bluuebunny/crossref_metadata_2025 huggingface dataset:
Alterations in oxidative phosphorylation complexes in arrhythmogenic cardiomyopathy using desmosomal KO cell model
Authors: Artigas Meleiro E , Vallverdu-Prats M , Brugada R , Alcalde M
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III (Fondo Investigación Sanitaria FIS)

Introduction

Arrhythmogenic cardiomyopathy (ACM) is an inherited disease that predominantly affects the right ventricle characterized by progressive cardiomyocyte loss and the replacement of the myocardium by fibro-fatty tissue, which can lead to sudden cardiac death (1). The main genetic causes of ACM are variants in desmosomal genes (PKP2, DSP, DSG2, DSC2 and JUP), representing up to 50% of patients (2).

We lack sufficient understanding at molecular level of the pathophysiological mechanisms that may trigger ACM and determine its progression. Recent studies have proposed a deficit in transcripts encoding for proteins from the electron transport chain, which may be a potential ACM molecular signature in samples from patients with ACM carrying mutations in PKP2 (3).

Purpose

Our study aims to deepen into the knowledge of this proposed mechanism in PKP2 deficiency using edited cell model and elucidating whether these mechanisms are also triggered in DSG2 and DSC2 deficiency, being a potential common ACM pathomechanism, or they are gene specific.

Methods

HL-1 edited cell lines for desmosomal genes previously generated by CRISPR/Cas9 (PKP2-KO, DSC2-KO and DSG2-KO) in our research group were used to perform our analysis (4). Expression profiling was performed by RNA-seq analysis.

To experimentally validate the RNA-seq analysis results, we performed western blot assays using total protein lysate to test protein levels of the five oxidative phosphorylation (OXPHOS) complexes for the PKP2-KO, DSC2-KO and DSG2-KO groups. To test if these differences have an impact at functional level, XFp cell Mito Stress Test (Agilent Technologies) was performed.

Results

RNA-seq analysis results showed numerous downregulated pathways related to aerobic respiration, the electron transport chain, oxidative phosphorylation and ATP synthesis in all three desmosomal KO lines.

Our experimental results showed a significant reduction of the OXPHOS complexes of the mitochondrial respiration chain at protein level in all PKP2-KO, DSC2-KO and DSG2-KO (Fig. 1).

At functional level, our preliminary results of Mito Stress Test suggest differences in basal and maximal respiration, ATP production, non-mitochondrial oxygen consumption and spare respiratory capacity between experimental groups.

Conclusions

Our results support previously published studies describing oxidative phosphorylation as a pathomechanism for PKP2 deficiency and also suggest that these mechanisms may be a common feature for desmosomal mutations in ACM.Total expression of OXPHOS complexes
Human in vitro disease modeling of ATTR cardiac amyloidosis
Authors: Qin J , Dokter I , Qiang Y , Lei Z , Sluijter J , Oerlemans M , Van Mil A
Published: May 2024

Funding Acknowledgements 

Type of funding sources: Private company. Main funding source(s): Pfizer Alnylam

Transthyretin amyloid cardiomyopathy (ATTR-CM) is a progressive disease with deposition of TTR amyloid fibrils in the myocardium and cardiac dysfunction with dismal prognosis. In ATTR-CM, either genetic mutations (variant; v) or ageing (wild-type; wt) leads to TTR instability. Late diagnosis and a lack of human disease models limit disease understanding and therapeutic discovery.

This study aimed to model ATTR-CM in vitro by exposing various cardiac cell types and multicellular 3D cardiac spheroids to TTR fibrils, characterizing disease phenotypes and providing insights into the cellular mechanisms of disease progression.

Human recombinant TTR (WT, V30M, V122I) and acid induced fibrils were generated and characterized. Seeding hiPSC-cardiomyocytes (CMs) and endothelial cells (ECs) on TTR fibrils resulted in cell viability reduction. Confocal microscopy revealed the extracellular attachment of TTR fibrils, leading to sarcomere disorganization, prolonged calcium transients and disrupted electromechanical coupling in hiPSC-CMs, and declined migration capacity of ECs showing aberrant cell morphology. Similarly, cardiac spheroids incorporating TTR fibrils resulted in decreased cell viability and prolonged calcium transients with lower amplitude.

Our models show the discovery of early cellular ATTR-CM phenotypes, providing a novel approach to study the disease progression, potentially facilitating the identification of novel therapeutic targets and biomarkers.Cytotoxicity of TTR fibrils on hiPS-CMs
High-throughput proteomic analysis unveils metabolic and mitochondrial signaling rewiring by chronic ivabradine treatment in duchenne muscular dystrophy
Authors: Dostal C , Szabo P L , Marksteiner J , Podesser B K , Hilber K , Kiss A
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): Austrian Science Fund (FWF)

Objective

Ivabradine (IVA) is indicated in symptomatic treatment of chronic stable angina, heart failure, and also in those who are unable to tolerate or have contraindications to the use of beta-blockers. It has been showed IVA may have cardiovascular benefits in Duchenne muscular dystrophy (DMD) patients. This study was aimed to investigate the sustained impact of chronic ivabradine (IVA) administration on cardiac function and potential underlying mechanism using high throutput proteomic analysis.

Methods

DMDmdx male and Sprague–Dawley wt (Sprague–Dawley) male rats were randomly allocated to vehicle (n = 6) or IVA (n=6; 10 mg/kg/day via drinking water for four months). Transthoracic echocardiography and unbiased proteomic analysis were performed to assess cardiac function and left ventricular (LV) tissue, respectively. Protein-protein interactions were graphically represented, and cluster analysis with subsequent enrichment analyses was conducted.

Results

Chronic IVA treatment significantly enhanced the LV ejection fraction (p<0.05 compared to vehicle-treated DMDmdx rats). Proteomic data identified possible transcription factors (SPI1, IRF1, PPARA), consistent with previous studies. The reduction in copper metabolic changes associated with DMD can be attributed to the upregulation of Atox1 following IVA treatment. Mitochondrial dysfunction was effectively mitigated by IVA, as indicated by the different abundance of mitochondrial proteins. In addition, intercellular adhesion molecule 1 showed a reduction in the number of animals treated with ivabradine, indicating a further positive effect of the treatment.

Conclusion

In summary, this study demonstrated the beneficial effects of IVA in DMDmdx rat hearts. Cluster analysis of proteomic data revealed notable changes in cardiac metabolism, inflammation, and mitochondrial function. Ivabradine has emerged as a potential therapeutic approach to address these shifts, while proteomic pathway analysis may uncover new drugable targets to alleviate cardiomyopathy progression in DMD.Interaction of IvabradinProtein-protein interaction network
Zebrafish dspb-/- mutant as model for arrhythmogenic cardiomyopathy - impact of physical activity
Authors: Munteanu S , Wagih Gomez J , Nicolas Rocamora E M , Gil Ortuno C , Bernabe Garcia A , Judez Serrano A , Martinez Perez C , Gimeno Blanes J R , Cayuela Fuentes M L , Sabater Molina M
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III

Background

Zebrafish has been reported as a model to study the Arrythmogenic cardiomyopathy, which is generally associated with desmosomal gene mutations. We have identified a founder non-sense human variant in patients from our region (DSP p.Q447*) associated with left ventricle Arrythmogenic cardiomyopathy and we have selected it for modelling in zebra fish by CRISPR/Cas9 method, choosing exon 10 as the region of homology to be mutated and obtaining the p.T449fs* variant that determines a premature stop codon.

Purpose

Our aim was the characterization of dspb-/- mutants, studying the impact of physical activity in development of the disease.

Methods

The characterization of phenotype was performed through viability assays, cardiac function measurements and gene expression assays in larvae (3 days post fertilization) and adult (8 months old) individuals. The impact of physical activity was determined through moderated training periods (1 month) and endurance assays using a swimming tunnel.

Results

Viability assays showed that dspb mutants suffer a premature death, with significant higher mortality rates than the wildtype group (survival WT 89.7% vs dspb-/- 61.3%, p<0.01). Larvae cardiac function showed dspb mutants had a higher heart rate (WT 106.8 ± 26.8 vs dspb-/- 141.0 ± 7.2, p<0.01) while echocardiography showed differences in heart rate (WT 127.2 ± 49,8 vs dspb-/- 219.2 ± 26.8, p<0.05), systolic (WT 26.9 ± 5.3 vs dspb-/- 18.5 ± 5.2, p<0.05) and diastolic (WT 37.6 ± 8.4 vs dspb-/- 23.7 ± 5.4, p<0.05) areas. Gene expression analysis in adult heart showed dspb expression drastically decreased (WT 0.9 ± 0.1 vs dspb-/- 0.4 ± 0.01, p<0.01), whilst pkp2 expression increased (WT 0.9 ± 0.1 vs dspb-/- 2.59 ±0.22, p<0.001) as well as dspa gene expression (wt 0.9 ± 0.1 vs dspb-/- 4.41 ± 0.18, p< 0.001) as a compensation mechanism. Moderated long term physical activity had a positive impact in heart rate (dspb-/-pre 219.2 ± 26.8 vs dspb-/-post 138 ± 7.5, p<0.01) and diastolic area (dspb-/-pre 24 ± 5 vs dspb-/-post 32 ± 2.1, p<0.05). Adult mutants showed a better endurance showing improvement in immobile cumulative duration (dspb-/-pre 849.3 ± 29.3 vs dspb-/-post 179.8 ± 29.7, p<0.01) and immobile frequency (dspb-/-pre 5399.5 ± 1127.8 vs dspb-/-post 1310 ± 481.9, p<0.01).

Conclusion

The dspb-/- zebrafish presented pathological cardiac phenotype at larvae and adult stages, and less endurance in intensity exercises. The moderate exercise has turned out to be a beneficial modulator of the phenotype.
Antisense oligonucleotides as a potential therapeutic strategy in hypertrophic cardiomyopathy caused by MYBPC3 variants
Authors: Munteanu S , Nicolas Rocamora E M , Martinez Perez C , Bernabe Garcia A , Nicolas Villaescusa F J , Gimeno Blanes J R , Arechavala Gomeza V , Sabater Molina M
Published: May 2024

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): Sociedad Española de Cardiología

Background/Introduction

Non-sense or frameshift mutations in the MYBPC3 gene encoding cardiac myosin-binding protein C (cMyBP-C) are a major cause for inherited forms of Hypertrophic Cardiomyopathy (HCM), the most frequent inherited cardiac disease, affecting 0.2% of the general population and represents the leading cause for sudden cardiac death in young athletes. During the last decade, RNA-based therapeutic approaches have been studied for neuromuscular disorders and recently became to be considered to envision causal therapies for cardiomyopathies, utilizing antisense oligonucleotide mediated exon skipping, which attempts to reframe mutated mybpc3 transcripts, resulting in shortened but functional protein. MYBPC3 gene has a total of 34 coding exons and, exons 2, 22, and 24-27 are ideal targets for skipping.

Purpose

We aim to design and evaluate antisense oligonucleotides (AONs) for efficient exon-skipping in targeted MYBPC3 exons.

Methods

We designed two specific AONs to mask the MYBPC3 exon 26 and confirmed successfully mediated exon skipping without disrupting the MYBPC3 reading frame. We chose a previously identified MYBPC3 c.2671insG/p.R891Afs*160 variant in exon 26, which causes a frameshift mutation leading to truncated MYBPC3 in HCM. Primary Human Cardiac Myocytes (PHCM) (Promocell) were transfected with the splice-site targeting AONs.

Results

In this study we designed two chemically modified AONs (AON1 and AON2) through the addition of a 2′-O-methyl RNA phosphorothioate backbone, targeting exonic splicing enhancers (ESEs) or splice sites (SSs) can induce skipping of MYBPC3 exon 26.

We evaluated AON1 (i26-3SS) and AON2 (ESE)-treated Primary Human Cardiac Myocytes (PHCM) show 26-27 exons skipping in comparison to control samples. The identity of the exon 26-27 skipped bands detected by RT-PCR was confirmed by Sanger sequencing and preservation of both nucleotide sequences of MYBPC3 exons 25 and 28 were observed.

These results indicate that AONs specifically for mutated MYBPC3 target exons, expanding the framework of future advancements in the therapeutic potential of antisense-mediated exon skipping MYBPC3-based HCM. In addition, skipping exons 26 and 27 we eliminate a total of 22 possible exonic mutations, which represent 7.2% of the total known MYBPC3 exonic mutations.

Conclusion(s)

These results demonstrate that disruption of the MYBPC3 reading frame due to a truncating HCM mutation can be restored by exon skipping in cardiomyocytes in vitro indicating RNA-based strategies as a potential treatment option for HCM.Exon skipping scheme
Connecting the dots: molecular pathways in bicuspid aortopathy explored
Authors: Pozo-Vilumbrales B , Soto-Navarrete M T , Martin-Chaves L , Lopez-Unzu M A , Pavon-Moron J , Rodriguez-Capitan J , Fernandez B
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Junta de Andalucía, Ministerio de Ciencia e Innovación

Background

Bicuspid aortic valve (BAV) is the most common congenital cardiac malformation. It predisposes to thoracic aortic dilatation (TAD), aneurysm and dissection. The current consensus is that the genetic defect that leads to the aortic valve malformation also causes structural anomalies of the ascending aorta. Altered hemodynamics caused by the abnormal valve morphology would accelerate aortic media degeneration. Molecular markers and pathways involved in the aetiology and pathophysiology of bicuspid aortopathy are poorly understood.

Objective

To elucidate the molecular and cellular mechanisms of the disease and to identify potential predictive molecular markers using a well-established isogenic hamster model (T strain) with a high (∼40%) incidence of BAV TAD.

Methods

Comparative quantitative proteomics combined with Western blot and morpho-molecular analyses in the ascending aorta of tricuspid aortic valve (TAV) and BAV animals from the T strain and TAV animals from a control strain. This strategy allows exploring independently the genetic and hemodynamic effects on the aorta in genetically homogeneous populations.

Results

The major molecular defect in the aorta of genetically homogeneous BAV individuals is PI3K/AKT overactivation caused by alterations in the EGF, ANGII and TGF-β signalling pathways. PI3K/AKT affects the downstream eNOS, MAP2K1/2, NF-κB, mTOR and WNT pathways. Most of these alterations are seen in independent patient studies with different clinical presentations, but not in TAV hamsters from the T strain, which mainly show downregulation of the WNT pathway.

Conclusions

We identify a combination of defective interconnected molecular pathways, directly linked to the central PI3K/AKT pathway, common to both BAV-associated TAD patients and hamsters. The defects indicate a shift of smooth muscle cells towards the synthetic phenotype induced by endothelial-to-mesenchymal transition, oxidative stress and inflammation. WNT signalling represents a genetic factor that may cause aortic structural abnormalities and aneurysm predisposition, whereas hemodynamics is the main trigger of molecular changes, likely determining the progression of aortopathy. We identify twenty-seven novel potential biomarkers with high predictive value.
Short- and long term preservation of cardiac tissue-engineered constructs
Authors: Janssen J , Chirico N , Ainsworth M , Viola M , Cedillo-Servin G , Vermonden T , Doevendans P , Serra M , Voets I , Malda J , Castilho M , Laake L , Sluijter J , Sampaio-Pinto V , Van Mil A
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This research was financially supported by the Gravitation Program ‘‘Materials Driven Regeneration,’’ funded by the Netherlands Organization for Scientific Research (024.003.013) and the EU-funded project BRAV∃ (H2020, ID:874827).

Background

Cardiac tissue engineering (cTE) has already advanced towards the first clinical trials, investigating safety and feasibility of cTE construct transplantation in failing hearts. However, the lack of well-established preservation methods poses a hindrance to further scalability, commercialization, and transportation, thereby reducing their clinical implementation.

Purpose

In this study, hypothermic (short-term) preservation (4°C), and slow and fast freezing methods for cryopreservation, at -196°C and -150°C respectively, were investigated as potential solutions to extend the cTE construct implantation window and increase their clinical applicability.

Material and Methods

The cTE model used consisted of induced-pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts embedded in a natural-derived hydrogel and supported by a polymeric melt electrowritten hexagonal scaffold. Constructs, composed of cardiomyocytes of different maturity, were preserved for three days, using several commercially available preservation protocols and solutions. Their viability, function (BPM and calcium handling), and metabolic activity were investigated after rewarming.

Results

Our observations suggest that cardiomyocyte maturation did not influence post-rewarming viability, however, aging positively influenced construct function pre- and post-storage. Hypothermic preservation in Hypothermosol ensured retained construct viability and function. Furthermore, fast-freezing outperformed slow-freezing, but both viability and function were severely reduced after rewarming.

Conclusion

In conclusion, whereas long-term preservation remains a challenge, hypothermic preservation in Hypothermosol represents a promising solution for cTE construct short-term preservation and potential transportation, aiding in off-the-shelf availability, ultimately increasing their clinical applicability.Graphical abstract
Lower activity of cholesteryl ester transfer protein (CETP) and the risk of dementia: a Mendelian randomization analysis
Authors: Schmidt A , Davidson M H , Ditmarsch M , Kastelein J J , Finan C
Published: May 2024

Funding Acknowledgements

Type of funding sources: Private company. Main funding source(s): New Amterdam Pharma

Background

Elevated levels of low-density lipoprotein cholesterol (LDL-C) are linked to dementia risk, and conversely, increased plasma concentrations of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein-A1 (Apo-A1) associate with decreased dementia risk. Inhibition of cholesteryl ester transfer protein (CETP) meaningfully affects the concentrations of these blood lipids and may therefore provide an opportunity to treat dementia.

Methods

Drug target Mendelian randomization (MR) was employed to anticipate the on-target effects of lower CETP concentration (µg/mL) on plasma lipids, cardiovascular disease, Lewy body dementia (LBD), as well as Parkinson’s dementia. Replication was sought by performed Apo-A1 and Apo-B weighted MR analyses of CETP variants. Genetic associations with protein expression in cerebrospinal fluid (CSF) and brain were consulted to identify potential associations of plasma CETP with dementia implicated proteins.

Results

MR analysis of lower CETP concentration (per µg/mL) recapitulated the blood lipid effects observed in clinical trials of CETP-inhibitors, as well as protective effects on CHD (odds ratio (OR) 0.92, 95% confidence interval (CI) 0.89; 0.96), heart failure, abdominal aortic aneurysm any stroke, ischemic stroke, and small vessel stroke (0.90, 95%CI 0.85; 0.96); Figure. Consideration of dementia related traits indicated that lower CETP concentrations were associated higher total brain volume (0.04 per standard deviation, 95%CI 0.02; 0.06), lower risk of LBD (OR 0.81, 95%CI 0.74; 0.89) and Parkinson’s dementia risk (OR 0.26, 95%CI 0.14; 0.48). APOE4 stratified analyses suggested the LBD effect was most pronounced in APOE-ε4+ participants (OR 0.61 95%CI 0.51; 0.73), compared to APOE-ε4- (OR 0.89 95%CI 0.79; 1.01); interaction p-value 5.81×10-4. The aforementioned associations were replicated using Apo-B and Apo-A1 weighted analyses; Figure. Additionally, MR was employed to link plasma CETP concentration to the levels of CSF and brain proteins such as EPHA2 and CSF-1, which are in development as dementia drug targets.

Conclusion

These results suggest that inhibition of CETP may be a viable strategy to treat dementia.
Deep proteome profiling of reticulated platelets in patients with chronic coronary syndrome using mass cytometry
Authors: Han J , Kirmes K , Klug M , Bernett J , Manz Q , Arend L , Lazareva O , Rosenbaum M , Ruland J , Laugwitz K L , List M , Bernlochner I , Bongiovanni D
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Else Körner Fresenius Stiftung (EKFS)

Background

Immature or reticulated platelets (RPs), a subgroup of larger and RNA-rich platelets, which are newly released by megakaryocytes from the bone marrow into blood, have gained rising interest because of the further decryption of RPs’ prothrombotic biology and the potential as biomarkers for predicting adverse events in cardiovascular disease.

Purpose

The biological features of RPs in patients with chronic coronary syndrome (CCS) are largely unknown. With this project, we aimed to perform the first high resolution proteomic characterisation of reticulated platelets using mass cytometry(CyTOF).

Method

Two samples of thrombocytes from peripheral blood (non-stimulated, TRAP-stimulated) were prepared from each patient with a custom-made CyTOF-antibody panel of 20 antibodies targeting essential transmembrane proteins(anti-CD9, anti-CD29, anti-CD31, anti-CD36-, anti-CD40, anti-CD41, anti-CD42a, anti-CD42b-, anti-CD47, anti-CD61, anti-CD62P-, anti-CD63, anti-CD69, anti-CD107a, anti-CD154, anti-GPVI, anti-GPIIbIIa complex, anti-Par1, anti-PEAR-1 and the negative control anti-CD3 coupled with different metal isotopes). RPs and mature platelets (MPs) were detected based on their RNA content. Earth mover’s distance (EMD) was assessed as a measure of differential expression.

Results

CyTOF detected an upregulation of important transmembrane receptors in RPs compared to MPs in quiescent platelets (Fig.1A and B). The collagen receptor GPVI (EMD 33.95), the collagen integrin receptor unit ITGB1 (EMD 32.37), the adhesion protein CD9 (EMD 31.78) and the von Willebrand receptor unit CD42b (EMD 30.08) were upregulated in RPs which all serve as crucial regulators of platelet activation and aggregation. RPs show higher median signal intensities of typical activation markers compared to MPs upon TRAP stimulation (Fig.1C).

Conclusion

We provide the first proteomic characterization of RPs’ pro-thrombotic profile offering the first biomolecular explanation of their hyperactivity and detrimental role in CCS patients.
Effect of diabetes on hypoxic microvascular remodeling and muscle damage in chronic limb threatening ischemia
Authors: Juusola G , Wirth G , Ord T , Kaikkonen-Maatta M , Hakovirta H , Yla-Herttuala S , Korpisalo P
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Finnish Academy/Research Council of Finland. The Finnish Foundation for Cardiovascular Research

Background

Hypoxia-induced arterialization of skeletal muscle capillaries has recently been identified as an important pathological feature that aggravates ischemic damage in chronic limb threatening ischemia (CLTI). Diabetes is also known to worsen the outcome of CLTI patients but its contribution to capillary arterialization and skeletal muscle pathology under ischemia is still largely unknown.

Purpose

To study how diabetes affects capillary arterialization and skeletal muscle pathology in CLTI.

Methods

Amputation muscle samples from CLTI patients (n=21 patients x3 samples) were histologically analyzed, grouped according to microvascular changes and RNA sequencing was performed. Differentially expressed genes, differentially regulated signaling pathways and histology between diabetics and non-diabetics were studied among corresponding microvascular groups. Also, spatial transcriptomics (n=2 patients) was applied to understand the mechanisms through which diabetes alters differential capillary responses on the tissue level.

Results

Diabetes differentially regulated gene expression of different angiogenic and arteriogenic factors including HIF1A, VEGFA, FLT1, NOTCH1, NOTCH3, NOS1 and ITGB3 in the CLTI muscles. This differential transcriptomic regulation translated as increased sprouting on the level of muscle capillaries, and also affected myofiber pathology. Muscle samples in diabetic patients often presented myofiber swelling and cell death that were uncommon in samples of non-diabetic patients.

Conclusion

Diabetic patients on the level of muscle transcriptional regulation show changes that can explain observed differential microvascular remodeling and worsened myofiber pathology. These results broaden our understanding of the causes for the exceptionally bad outcome of diabetics among CLTI patients.
MicroRNA-26b as novel regulator of cardio-metabolic diseases
Authors: Van Der Vorst E , Rakateli L , Peters L , Bidzhekov K , Jans A , Maas S , Gijbels M , Jankowski J , Biessen E , Weber C , Doering Y , Bartneck M
Published: May 2024

Funding Acknowledgements 

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): IZKF

Increasing evidence has shown that microRNAs (miRs) are fundamental players in cardio-metabolic diseases. MiR-26b has been associated with the development of metabolic dysfunction-associated steatohepatitis (MASH) and atherosclerosis, although causality has not been demonstrated yet. Therefore, we aimed to determine the causal role of miR-26b in MASH and atherosclerosis.

Apoe-/-Mir-26b-/- mice were fed a 4- or 12-week western type diet (WTD). Plasma and liver samples were subjected to various assays to investigate the effects on lipid metabolism, inflammation and fibrosis. Additionally, miR-26b mimic-loaded lipid nanoparticles (LNPs) were used to rescue the phenotype.

A 1.5-fold increase in hepatic lipid levels was observed in Apoe-/-Mir26b-/- mice, while plasma cholesterol/triglyceride levels remained unchanged. These results were further confirmed by an increase in steatosis. Additionally, the proinflammatory cytokines TNF-α, IL-6 and CXCL1 were significantly elevated, which coincided with increased macrophage infiltration into the liver. Moreover, loss of miR-26b resulted in more hepatic fibrosis. Finally, a treatment with miR-26b mimic-loaded LNPs reduced hepatic lipid levels, rescuing the observed phenotype. Additionally, Apoe-/-Mir26b-/- mice demonstrated a striking 10-fold increase in atherosclerotic lesion size. Consistent with a more advanced plaque phenotype, collagen content and relative necrotic core area were significantly increased in plaques from mice lacking miR-26b. Intriguingly, this increased atherosclerotic lesion size could also be rescued by repetitive injections of miR-26b mimic-loaded LNPs.

Overall, our results demonstrate a protective role of miR-26b in cardio-metabolic diseases, thereby opening doors to future research and potential new treatment options using LNP technology.
Role of miRNAs regulation of Orai1 and store operated Ca2+ entry in vascular restenosis
Authors: Martin Bornez M , Dominguez Liste B , Falcon Boyano D , Smani T
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación

Background

The main cause of restenosis is attributed to the excessive proliferation and migration of vascular smooth muscle cells (VSMCs). Orai1 is one of the main molecular components of Store-Operated Calcium Entry (SOCE), playing a key role in VSMCs proliferation during neointima formation and vascular remodeling. Recent studies have demonstrated the implication of microRNAs (miRNAs) in VSMCs proliferation and migration, however, the role of these miRNAs as regulators of SOCE during vascular remodeling has not been described.

Purpose

This study aims to determine whether miRNAs regulate the intracellular Ca2+ handling, focusing on SOCE, during neointima formation.

Methods

Experiments were conducted in an animal rat model following the "Principles of laboratory animal care" and according to the Spanish law. The neointima development was assessed after balloon injury of carotid arteries and confirmed by haematoxylin and eosin staining, and αSMA staining of tissue sections up to 3 weeks after surgery. miRNAs array and RT-qPCR was used to examine the expression of miRNAs. Dual-Luciferase reporter assay was performed in A7R5 VSMCs line.

Results

Injured arteries showed significant increase in the expression of Orai1, as compared to non-injured arteries, indicating their possible participation in the neointima layer formation. Microarray of miRNAs analysis of the injured carotids compared to control carotids showed significant increase in the expression of miRNAs related to signalling pathways involving cell proliferation and migration, cell differentiation, or VSMCs contraction. Of these, we confirmed the significant increase of miR-18a-5p, miR-20a-5p, miR-20b-5p and miR-17-5p, which belong to widely studied miR-17-92 cluster involved in different cell proliferation. Moreover, we found that mimics of those miRNAs were able to regulate differentially the activity of the Orai1 promotor in A7R5 cell line, indicating that Orai1 can be targeted by miRNAs during in VSMCs.

Conclusions

In conclusion, our data confirms that Orai1 is upregulated during neointima formation associated with miRNAs dysregulation. Moreover, we found that Orai1-dependent SOCE can be finely regulated by miRNAs during restenosis.
FHL2 is a novel target in pulmonary arterial hypertension
Authors: Nadeem C , Abbas A L C , Verhoeven D T B , Willems L , Goumans M J , Kurakula K
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): - Longfonds Nederland

Background/ Objectives

Pulmonary arterial hypertension (PAH) is a fatal disorder characterized by excessive proliferation of endothelial (EC) and smooth muscle cells, and impaired BMP signaling in small lung arteries. The LIM-only protein FHL2 is expressed in the vessel wall in ECs and smooth muscle cells and plays a crucial role in vessel wall homeostasis. However, the molecular mechanisms how FHL2 exerts its effects and its role in pulmonary artery EC function remain unclear. Here, we investigated the effect of FHL2 on EC proliferation and unraveled the underlying mechanisms involved.

Methods/Results

We found that FHL2 is highly expressed in lungs of PAH rats compared to control rats as evidenced by qPCR and western blot analysis. Human microvascular ECs (MVECs) derived from lungs of PAH patients displayed enhanced expression of FHL2. Ectopic expression of FHL2 in MVECs significantly increased mRNA levels of BMPR2 and Id1. To substantiate these results, luciferase assays were performed using BMP response element reporter construct and found that FHL2 increases Id1 expression. Furthermore, overexpression of FHL2 decreased TNF-α induced expression of pro-inflammatory cytokines. Finally, ectopic expression of FHL2 reduced proliferation of ECs.

Conclusion

Our data indicate that FHL2 is a novel negative regulator of MVEC proliferation through enhancing BMP signaling and inhibiting inflammation. Modulation of FHL2 may potentially represent a novel therapeutic strategy for the treatment of PAH.
BMP9 attenuates IL-33 signaling and prevents endothelial-to-mesenchymal transition in pulmonary arterial hypertension
Authors: Becher C , Sanchez-Duffhues G , Goumans M J
Published: May 2024

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): Dutch Cardiovascular Alliance - PHAEDRA IMPACT

Background

Pulmonary Arterial hypertension (PAH) is a rare and progressive disease characterized by increased vascular resistance and remodeling of the pulmonary arteries. Disturbed Transforming growth factor (TGF)-β signaling, often associated with mutations in the gene encoding for the bone morphogenic protein receptor type II (BMPR2) and endothelial-to-mesenchymal transition (EndMT), emerges as a central culprit in the pathogenesis of PAH. Interleukin-33 (IL-33) has been identified as an inducer of EndMT, and its soluble receptor ST2 (sST2) has emerged as a potential biomarker for PAH. Bone morphogenic protein (BMP) 9, a BMPR2 ligand, showing a high affinity for endothelial cells, is being explored as a therapeutic approach.

Purpose

We hypothesize that a crosstalk between the TGF-β pathway and IL-33, a known promoter of vascular remodeling, occurs in PAH. This study aims to unravel the protective role of BMP9 in PAH, with a specific focus on the involvement of EndMT, a key player in the pathogenesis of vascular diseases.

Methods

The expression of IL-33 protein levels was assessed in Pulmonary Arterial Endothelial Cells (PAEC) and in lung tissues from experimentally induced PH (Sugen/Hypoxia (SuHx)) or control mice. Signal transduction and EndMT were investigated in PAECs exposed to IL-33, BMP9 and sST2, by gene expression analysis (qPCR), western blotting, ELISA and immunostaining. BMP9 and sST2 levels were measured in plasma from PAH patients by ELISA.

Results

Elevated IL-33 expression was observed in PAEC from PAH patients and lung tissues from SuHx mice compared to healthy controls. In vitro, BMP9 significantly induced the expression of the IL-33 soluble receptor ST2, while concurrently reducing IL-33 target gene expression. IL-33-induced EndoMT in PAEC was effectively mitigated by pre-incubation with BMP9, proving more efficacious than ST2 neutralizing antibody. Furthermore, a correlation between BMP9 and sST2 levels in plasma from PAH patients was identified.

Conclusion

Our findings demonstrate that BMP9 offers superior protections against IL-33-induced EndMT in PAECs compared to a ST2 neutralizing antibody. BMP9 induces expression of sST2, leading to IL-33 neutralization and subsequent EndMT inhibition. Therefore, targeting the IL-33 pathway in PAH patients presents a promising therapeutic option for preventing EndMT. This study provides novel insights into the pathogenesis of PAH, revealing a potential therapeutic target for this rare disease.BMP9 inhibits IL-33 downstream signalingBMP9 protects from IL-33-induced EndMT
Selective inhibition of Histone deacetylases reverses vascular remodeling and improves right ventricle function in pulmonary hypertension
Authors: Kurakula K B , Sun X , Berger R M F , Bonnet S , Bogaard H J , Goumans M J
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Longfonds and CVON

Background

Pulmonary arterial hypertension (PAH) is a progressive fatal disease characterized by abnormal remodeling of pulmonary vessels, leading to increased vascular resistance and ultimately right ventricle (RV) failure. Disturbed BMPR2/TGFβ signaling is a driver of disease progression. Inhibition of Histone deacetylases (HDACs) using small molecule inhibitors attenuated vascular remodeling, however, they worsened RV function in animal models of PAH.

Objectives

The aim of this study is to validate if the selective HDAC inhibitor Quisinostat reverses pulmonary vascular remodeling and improves RV function.

Methods and results

We found that protein levels of HDAC1 are increased in the lungs of PAH patients. Treating microvascular endothelial cells (MVECs) and smooth muscle cells of PAH patients with Quisinostat significantly decreased TGF-β signaling and strongly decreased cell proliferation and the levels of inflammatory cytokines. In the MCT-shunt and Sugen Hypoxia rat models of severe angioproliferative PAH, Quisinostat reversed abnormal vascular remodeling and improved RV function. Furthermore, Quisinostat could be combined safely with the contemporary PAH standard of care. Finally, Quisinostat improved the RV function in rats with RV pressure load induced by pulmonary artery banding, and no systemic or cardiotoxic effects were observed.

Conclusions

Our data demonstrate that selective inhibition of HDACs reverses pulmonary vascular remodeling and improves RV function. These data support the establishment of a clinical trial with Quisinostat in patients with PAH.
Exploring PD-1+ and tissue resident memory T cells in atherosclerotic diseases
Authors: Van Dijk K , De Jong A , Quax P H A , De Vries M R
Published: May 2024

Funding Acknowledgements

Type of funding sources: Other. Main funding source(s): Health Holland

Visual Sonics

Background

Checkpoint inhibitor therapy (ICI), a potent oncologic treatment, has been associated with an enhanced risk for atherosclerotic cardiovascular events and increased plaque progression. Among the key players affected by ICI are tissue resident memory T cells (Trm), a subset residing in tissues and characterized by CD44, CD69, PD-1, and CD103. Although Trms have been associated with chronic inflammatory conditions, their role in atherosclerosis and accelerated atherosclerosis, as observed in postinterventional diseases such as vein graft disease, remains elusive Here, we present an investigation into the role of PD-1 positive cells and Trms in accelerated atherosclerosis as found in vein graft disease.

Methods

Human carotid atherosclerotic plaques were stained with a MOVAT stain and scored to determine plaque stability. 25 Stable and 25 unstable plaques were stained for CD3+ T cells and PD-1.

Hypercholesterolemic ApoE3*Leiden mice underwent vein bypass surgery and were sacrificed at t7, t14, t28 (n=6 per time point). The vein grafts were processed for flow cytometry to quantify the Trms population, using markers including CD4+, CD8+, CD44+, CD25-, CD69+, CD103+ and PD-1+.

Hypercholesterolemic ApoE3*Leiden mice underwent vein bypass surgery. Biweekly, mice received ICI (anti-PD-L1) or control antibody (n=7). After 28 days vein graft morphology was assed using MOVAT staining and vein graft inflammation by staining MAC3 and CXCL10.

Results

Within human atherosclerotic plaques, PD-1-expressing T cells were predominantly located in the shoulder regions of the necrotic core. Unstable lesions exhibited significantly higher levels of PD-1 expressing CD3+ T cells compared to stable lesions (p=0.001). Furthermore, the percentage of PD-1 expression positively correlated with immune cell infiltration scores (p=0.019).

In murine vein grafts, Trms were identified through flow cytometry. A trend was observed indicating an increase in the number of CD4 and CD8 Trms at t14 compared to t7 Trms (CD8+, CD44+, CD69+/CD103+). At t28, the number of CD4 and CD8 Trms decreased compared to t14, with PD1+ expressing Trms showing a similar trend, peaking at t14.

Vein grafts in mice receiving anti-PD-L1 mAb showed no morphological differences compared to the control group. However, a significant difference in inflammation was noted in the anti-PD-L1 treated group, characterized by a higher number of macrophages (p=0.02) and increased expression of CXCL10 (p=0.008).

Conclusion

Here we demonstrate the association of PD-1+ T cells with unstable lesions and reveals that Trms increase during the inflammatory phase and decrease during the stabilizing phase of accelerated atherosclerosis. Trms emerge as potential detrimental targets of ICI therapy, which is characterized by increased inflammation.
Untangling the inflammatory responses of liver x receptor (LXR) activation in human macrophages
Authors: Zijlstra L F , Kempkes R W M , Giera M A , Rensen P C N , Kooijman S , De Winther M P J , Neele A E
Published: May 2024

Funding Acknowledgements 

Type of funding sources: Public Institution(s). Main funding source(s): Rembrandt Institute for Cardiovascular Science (grant 2022)

The regulation of cholesterol and fatty acid homeostasis is essential and the Liver X Receptor (LXR) plays a crucial role in this process. When activated, LXR promotes cholesterol efflux and suppresses the transcription of pro-inflammatory genes in mouse macrophages, making it a promising therapy against atherosclerosis. Studies indeed have shown that LXR agonism is effective in pre-clinical atherosclerotic mouse models. However, the limited data on human macrophages suggests that LXR activation may have additional pro-inflammatory effects in humans, and the mechanisms contributing to this are not yet fully understood.

To investigate the inflammatory properties of LXR activation in human macrophages, monocytes were isolated from buffy coats of healthy donors and differentiated into macrophages using M-CSF. The macrophages were exposed to synthetic and endogenous LXR ligands, such as GW3965, T0901317, and desmosterol, respectively, followed by stimulation with lipopolysaccharide (LPS) for six hours. We used qPCR and ELISA to analyze experiments and plan to supplement our data with RNA-Seq and multiplex assays.

Preliminary results show that LXR activation reduces LPS-induced activation of interferon-mediated genes like CXCL9 and CXCL10, similar to mice. However, it promotes the transcription of NF-kB target genes like IL1B and IL12B upon LPS activation. These findings suggest that LXR activation in human macrophages not solely suppresses inflammatory responses in macrophages and that LXR activation has different effects on the regulation of genes mediated by interferon or NF-kB. Our research aims to further uncover the regulatory mechanisms behind LXR activation in human macrophages as central regulators in atherosclerosis.
Liraglutide prevents hyperglycemia-induced senescence and proinflammatory monocyte differentiation in CD34+ hematopoietic stem cells
Authors: Damiano G , Rinaldi R , Pompilio G , Vinci M C
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health- Ricerca Finalizzata

Background

Diabetes mellitus (DM) is known to promote aberrant activation of immune cells that contribute to the onset and progression of chronic inflammation and atherosclerosis in DM patients (1). This phenomenon, termed 'trained immunity', appears to be initiated in hematopoietic stem/progenitor cells (HSPCs). In recent years, there has been great interest in investigating the pleiotropic effects of glucagon-like peptide-1 receptor agonists (GLP-1RAs), including anti-inflammatory and immunological properties. We have recently demonstrated that HSPCs express the GLP-1R whose stimulation with the agonist liraglutide (LIRA) prevents oxidative stress and functional impairment induced by hyperglycemic conditions (HG) (2). On this basis, we hypothesized that LIRA treatment can also avert HG-induced senescence and proinflammatory myeloid differentiation of HSPCs.

Purpose

To evaluate senescence and inflammatory myeloid differentiation of CD34+ HSPCs after HG exposure in the presence or not of GLP1R agonist.

Methods

CD34+ HSPCs derived from cord blood of healthy donors were expanded in normal-glucose (NG; with 30 mM mannitol for osmotic control) or high-glucose (HG; 30 mM) and treated or not with LIRA (100nM) in serum-free medium plus cytokines (FLT3, SCF, IL3 and IL6) for up to 20 days. The expression of p21, p27, IL6, TNFα, and NFkB-p65 genes was assessed by qPCR. ROS production and CD34+ HSPC-derived monocyte subsets were evaluated by flow cytometry whereas cytokine secretion after LPS stimulation was measured by ELISA assay.

Results

LIRA treatment showed to significantly prevent the impairment of CD34+ HSPC proliferation as well as the upregulation of senescence-associated secretory phenotype (SASP) genes such as p21, p27, NFkB-p65, IL6, and TNFα in HG-CD34+ HSPCs when compared to HG cell alone (one-way ANOVA; p≤0.05). Interestingly, these pharmacological effects were associated with a significant reduction in HG-induced ROS production. Myeloid differentiation in vitro of HG-CD34+ HSPCs generated monocytes composed mainly of an inflammatory and highly reactive intermediate (CD14++CD16+) subpopulation characterized by higher LPS-induced release of IL6 and TNFα. LIRA treatment significantly prevented both the accumulation of intermediate (CD14++CD16+) monocyte subpopulation from differentiating HG-CD34+ HSPCs and their inflammatory phenotype (one-way ANOVA; p≤0.05).

Conclusions

These results demonstrate that HG exposure alters the differentiation program of CD34+ HSPCs towards more inflammatory immune cell components and suggest that GLP1R agonists are endowed with immunomodulatory properties that could be further developed and exploited for the treatment of immune system dysregulation in diabetes.
The effects of siRNA-mediated knockdown of AP-1 on pulmonary arterial and right ventricular dysfunction associated with cardiac and pulmonary fibrosis in cardiopulmonary disease
Authors: Comarita I K , Anghelache I L , Tanko G , Georgescu A
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Romania’s National Recovery and Resilience Plan, PNRR-III-C9-2022-I8, CF 93/15.11.2022, Financing Contract no. 760063/23.05.2023, and by the Romanian Academy

Background

Pulmonary hypertension (PH) is a complex multifactorial vascular pathology characterized by an increased pulmonary arterial pressure, vasoconstriction, remodelling of the pulmonary vasculature, thrombosis in situ and inflammation associated with right-side heart failure.

Purpose

Herein, we explored the potential beneficial effects of treatment with siRNA AP-1 on pulmonary arterial and right ventricular dysfunction and perivascular and interstitial fibrosis in pulmonary artery (PA), right ventricle (RV) and lung in an experimental animal model of monocrotaline (MCT)-induced pulmonary arterial hypertension.

Methods

Golden Syrian hamsters were divided into: (1) C group; (2) MCT group obtained by a single subcutaneous injection of 60 mg/kg MCT at the beginning of the experiment; (3) MCT- siRNA AP-1 group received a one-time subcutaneous dose of MCT and subcutaneous injections containing 100nM siRNA AP-1, every two weeks. All animal groups received water and standard chow ad libitum for 3 months.

Results

In comparison with the MCT group, siRNA AP-1 treatment had significant beneficial effects contributing to: (1) a reduction in TGF-β1/ET-1/IL-1β plasma concentrations; (2) a reduced level of cytosolic ROS production in PA, RV and lung, and ultrastructural improvements of these tissues; (3) a decrease of inflammatory and fibrotic marker expressions in PA (COL1A, Fibronectin, Vimentin, α-SMA, CTGF, Calponin, MMP-9), RV (COL1A, CTGF, Fibronectin, α-SMA, F-actin, OB-cadherin) and lung (COL1A, CTGF, Fibronectin, α-SMA, OB-cadherin), and an increase of endothelial marker expressions (CD31/VE-cadherin) in PA; (4) structural and functional recoveries of PA (reduced VTI/Vel, restored vascular response) and RV (enlarged internal cavity diameter in diastole, increased TAPSE/PRVOFs) associated with a decrease in systolic/diastolic blood pressure, and heart rate; (5) a reduced protein expression profile of AP-1S3/ pFAK/FAK/pERK/ERK and a significant decrease in the expression levels of miRNA-145, miRNA-210, miRNA-21, and miRNA-214 along with an increase of miRNA-124 and miRNA-204.

Conclusion

The siRNA AP-1-based therapy led to an improvement of pulmonary arterial and right ventricular function accompanied by a regression of perivascular and interstitial fibrosis in PA, RV and lung and a down-regulation of key inflammatory and fibrotic markers in MCT-treated hamsters.