/- mice exposed to AAA-1 were used for lipid quantification and conditional protein expression. Randomly selected sera from 312 subjects of a general population cohort were used to measure AAA-1. A Fatty Liver Index (FLI) ≥60 and a 10-year Framingham Risk Score (FRS) ≥20% were used as proxy of MAFLD and high CVD risk, respectively.

Results

In-vitro and mouse models showed tha-t AAA-1 interfered with triglyceride synthesis leading to steatosis. AAA-1 generated pro-inflammatory and pro-fibrotic responses. In the 112 participants with FLI≥60, AAA-1 were associated with higher FRS, alkaline phosphatase levels, lower HDL cholesterol and tended to display higher FLI values. Univariate linear and logistic regression analyses (LR) confirmed significant associations between AAA-1, FLI and FRS≥20%, while in adjusted LR, FLI was the sole independent predictor of FRS≥20% (OR:1.05,95%CI:1.01-1.09, P=0.003) and AAA-1 was not found to be an independent FLI predictor.

Conclusion

Despite inducing a MAFLD-compatible phenotype on in vitro and in mouse models and being associated with an increased CVD risk according to FRS, the present results suggest that the AAA-1 association with CVD risk in the general population is dependent on hepatic steatosis.
Genetic Deletion of Hepatic NCOR1 regulates the bile acid metabolism and leads to reduction in atherosclerosis
Authors: Geiger M , Oppi S A , Stein S N , Costantino S , Mohammed S A , Ruschitzka F , Paneni F , Stein S
Published: November 2023

Background

The nuclear receptor corepressor 1 (NCOR1) plays an important role in the regulation of gene expression in immunometabolic conditions by connecting chromatin-modifying enzymes, coregulators and transcription factors. NCOR1 has been shown to be involved in cardiometabolic diseases. Since NCOR1 transrepresses the function of several nuclear receptors involved in hepatic lipid transport and synthesis, we hypothesized that genetic NCOR1 deletion in the liver alters lipid metabolism and atherosclerosis development.

Methods

To test this hypothesis, we generated hepatocyte-specific Ncor1 knockout mice on a Ldlr-/- background. Besides assessing the progression of the disease in thoraco-abdominal aortae en face, we analyzed hepatic cholesterol and bile acid metabolism at expression and functional level.

Results

This study findings demonstrated that specific Ncor1 knockout mice on an atherosclerosis-prone background develop less atherosclerotic lesions than controls. Interestingly, under chow diet plasma cholesterol levels of liver-specific Ncor1 knockout mice were slightly higher compared to control, but strongly reduced compared to control mice after feeding them an atherogenic diet for 12 weeks. Moreover, hepatic cholesterol and triglyceride contents were decreased in liver-specific Ncor1 knockout compared to control mice. Our mechanistic data revealed that NCOR1 reprograms the synthesis of bile acids towards the alternative pathway, which in turn reduce bile hydrophobicity and enhances fecal cholesterol excretion.

Conclusions

Our data suggest that hepatic Ncor1 deletion in mice decreases atherosclerosis development by reprograming bile acid metabolism and enhancing fecal cholesterol excretion. Hepatocyte NCOR1 may therefore provide a novel target for development of anti-atherosclerotic drugs.
Bile acids promote endothelial cell homeostasis via the regulation of cell metabolism and quiescence
Authors: Dietrich E , Lettlova S , Jomard A , Wasilewski G , Osto E
Published: November 2023

Background

Endothelial cell (EC) homeostasis is characterized by a quiescent and low-metabolic state. Obesity leads to EC dysfunction and CVD. We demonstrated that bariatric surgery improved obesity-induced EC dysfunction and increased fasting and post-prandial plasma levels of bile acids (BAs)(in particular, cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)). BAs are emerging as metabolic signaling molecules in several tissues, but their effect on ECs has not been clearly established yet.

Purpose

We investigate the effect of BA on the maintenance of EC quiescence.

Methods

Human Aortic Endothelial Cells (HAECs) were treated for 24h with vehicle (0.1% DMSO), CA, CDCA and DCA (50uM each) in 2%FBS EGM-2 media. Percentage of apoptotic cells was assessed with AnnexinV/PI. Seahorse Mitostress test, Mitoplate and glucose uptake with 0.5µCi/ml of 14C-2-deoxy-D-glucose were used to evaluate changes of HAEC metabolism. Immunostaining for Ki67, Crystal Violet staining and scratch assay were performed to assess HAEC proliferation and migration. NO production and endothelial NO synthase (eNOS) activity were measured via DAF-2 probe and Western Blot, respectively. By qPCR genes involved in Notch pathway were evaluated. Mesoscale vascular injury panel, scratch assay, Ki67 immunostaining and glucose uptake were also performed in HAECs pre-treated with 50uM of BAs and stimulated with 0.5ng/mL TNF-α for 24h.

Results

HAECs treated with BAs increased NO production compared to vehicle and a reduction in both basal and maximal mitochondrial respiration and extracellular acidification rate was shown by Seahorse Mitostress test. Mitoplate assay displayed an overall reduction in the metabolic rate of HAECs treated with CA and DCA and a peculiar metabolic pattern in cells treated with CDCA. Reduction in glucose uptake was also observed after BA treatment, suggesting that BAs are able to decrease EC metabolism. Immunostaining for Ki67 and Crystal Violet highlighted that BAs reduced cell proliferation. The scratch assay also displayed a reduction in EC migration after BA treatment. BAs also reduced EC apoptosis, as significant differences in the percentage of AnnexinV/PI positive cells was observed in BA-treated cells compared to vehicle. qPCR showed increased expression of genes involved in Notch pathway promoting quiescence after BA treatment. Glucose uptake, scratch assay and Ki67 immunostaining performed in TNF-α stimulated HAECs suggested a BA-dependent endothelial protection in conditions of EC pro-inflammatory activation. We did not observed a reduction in pro-inflammatory biomarkers released in the supernatant of HAECs pre-treated with BA and stimulated with TNF-α.

Conclusions

Our results suggest that BAs may be involved in the maintenance of EC quiescence by regulating EC metabolism and gene expression profile. These findings highlight a potential role of BAs as endothelial-protective molecules.
Human platelets and megakaryocytes express the BPIFB4 protein and their stimulation with the recombinant Longevity-associated variant LAV-BPIFB4 displays anti-aggregating activity
Authors: Ciaglia E , Montella F , Lopardo V , Esposito M R , Carrizzo A , Vecchione C , Maciag A , Barbieri S S , Puca A A
Published: November 2023

Platelets are not only the main players in primary haemostasis and cardiovascular disease, but recent advances attribute them a key role in immune defence mechanisms via the release of plasma mediators of potential clinical relevance.

The bactericidal/permeability-increasing fold-containing family B, member 4 (BPIFB4) belongs to a family protein participating in the innate mechanism of host defence.

It is a secreted protein and its plasma levels inversely correlated with COVID-19 and coronary artery disease (CAD) severity and the degree of carotid stenosis and intima media thickness in human patient cohorts. Interestingly, Long Living Individuals (LLIs) displayed high BPIFB4 circulating levels and an enrichment of the 4 SNPs missense haplotype at BPIFB4 locus, the Longevity-Associated Variant (LAV). LAV-BPIFB4 genotype correlate with higher BPIFB4 plasma levels and the transfer of LAV-BPIFB4 gene delayed major age-related diseases, mainly rescuing the immunesenescence and the endothelial and cardiac dysfunctions, yet the critical assessment of the BPIFB4 protein as a platelet release product is lacking.

Herein we observed BPIFB4 release by calcium activated human platelets, a process that is amplified in LAV allele carriers donors. Furthermore, LAV gene transfer resulted in an enhanced protein enrichment in bone marrow megakaryocyte precursor cells and in a more efficient process of platelets formation in vitro. At functional level, LAV genotype was mimicked by the rhLAV-BPIFB4 platelet stimulation that induced endogenous BPIFB4 release similarly to calcium stimuli.

In a small cohort of treatment-naive patients with type 2 diabetes, while ADP and Collagene agonist stimulation induced high aggregation, the co-treatment with the rhLAV-BPIFB4 significantly reduced the activation capacity of patients’ platelets.

Accordingly, an administration of rhLAV-BPIFB4 24 hours before tromboembolism induction partly protected from mortality in a murine model of lethal pulmonary thromboembolism in vivo. As a result of reduced platelets’ activation, lower level of circulating leukocyte-platelets aggregates (LPA) was found in rhLAV-BPIFB4-treated mice compared to control.

In summary, our data report for the first time that both, human platelets and megakaryocytes, can express BPIFB4 and unravel a novel rhLAV-BPIFB4 therapeutic action in platelet iperaggregation and thrombocytopenia.