bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract

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) &lt;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

Abstract

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

Abstract

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

Abstract

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 &gt;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 &lt;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

Abstract

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

Abstract
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 &lt; 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

Abstract

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

Abstract

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 &gt; 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&lt;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.