Funding Source Research Articles

Circulating LIGHT/TNFSF14 levels are negatively associated with cardiovascular mortality, while intra-plaque levels are associated with symptomatic cerebrovascular disease

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): The Swedish Heart and Lung Foundation, Åke Wiberg's Foundation Background Plaque rupture is the underlying cause of most cardiovascular events, such as stroke and myocardial infarction. Co-stimulatory molecules, the immune checkpoints, are emerging as strong candidates to battle atherosclerosis progression. The co-stimulatory molecule LIGHT (tumour necrosis factor 14; TNFSF14) exists in foam cell-rich regions of atherosclerotic plaques, but whether it is associated with a high-risk plaque phenotype and cardiovascular events is not known. Purpose This study investigates the association between intra-plaque and circulating LIGHT levels and cardiovascular disease. Method Plaque levels of LIGHT/TNFSF14 were measured in plasma and homogenised carotid endarterectomy plaques by Proximity Extension Assay in subjects from the Carotid Plaque Imaging Cohort (CPIP; N=557 and 201, respectively). Homogenates were further examined by multi-plex cytokine analysis and ELISA, and tissue plaque sections through immunohistochemistry. Multivariate Cox proportional hazards analysis was performed on normalized (logarithmically transformed) values. Results Plaque, but not circulating, levels of LIGHT were associated with the occurrence of pre-operative cerebrovascular symptoms (p=0.00014). Furthermore, intra-plaque levels of LIGHT correlated strongly with the histological plaque vulnerability index (p=5x10-15): a ratio between the sum of destabilising versus stabilising plaque components (i.e. macrophages, intra-plaque haemorrhage and lipids versus smooth muscle cells and collagen) as well as with necrotic core size and the inflammatory cytokines interleukin(IL)-1β, IL-6, chemokine (C-C motif) ligand (CCL)-2, CCL4 and CCL5. Moreover, plaque LIGHT levels correlated with several components of the extracellular matrix turnover machinery, including cleaved collagen, fibromodulin, ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs-7) and matrix metalloproteinases 1, 2, 9 and 10. While plaque LIGHT levels were not predictive of cardiovascular death, circulating LIGHT levels were negatively associated with risk of cardiovascular death (after 6 ½ years) after correcting for the possible confounders age, sex, cerebrovascular symptoms, diabetes, high-sensitivity C-reactive protein, inflammatory disease, hypertension and smoking (hazard ratio=0.032, confidence interval=0.420-0.951, p=0.028). Conclusion Though intra-plaque LIGHT levels are associated with a rupture-prone plaque phenotype and pre-operative symptoms, circulating levels are associated with a lower risk of future cardiovascular mortality. This suggest that upregulation of LIGHT in atherosclerotic plaques may occur as part of a reparative rupture response and, systemically, reflect efficient wound-healing capacity.

Nuclear eNOS interacts with and S-nitrosates ADAR1 to modulate endothelial gene expression

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Collaborative Research Centre (CRC 1366); Chinese Scholarship Council (CSC) Introduction and Aim Nitric oxide (NO) generated by the endothelial NO synthase (eNOS) regulates vascular tone and endothelial homeostasis to counteract vascular inflammation. The effects of NO are attributable to its interaction with heme-containing proteins e.g., soluble guanylyl cyclase, or to the S-nitrosation of proteins. Most eNOS is localized to the cell membrane or the Golgi apparatus, however, it has also been detected in the nucleus. In this study, we assessed the role of nuclear eNOS in endothelial cells. Methods and Results Confocal microscopy studies confirmed the presence of eNOS in the nucleus of unstimulated human and murine endothelial cells (ex vivo and in vitro) and stimulation with vascular endothelial growth factor (VEGF) increased eNOS nuclear translocation. Co-immunoprecipitation studies coupled with proteomics revealed the VEGF-dependent association of nuclear eNOS with 81 proteins involved in RNA binding and processing. Among these, nuclear eNOS bound double-stranded RNA-specific adenosine deaminase 1 (ADAR1), an enzyme involved in RNA editing via the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). ADAR1 was S-nitrosated in human endothelial cells and the knockdown of eNOS was associated with an increase in dsRNA (immunofluorescence) and altered ADAR1-mediated A-I editing (RNA seq). The accumulation of dsRNA in human endothelial cells lacking eNOS led to the activation of the interferon α/β signalling pathway (e.g., MX2, OASL, OAS1/2/3, RNASEL) and the downregulation of cell cycle-related genes. As a result, growth factor-stimulated cell proliferation was abrogated and basal as well as tumour necrosis factor- or H2O2-induced cell death were increased. Furthermore, the increased dsRNA in endothelial cells lacking eNOS elicited the activation of interferon-induced, dsRNA-activated protein kinase, which in turn phosphorylated the eukaryotic translation initiation factor 2A. Accordingly, new protein synthesis was inhibited following eNOS knockdown (immunofluorescence and mass spectrometry). Conclusion These results demonstrated that eNOS/NO signalling regulates endothelial type I interferon signalling and affects translation, cell cycle and cell death through S-nitrosation of ADAR1.

Cardiomyocyte-stromal cell interplay modulates the pathogenesis of arrhythmogenic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Transnational Research Projects on Cardiovascular Diseases Background Arrhythmogenic Cardiomyopathy (ACM) is an inherited heart disorder characterized by a high incidence of sudden death at young age. Mutations linked to ACM occur primarily in desmosomal genes (e.g. PKP2) [1]. Different cell types individually contribute to set the ACM phenotype with either functional abnormalities (cardiomyocytes; CM) [2]; or fibro-fatty substitution (cardiac mesenchymal stromal cells; cMSC) [3]. The relative contribution of the different cell type derangements to determine disease evolution and electrical instability is still unclear. Purpose We hypothesize that the use of a multicellular cardiac system, modelling the functional interaction between CM and cMSC, will allow the understanding of the relative contribution of the different cell types to the ACM phenotypes. Methods Primary cMSC (carrier of the PKP2 mutation c.2013delC) have been collected from a right ventricle biopsy sample; control cMSC obtained from a biobank. iPSC reprogrammed from the same ACM patient and the isogenic line in which the PKP2 mutation was corrected [4] were used. Cardiac cocultures (cc) were assembled by combining 85% iPSC-CM and 15% primary cMSC (as in Figure 1A), either mutated or control, in different combination (as in Figure 1B). Fibro-fatty accumulation have been evaluated by immunofluorescence analysis. Movies of contracting clusters have been collected and analysed by the MUSCLEMOTION tool [5]. Cell-cell communication was studied in silico by InterCellar analysis [6] on the single culture transcriptomes. The assessment of the paracrine interactions was performed on the cc secretome by Olink technology. Results Immunofluorescence analysis highlighted that ACM cc (cc 4) accumulate more lipids and collagen than healthy control (HC) ones (cc 1). Since intermediate fibro-fatty levels were observed in ‘mixed cc’ (cc 2 and 3), we conclude that ACM iPSC-CM are able to influence fibro-adipo-commitment of cMSC. Contraction studies showed a high propensity for cc monolayers which include ACM iPSC-CM (cc3 and cc4) to display arrhythmic events. An increase in the percentage of arrhythmic events was triggered when the ACM cMSC were replacing HC cMSC, indicating that ACM cMSC can affect iPSC-CM rhythm. The most abundant ligand-receptor couplets regarded cell-cell adhesion, cell-matrix coupling, cell differentiation and inflammation. Differential abundance between the four cc was observed mainly for secreted factors involved in cardiac fibrosis, inflammation, adipogenesis / lipid metabolism and heart failure. In particular, DLK-1, secreted only by HC CM, is validated as novel inhibitor of ACM fibro-adipose differentiation. Conclusion Overall, our novel simple multicellular ACM cell model can recapitulate different phenotypes of ACM and contributed to understand the relative contribution of the different cell types to ACM features (e.g. fibro-adipose replacement, contractile defects and proarrhythmic events).

LMNA p.Arg624His missense variant reduces lamin expression at mRNA level: elucidating molecular pathways toward cardiac involvement in laminopathies

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science and Innovation LMNA is a gene encoding lamin A/C proteins of the cell nuclear lamina. Pathogenic variants in LMNA cause a group of diseases known as laminopathies, a common cause of dilated cardiomyopathy (DCM). LMNA is the second most frequently mutated gene in DCM. Although molecular mechanisms underlying the cardiac involvement are unclear, previous studies have shown a reduction of the lamin expression in LMNA heterozygous mutation carriers. We identified a missense variant in LMNA, c.1871G>A (p.Arg624His), in a family affected with DCM. In this study, we analysed the impact of the p.Arg624His variant on the mRNA expression through quantitative PCR (qPCR) to demonstrate its pathogenicity. RNA was isolated from patient’s peripheral blood lymphocytes. Quantitative reverse transcription PCR (RT-qPCR) with LMNA specific primers was performed to measure lamin RNA levels. Whole-exome sequencing (WES) through next-generation sequencing (NGS) yielded an heterozygous missense variant in the LMNA gene (NM_170707.4(LMNA): c.1871G>A; p.Arg624His; ClinVar ID 66870) classified as variant of uncertain significance (VUS) in five members of a family affected with DCM. Computational (In-Silico) programs predict a damaging effect and variant is absent from controls in population databases. RT-qPCR demonstrated a significant reduction in the LMNA mRNA expression between the control (II.1) and affected family members (II.3, III.1) (Figure 2). According to evidence of co-segregation, variant frequency, results of in-silico predictors and mRNA reduction, LMNA p.Arg624His variant has been reclassified as pathogenic. In this study we reclassified to pathogenic a variant of uncertain significance in LMNA which reduces mRNA expression and leads to cardiac laminopathy. Further investigations are needed to determine the effect on the protein.

Identifying novel small molecule integrin agonists to mimic the favourable effects of the RGD domain of IGFBP-1 to treat cardiometabolic disease

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): British Heart Foundation (BHF) 4-Year PhD Programme Background Insulin-like growth factor binding protein (IGFBP)-1 has previously been shown to be associated with reduced type 2 diabetes (T2D) progression and preferable cardiovascular profiles in patients. Our laboratory previously demonstrated that IGFBP-1 is an insulin sensitiser in mouse models, in addition to having favourable vascular effects, independently of IGFs. IGFBP-1 has an Arg-Gly-Asp (RGD) domain which is thought to facilitate the direct interaction with the heterodimeric cell-surface receptor, integrin α5β1, inducing downstream cell signalling. We then showed the positive effects of IGFBP-1 could be mimicked with an RGD-containing linear peptide, GRGDTP, however this peptide is not a suitable therapeutic as it is rapidly metabolised in vivo. To our knowledge, RGD-based therapeutics have not been explored for cardiometabolic disease. Purpose There are currently few therapeutics which have been specifically designed to address both insulin resistance and the high risk of macrovascular disease in T2D. Therefore, exploring RGD-based integrin agonists will be essential in developing new insulin sensitising therapeutics which prevent cardiovascular complications. Methods The ROCS high speed ligand-based virtual screening tool was used to screen commercially available small molecule libraries mimicking the chemical structure of the RGD sequence of IGFBP-1. Additionally, we performed in silico modelling to predict the binding interactions of these molecules with the integrin α5β1 headpiece to select candidates. We then used a LIVE/DEAD cell viability assay to initially profile any cytotoxic effects of our selected compounds in integrin-expressing human umbilical vein endothelial cells (HUVECs) and C2C12 skeletal muscle myotubes. We further assessed the effects of compounds on relevant HUVEC functions using a high-throughput wound closure assay. Results The structure of the RGD domain of IGFBP-1 can be mimicked with small molecules containing amino acid moieties. These molecules are predicted to bind with micromolar affinity to the integrin, promoting beneficial interactions for integrin agonism. We selected 17 structurally diverse small molecules with the best predicted binding interactions. Initial in vitro profiling demonstrates that the molecules are not cytotoxic. In preliminary analyses, three of the selected compounds show a trend towards inhibition of HUVEC migration and proliferation, while six show a trend towards increased migration and proliferation. Conclusion The RGD domain of IGFBP-1 can be mimicked with novel small molecules, of which we have selected 17 ligands. Our compounds do not affect cell viability in relevant cell types, so can be further profiled. Our initial findings indicate that the compounds have different functional effects on HUVECs which allows us to prioritise hits having favourable effects in the context of T2D, such as enhancing angiogenic properties of endothelial cells.

Circulating cluster miR-99b/let-7e/miR-125a expression associates with endothelial function, cytokine, and chemokine levels in myocardial infarction patients

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Science and Innovation (ISCIII) co-financed by the European Regional Development Fund (ERDF), and by the Generalitat Valenciana Introduction During vascular remodelling following a myocardial infarction, miRNAs can modulate endothelial function in a post-transcriptional manner. Our study is focused on the cluster miR-99b/let-7e/miR-125a, examining its serum expression during the acute phase of a Non-ST-segment Elevation Myocardial Infarction (NSTEMI) and its expression one year later. Purpose All the miRNA miR-99b/let-7e/miR-125a cluster is hypothesized to impact endothelial function, specifically influencing cellular adhesion, proliferation, and vasculogenesis. Furthermore, it is hypothesized an association exists between the levels of miRNAs from this cluster during the acute phase of myocardial infarction and the serum levels of inflammatory cytokines and chemokines implicated in the pathology. Methods Serum RNA was extracted from control (n=41), acute NSTEMI phase (n=46), and one-year follow-up (n=24) patients. miRNA expression was quantified by qRT-PCR and miR-484 was used as endogenous control. Endothelial function was assessed by analyzing adhesion, proliferation, and vasculogenesis in human umbilical vein endothelial cells (HUVEC) and transfected with miRNA inhibitors and mimics. Serum levels of proinflammatory cytokines (IL-1β, IL-6, TNF-α, and VEGF-A), anti-inflammatory cytokines (IL-10 and IL-13), and chemokines (MCP-3, MDC, IL-8, and MIP-1α) were determined. Results The miRNA cluster components exhibited decreased expression during the acute phase of the infarction (p< 0.05) and returned to control levels after one year of follow-up. All the cluster components were high expressed in endothelial cells, and we determined the effect of these miRNAs or their inhibition on endothelial function. let-7e mimic increased (p< 0.05) cell adhesion, as well as the formation of tubular networks (p< 0.001). miR-125a inhibitor decreased cell proliferation (p< 0.05). Neither the mimic nor the inhibitor of miR-99b had effects on this endothelial function. let-7e exhibited a negative correlation with the proinflammatory cytokine TNF-α (r= -0.19, p= 0.0386) and a positive correlation with the levels of the anti-inflammatory cytokine IL-13 (r= 0.22, p= 0.0416). miR-99b showed a positive association with the chemokine MDC (r= 0.22, p= 0.0195), while the levels of miR-125a did not correlate with any of the analysed mediators. Conclusions miR-99b/let-7e/miR-125a cluster is implicated in NSTEMI and exhibits a consistent expression pattern in patients' serum, decreasing during the acute phase and returning to control levels one year after. Each of these miRNAs, especially let-7e and miR-125a, differentially affects cellular processes such as endothelial proliferation and angiogenesis. Additionally, let-7e and miR-99b miRNAs differentially impact the inflammation generated in NSTEMI. These results suggest the involvement of miRNAs from the same cluster in various processes that are coordinately activated in inflammatory situations, such as acute coronary syndrome.

Generation of isogenic allelic series of LMNA-mutated hiPSC lines using the novel and highly-efficient targeting platform, STRAIGHT-IN

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Netherlands Organisation for Health Research and Development ZonMW Background Human induced pluripotent stem cells (hiPSCs) have demonstrated enormous potential for advancing in vitro disease modelling and genetically modified hiPSCs carrying disease-associated variants are particularly useful tools for this purpose. Until now, strategies for efficient and rapid integration of large DNA payloads (>10 kb) into specific genomic regions are still limited, thus precluding efficient and rapid targeting of hiPSC lines. Purpose and Methods To overcome this, the STRAIGHT-IN platform (Serine and Tyrosine Recombinase Assisted Integration of Genes for High-Throughput INvestigation) was developed that combined different classes of site-specific recombinases with CRISPR-Cas9 mediated homologous recombination and allowed stringent site-specific replacement of large genomic fragments in hiPSCs. Here, we use STRAIGHT-IN to simultaneously generate a library of genetically matched hiPSC lines carrying multiple heterozygous mutations in LMNA gene. Mutations in LMNA, encoding Lamin A/C protein, have been associated with 5-10% cases of dilated cardiomyopathy (DCM) with conduction defects. Results Through detailed online searches of the LMNA database and literature, we identified and selected 11 LMNA mutations to insert into a wild-type hiPSC line based on the following criteria: (i) mutations associated with cardiac abnormalities, in terms of structural (DCM/heart failure) and arrhythmic phenotypes; (ii) mutations with known familiar/pedigree-relationship; (iii) different types of mutations (missense and nonsense). We then applied the workflow of STRAIGHT-IN consisting of: (1) replacing the entire LMNA genomic locus (33 kb) on one allele with a Landing Pad (LP) cassette containing Serine Recombinase (Bxb1) recognition sites; (2) reintroducing LMNA gene into the LP cassette through integration of Bxb1-Donor plasmids carrying the LMNA variants of interest via Bxb1 expression; (3) expressing a tyrosine recombinase (Cre) to excise the majority of the auxiliary exogenous DNA sequences. Conclusions STRAIGHT-IN allows simultaneous generation of a panel of 11 isogenic hiPSC lines carrying selected LMNA mutations rapidly (5-6 weeks), efficiently and cost-effectively, thus facilitating the production of specific mutated hiPSC lines for disease modelling and personalized medicine applications.

Therapeutic inhibition of biomechanically-activated YAP-TAZ signaling prevents vein graft atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Rembrandt Institute for Cardiovascular Sciences Background Venous bypass grafts may have limited patency due to excessive intimal hyperplasia and accelerated atherosclerosis. YAP-TAZ signaling regulates cellular responses to biomechanical cues, such as disturbed flow or vessel distention as seen in vein grafts. YAP activation leads to inflammation and angiogenesis, which both contribute to vein graft atherosclerosis. Inhibition of YAP-TAZ attenuates naive atherogenesis, but its potential to inhibit intraplaque angiogenesis and reduce vein graft atherosclerosis has not been explored. Methods Saphenous vein progenitor cells (SVPs) were subjected to mechanical strain in vitro to assess YAP activation. IHC to assess YAP-expression was performed on vein grafts from pigs as well as ex vivo cultured human saphenous veins. Furthermore, YAP-IHC was performed on murine vein grafts from hypercholesterolemic ApoE3*Leiden mice harvested at early, mid- and late-stage disease timepoints to assess optimal treatment window. In a separate experiment, mice (n=11-13/group) underwent bypass surgery and were treated with the FDA-approved YAP-TAZ inhibitor Verteporfin (50 mg/kg) or vehicle 3 times/week from day 10 until sacrifice (day 28). Ultrasound was used to longitudinally quantify vascular remodeling. Vein grafts were harvested and processed for morphometric and compositional analysis. Results Mechanical strain induced activation of YAP and its downstream targets (Ctgf, Cyr61) in SVPs, which was inhibited by Verteporfin (confirmed by WB, IP and qPCR). Moreover, YAP was abundantly expressed in pig vein graft lesions, whilst Verteporfin blocked YAP expression in ex vivo cultured human saphenous veins after exposure to disturbed flow. In addition, YAP was increasingly expressed during vein graft lesion development in mice, reaching its peak around day 14, which coincides with the initiation of intraplaque angiogenesis. Furthermore, YAP was predominantly expressed in luminal and adventitial endothelial cells. Analysis of ultrasound demonstrated that Verteporfin prevented vein graft thickening in mice over time. End-point histology revealed that the intima/media ratio was significantly decreased by Verteporfin treatment (37%, p=0.001). Intraplaque angiogenesis was also reduced (34%, p=0.002), whilst neovessel maturation was improved upon verteporfin treatment. The vessel wall ACTA2 content was reduced (42%, p=0.023), whilst collagen was not altered between both groups, indicating that Verteporfin diminished the fibroproliferative response without affecting vessel wall stability. Conclusion Verteporfin inhibited YAP-TAZ activation induced by mechanical strain in vitro (SVPs) as well as ex vivo (cultured human saphenous veins). In vivo, Verteporfin treatment resulted in a significant decrease of vein graft atherosclerosis and intraplaque angiogenesis. Therefore, therapeutic targeting of YAP-TAZ using Verteporfin might be a new candidate to improve vein graft patency.

Increased oxidative stress alters coronary microvascular tone in exercising swine with multiple comorbidities

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Cardiovascular Alliance Background/Objectives We have previously shown, in female swine that the combination of three cardiovascular risk factors (diabetes mellitus (DM), hypercholestolemia (HC) and chronic kidney disease (CKD)) induces systemic inflammation, oxidative stress, coronary endothelial dysfunction and impaired myocardial perfusion due to impaired nitric oxide (NO) bioavailability. In the present study we hypothesized that the reduced bioavailability of nitric oxide (NO) is the result of increased production of reactive oxygen species (ROS) and ROS scavenging will restore myocardial oxygen balance thereby improving myocardial perfusion. Methods 12 female swine, with DM (streptozotocin 3x50mg/kg iv), CKD (renal embolization), and HC (high fat, high sugar, high salt diet) and 9 female healthy swine on normal pig chow (Normal) were included. The effect of ROS scavenging on the coronary microvascular tone control was studied in vivo, at rest and during graded treadmill exercise before and after infusion of the ROS scavengers (MPG+Tempol). Additionally, vasodilation to bradykinin was studied in the presence of MPG and TEMPOL and after subsequent addition to catalase, in isolated left ventricle small coronary arteries, in vitro using wire-myography. Results 6 months of DM+HC+CKD resulted in hyperglycemia (18.0±1.3 vs 8.4±0.9mmol/L), hypercholesterolemia (13.4±2.1 vs 1.7±0.1mmol/l), renal dysfunction (plasma creatinine: 166±9 vs 119±3µmol/l), and systemic inflammation (TNFα: 63±8 vs 41±4pg/ml, all P<0.05) associated with impaired endothelium-dependent vasodilator response to bradykinin in vitro. Furthermore, myocardial oxidative stress was increased in DM+HC+CKD animals (8-isoprostane 13±1 vs 10±1pg/ml) and total antioxidant capacity was reduced (0.67±0.02 vs 0.72±0.01nmol Trolox eq./mg protein, both P<0.05). Surprisingly, in vivo ROS scavenging resulted in an even further increase in myocardial oxygen extraction in DM+HC+CKD while it had no effect in Normal, indicating the involvement of a vasodilator ROS (most likely H2O2) in the control of coronary microvascular tone. This was supported by increased activity of catalase in the myocardium of DM+HC+CKD animals (44±3 vs 31±4nmol/min/ml, both P<0.05) and the vascular relaxation response to bradykinin when catalase was added on top of the ROS scavenging in vitro. Conclusion In swine exposed for 6 months to multiple comorbidities, impaired myocardial perfusion is mediated by a loss of NO bioavailability due to increased oxidative stress which was partially compensated by increased H2O2-mediated coronary vasodilation.

HiPSC-derived cardiomyocyte cultivation and measurement of electrophysiological properties on gelatine/glucose/polypyrrole scaffold

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): EU Horizon 2020 Framework Programme (H2020) under Grant agreements No. 953138 (EMAPS-Cardio) Purpose Cardiotoxicity is one of the main side effects limiting development of new medicines and resulting in withdrawal of drugs from the market. There are different mechanisms of drug-induced cardiotoxicity, including arrhythmia, disruption of mitochondria function, apoptosis, altered growth factor signalling [1]. Heart on a chip model for drug testing seems attractive approach to evaluate the possible cardiotoxic effects on cell biological, but mostly electrophysiological properties. Our aim was to create a 3D system with a biocompatible scaffold, coated with a conductive layer, suitable for culturing and maturation of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) and compatible with electrophysiology parameter measurement techniques. Methods Elastic scaffold was fabricated by electrospinning of gelatin-glucose microfibers followed by the vapor-phase and electrochemical deposition of conductive polymer polypyrrole (PPy) to create electroconductive layer. hiPSC-CMs at different maturation levels (at 6 vs 14 weeks) were used to test biocompatibility of the scaffold. To increase cell attachment, scaffold was treated with 0.1 M HCl and coated with Geltrex. Cells were stained with fluorescent dyes (Calcein AM, DAPI). Electrophysiological properties were analysed using intracellular calcium imaging (Cal520) and action potential recordings with sharp electrodes. HiPSC-CMs on coverslips were used as a 2D control. Results Calcein AM staining showed that high number of hiPSC-CMs attached to gelatine/glucose/PPy scaffold proving that the scaffold is biocompatible and cells remain viable even after long term incubation (> 2 weeks). HiPSC-CMs at lower maturity (6 weeks) had better attachment properties to it, indicating that more mature cardiomyocytes (14 weeks) gradually lose their capacity to attach to PPy surface. Intracellular calcium imaging showed that both types of cells generated synchronous spontaneous calcium oscillations on gelatine/glucose/PPy scaffolds. Action potential recordings in 3D were similar to those in 2D, and confirmed differences in hiPSC-CMs maturation levels – at lower maturation level hiPSC-CMs had lower upstroke velocity as compared to more matured hiPSC-CMs. Conclusions Gelatine/glucose/PPy scaffold is biocompatible for hiPSC-CMs cultivation, and suitable for evaluation of cardiomyocyte electrophysiological properties. Such electromechanoactive scaffold can be further employed for development of heart-on-a-chip model.

Decreased atherosclerosis and abdominal aortic aneurysm in mice deficient in polymeric immunoglobulin receptor

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): La Caixa Banking Foundation and Comunidad de Madrid Background Polymeric immunoglobulin receptor (PIGR) is a transmembrane protein involved in the transcytosis of polymeric immunoglobulins that is widely expressed in mucosal epithelial cells. Recent data from our group described increased PIGR levels in plasma and tissue of atherosclerotic subjects. However, the role for PIGR in the mechanisms involved in vascular remodeling has not been addressed. Purpose To analyse the role for PIGR in experimental mouse models of atherosclerosis and abdominal aortic aneurysm (AAA). Methods We analyzed PIGR expression, secretion and distribution in early atherosclerotic plaques and AAA tissues compared to healthy aortic samples, as well as in macrophages derived from the human monocytic THP-1 cell line. Next, we studied the effect of global PIGR deficiency in experimental atherosclerosis (Ldlr-/- Pigr-/- mice fed with atherogenic diet for 10 weeks). In addition, the contribution of hematopoietic PIGR was assessed in both experimental atherosclerosis and AAA (Ldlr-/- mice fed with atherogenic diet and 1 µg/Kg/min angiotensin II infusion for 28 days) by bone marrow transplantation experiments. Finally, bone marrow-derived macrophages (BMDMs) from Pigr-/- and control mice were isolated to assess their inflammatory expression profile and modulation of polarization in response to oxidized LDL (ox-LDL), as well as their ability to promote foam cell formation. Results PIGR expression was increased in the intima of early human atherosclerotic lesions and showed colocalization with CD68+ expression. PIGR expression was also increased during the in vitro differentiation of THP-1 monocytes to macrophages with phorbol myristate acetate. Both global and hematopoietic-specific PIGR deficient mice displayed reduced plaque size in the aortic sinuses as well as a strong decrease in macrophage infiltration (CD68+ cells), while no changes in smooth muscle cells (α-SMA+ cells) and collagen content were observed. Regarding AAA, increased PIGR levels were found in human AAA tissues, associated with infiltrating CD68+ cells. PIGR deficiency in hematopoietic cells also showed significantly reduced AAA dilatation and reduced macrophage infiltration. In vitro experiments with BMDMs obtained from Pigr-/- mice and stimulated with ox-LDL showed reduced proinflammatory gene expression (TNF-a and IL-6) and M1 markers (iNOS and CD11c) as well as diminished foam cell formation. Conclusions PIGR deficiency in hematopoietic cells decreases atherosclerosis and AAA progression by modulating macrophage inflammatory responses, suggesting a novel proinflammatory role for PIGR in vascular pathologies. Future experiments will test the potential therapeutic effect of modulating PIGR to dampen the inflammatory profile in vascular diseases.Results in human and mouse studies

The RGK protein rrad regulates cardiac calcium levels in zebrafish

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science, Innovation and Universities, Spain Background/Introduction High voltage-activated Ca2+ (CaV) channels are essential for excitation-contraction coupling. RGK proteins (Gem, Rad, Rem, and Rem2) are small Ras-like G proteins that bind CaV β-subunits and profoundly inhibit CaV1/CaV2 channels. A recent study showed that Rad inhibits CaV1.2 channels in cardiomyocytes, thus mediating the enhanced inotropy and chronotropy by β-adrenergic agonists during the fight-or-flight response. The zebrafish has emerged as a useful in vivo animal model in cardiovascular physiology and pathophysiology. The role of zebrafish rrad, the orthologe of the human RAD, in the regulation of cardiac Ca2+ homeostasis in zebrafish has not been investigated. Purpose The aim of this study was to investigate the role of rrad in cardiac Ca2+ regulation in zebrafish. Methods We evaluated the cardiac Ca2+ dynamics, heart rhythm, and hemodynamics in transgenic zebrafish larvae that overexpress rrad in the heart, as well as in heterozygous rrad knockout larvae. Measurements were done in the beating heart of 3 dpf transgenic zebrafish larvae expressing the fluorescent Ca2+ biosensor Twitch-4. Results In some larvae, overexpression of rrad induced a severe enlargement of the atrium with pericardial edema and a high mortality rate. In addition, the ventricle displayed low Ca2+ levels and reduced contractility. The remaining larvae had no altered morphology, but the ventricular Ca2+ levels were reduced. On the other hand, preliminary results showed that down regulation of rrad in zebrafish larvae increased atrial and ventricular Ca2+ levels, like what was found in Rad knockout mice. Conclusion Taken together, these findings suggest that rrad in the zebrafish heart, as in mammals, is an inhibitor of the Ca2+ entry, presumably through CaV1/CaV2 channels, and demonstrate the zebrafish's utility as a model to study the involvement of rrad in cardiac function.

Deficiency of Abca1 and Abcg1 mediated cholesterol efflux pathways in smooth muscle cells enhances vasoconstriction but does not affect atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): VIDI grant from Netherlands Organization of Scientific Research (NWO). Rosalind Franklin Fellowship from the University Medical Center Groningen. Both to Marit Westerterp, PhD Smooth muscle cells (SMCs) regulate blood flow distribution and blood pressure via vasoconstriction mediated by α-adrenergic receptors (α-ARs). Plasma membrane cholesterol may affect α1-AR signaling, but consequences for SMC-mediated vasoconstriction are unclear. Cholesterol loading promotes SMC-to-macrophage transition in vitro, which may enhance atherosclerotic plaque vulnerability. The cholesterol transporters ATP Binding Cassette A1 and G1 (ABCA1/ABCG1) are highly expressed by SMCs and mediate cholesterol efflux to apolipoprotein A1 and high-density lipoprotein (HDL), respectively. The role of ABCA1/ABCG1-mediated cholesterol efflux pathways in SMC-mediated vasoconstriction and atherogenesis remains poorly understood. We generated mice with SMC-specific Abca1/Abcg1 deficiency on the low-density lipoprotein receptor deficient (Ldlr-/-) background by crossbreeding Abca1fl/flAbcg1fl/flLdlr-/- mice with Myh11-CreERT2 transgenic mice and feeding them tamoxifen-diet. To induce SMC cholesterol accumulation and atherogenesis, we fed Myh11-CreERT2Abca1fl/flAbcg1fl/flLdlr-/- and Myh11-CreERT2Ldlr-/- mice Western-type diet (WTD) for 16 weeks. Combined SMC-Abca1/Abcg1 deficiency increased vasoconstriction in aortic rings induced by the α1-AR agonist phenylephrine, with reversal by methyl-β-cyclodextrin, substantiating its cholesterol-dependency. Unexpectedly, SMC-Abca1/Abcg1 deficiency induced urinary bladder enlargement by >20-fold, resembling bladder outlet obstruction (BOO). This was reversed by the α1-AR antagonist tamsulosin, indicating its dependence on bladder neck SMC constriction. Moreover, SMC-Abca1/Abcg1 deficiency decreased SMC markers and increased macrophage- and fibroblast-markers in the bladder wall, enhancing collagen deposition, consistent with SMC transdifferentiation. However, after 16 weeks WTD, SMC-Abca1/Abcg1 deficiency did not affect atherosclerotic lesion size, fibrous cap thickness, necrotic core, collagen, or macrophage content, suggesting that SMCs in atherosclerotic plaques were not affected. This may be due to low Abca1/Abcg1 expression in intimal compared to medial SMCs. We uncover a new role of SMC cholesterol efflux pathways in suppressing α1-AR mediated vasoconstriction and bladder SMC transdifferentiation, decreasing BOO. Our data may provide a mechanistic link for the association between BOO and diabetes in humans, particularly because diabetes is associated with decreased cholesterol efflux. SMC-Abca1/Abcg1 deficiency did not affect atherosclerotic lesion size or plaque composition, presumably due to low Abca1/Abcg1 expression in intimal SMCs of atherosclerotic lesions, as shown in mice and humans.

Endothelial cellular response to oxidative stress key to proteasome inhibitor cardiovascular toxicity profile

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Karolinska Institutet Background/Introduction Proteasome inhibitors (PI) have contributed to longer survival in multiple myeloma although PI related cardiovascular adverse events are common. Associated to carfilzomib hypertension, coronary ischemic events and heart failure (both with reduced and preserved left ventricular ejection fraction). Here we explore the effect of PIs on endothelial gene expression. Methods Human aortic endothelial cells (HAEC) were incubated for 24 h with carfilzomib or bortezomib at different concentrations. Gene expression determined using using bcl2fastq (v2.20.0), and read quality was assessed using FastQC (v0.11.8). For each group the 100 genes with highest ratio were used for analysis of gene ontology (GO), biological processes using ClusterProfiler. The carfilzomib / bortezomib >1.5 was determined in the relevant GO groups. Results PIs induce gene expression related to inflammatory response and oxidative stress, more pronounced for carfilzomib. 44 / 31 genes related to "cytokine-mediated signaling pathway" and "response to oxidative stress pathway. NR4A3 was 15.5 fold above control while its increase with bortezomib was modest. Discussion Both carfilzomib and bortezomib induce genes related in inflammation and oxidative stress. Carfilzomib induced more pronounced upregulations in several genes, that stimulates expression of increased cellular respiration. Our result show a differentiated effect on endothelial cell gene expression by carfilzomib and bortezomib -a possible explanation to the differences in cardiovascular toxicity profile. Figure 1. Selected GO, Biological function characteristics calculated on the top 100 fold-change genes in each group. C2 high dose carfilzomib, B1 and B2 low and high dose bortezomib. C1 did not converge to any GO characteristics.

Fast but not furious: neonatal cardiac fibroblasts as promoters of regeneration after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Fundação para a Ciência e Tecnologia (FCT) Introduction Mouse neonates at postnatal day 1 (P1) regenerate their hearts after myocardial infarction (MI) but this capacity is transient. Seven day-old animals (P7) develop a reparative response similar to adults, with ischemic tissue being progressively replaced by a rigid fibrotic scar mainly produced by activated cardiac fibroblasts (CF). Recent works support that CF are likely to play an important role in mediating the regeneration-to-repair transition, but the current knowledge is limited. Aims Our final goal is to unravel CF-mediated mechanisms that confer regenerative potential to the neonatal heart and later reactivate these processes in the context of adult MI. Methods Ventricles from non-manipulated mice at different developmental stages were subjected to targeted RNA-seq. CF were isolated by FACS from P1 (regenerative) and P7 (reparative) hearts and characterized at the transcriptional, phenotypic and functional level in vitro. MI was induced by permanent ligation of the left anterior descending coronary artery at P1 and P7. CF and immune cell dynamics were assessed at different timepoints after MI by flow cytometry and immunofluorescence (IF). Transcriptomics of FACS-sorted CF from sham-operated and infarcted hearts at P1 and P7 were assessed by targeted RNA-seq. Intramyocardial injections of either P1 or P7 CF in MI-operated P1 hearts were performed immediately after surgery and hearts were analysed by IF at 7 and 14 days after injury. Results Severe transcriptional alterations on extracellular matrix (ECM) genes were found between P1 (regenerative) and P7 (reparative) hearts. Concomitantly, CF were shown to progressively infiltrate the myocardium and undergo a phenotypic shift that recapitulates transcriptional alterations observed for ECM-encoding genes in the heart. In vitro, CF at P7 show increased proliferation, migration capacity and TGF-β responsiveness. After MI at P1, CF participate in the regenerative response through a fast and transient recruitment to the ischemic site, returning to basal levels at day 7, a period in which CM proliferation and neovascularization are upregulated. Contrarily, MI at P7 resulted in permanent loss of CM, impaired neovascularization and formation of aberrant fibrosis as a result of exuberant and persistent CF recruitment and activation. Whole transcriptomic analysis further showed CF from MI at P7 are highly reactive to injury and engage on the expression of ECM components. The injection of CF resulted in a reduced heart weight and accumulation of cells in the injury site at 7 and 14 days after MI, which suggests a delay in regeneration. Conclusion Physiological CF development after birth entails a shift from a pro-regenerative to a pro-fibrotic phenotype that impacts on the regenerative capacity of the heart and determines the persistent CF activation and permanent scarring after P7.

Cardioprotective effect of cemtirestat treatment in male Zucker diabetic fatty rats: a pilot study

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Slovak Research and Development under the contract no. 21-0410 (APVV) and 20-0411 (APVV) and VEGA grant no. 2/0006/23 Introduction Type 2 Diabetes Mellitus (T2DM), the most common metabolic disorder in human population is caused by defective insulin secretion and reduced tissue responsiveness to insulin. T2DM is characterized by hyperglycaemia accompanied by oxidative stress and inflammation that results in organopathy. The Zucker diabetic fatty rats (ZDF) are established animal model for research of T2DM associated with obesity, and for efficacy of novel treatments. Myocardial electrical coupling protein, connexin43 (Cx43) is one of the key factors affecting heart function and arrhythmogenesis. We have previously shown upregulation of Cx43 in type 1 DM rats that was associated with reduced susceptibility to malignant arrhythmias. However, it is not known whether T2DM affects Cx43 expression and/or function in the heart as well as in brown adipose tissue (BAT). The purpose of our study was to examine expression of Cx43 in heart and BAT as well as the effect of treatment with highly selective and efficient aldose reductase inhibitor enriched with antioxidant activity, Cemtirestat. Methods: Forty 4-month-old male ZDF rats (fa/+ and fa/fa) were divided into 4 groups: 1) untreated control lean rats (fa/+), 2) control lean rats treated with Cemtirestat, 3) untreated diabetic fatty rats (fa/fa), 4) diabetic fatty rats treated with Cemtirestat (2,55 mg/kg/day). After 6 months of treatment, biometric parameters were registered and left ventricle (LV) + BAT were used for Western blot analysis of Cx43, PKCɛ (phosphorylates Cx43) and PKCδ (implicated in pro­apoptotic signaling). BAT and LV tissue sections were also used for immunolabeling of Cx43 to examine its topology. Results: Increase of body weight, heart weight, LV weight, adiposity, plasma triglycerides, cholesterol and blood glucose were registered in ZDF fa/fa rats. T2DM resulted in altered localisation of Cx43 that was not affected by treatment with Cemtirestat. Expression of Cx43 was significantly decreased in LV of fa/fa rats and restored with Cemtirestat. Moreover, BAT exhibited significantly lower expression of total Cx43 in fa/fa rats, that was moderately increased by Cemtirestat. The expression of PKCɛ was significantly decreased in both, LV and BAT of fa/fa rats with mild elevation of PKCɛ in LV of treated fa/fa rats. Cemtirestat slightly normalized increased expression of PKCδ in LV of fa/fa rats, but had no effect on lower PKCδ levels in BAT of fa/fa rats. Echocardiographic examination did not show significant difference between treated and untreated groups of rats and showed mild differences between diabetic and control rats. In conclusions, findings point out that T2DM downregulates Cx43 in the heart and BAT, however further studies are necessary for understanding the mechanisms implicated in every step in the development of T2DM cardiopathy and arrhythmogenesis as well as BAT alterations. Further research is required to determine factors involved in cardioprotective effects of Cemtirestat.

Meteorin-like regulates cardiac hypertrophy and associated metabolic plasticity during gestation and lactation

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de investigación y ciencia de España Introduction Pregnancy-induced cardiac hypertrophy is considered a physiological, reversible process (1, 2). However, cardiac remodeling after delivery and through lactation, as well as mechanisms driving hypertrophy reversion, remain undefined. In recent studies, we described the cardioprotective role of Meteorin-like (Metrnl) during pathological remodeling of the myocardium (3). Its role during gestation and lactation in the maternal heart is completely unknown. Purpose To characterize pregnancy-induced cardiac hypertrophy and its underlying mechanisms, and to determine the role of Metrnl in the cardiac remodeling associated to pregnancy and lactation. Methods C57BL/6J wild-type (WT) and Metrnl knockout (Metrnl-KO) female mice were studied during gestation, lactation and weaning. Echocardiographic measurements and cardiac samples were obtained from virgin control animals (V), pregnant mice at term (E18), lactating females at days 14 and 21 (L14 and L21), and mice 7 and 21 days after L21 weaning (W7 and W21). Results In WT females, pregnancy induces eccentric cardiac hypertrophy characterized by a major dilation of the left ventricle, a phenomenon that is maintained and further increased during lactation, reaching the highest degree of eccentric hypertrophy by L14. After weaning, hypertrophy is totally reversed (Figure 1A). By contrast, Metrnl-KO mice show a more pathological cardiac phenotype characterised by an exacerbated eccentric hypertrophy development accompanied by reduced ejection fraction. Post-weaning resolution is also delayed compared to WT. Next, we analysed cardiac metabolism since during the latest stages of gestation, the heart relies more on fatty acid utilization due to the increased glucose demands of the foetus (Figure 1B). We found that in WT mice, cardiac expression of genes linked to fatty acid oxidation (FAO; Acadm, Pdk4 and Cpt1b) is increased during gestation and repressed during lactation in parallel with cardiac Metrnl expression, recovering basal levels by W7. By contrast, in Metrnl-KO mice mitochondrial pyruvate transporter Mpc1 expression is increased during the whole process, while Pdk4 and Cpt1b protein levels are lower, suggesting a reliance of these hearts on glucose metabolism during pregnancy and post-partum. Conclusions The maternal heart develops an adaptive pregnancy-induced cardiac hypertrophy, a condition that initiates at gestation, peaks by day 14 of lactation and starts reverting upon weaning. Metrnl is necessary for the correct development of cardiac adaptations associated with pregnancy/lactation.Figure 1

Macrophage transcriptome changes in response to cardiac injuries are determined by the type of lesion rather than their ontogenetic nature

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): CRC1425 - DFG Introduction Resident macrophages account for 5% of the cells in the healthy heart. In response to cardiac injury, monocytes infiltrate and differentiate into recruited macrophages complementing the original resident macrophage population, and together orchestrate cardiac remodeling. We aim to identify the extent to which macrophage origin, tissue location and type of cardiac injury determine macrophage phenotypes in ischemic and non-ischemic cardiac injuries over time. Results and methods We use a reporter mouse line CX3CR1Yfp CreER/+:R26tdT/+ to visualize and quantify fluxes of resident and recruited macrophages and their respective localizations within the heart following ischemia and reperfusion (I/R) injury and pressure overload after transversal aortic constriction (TAC), respectively. Interestingly, macrophage numbers peaked during the first week post-surgery in both the local/acute and the global/continuous injury models. Initially, recruited macrophages outnumbered the resident macrophage pool within the infarct area but ultimately reached a near 1:1 equilibrium at 4 weeks post I/R injury. In the remote myocardium the 1:1 equilibrium established already during the first week. In TAC-injured hearts, the 1:1 ratio of recruited and resident hearts also established within the first week post-surgery and persisted up to 8 weeks of follow-up even as cardiac function deteriorated. In the infarct, recruited macrophages showed increased cytokine synthesis, defensive response, and cell adhesion pathways during the first week after injury. Many inflammatory pathways remained activated up to day 28 post MI despite simultaneous activation of the anti-inflammatory response. Formerly resident macrophages and their progeny also mounted an immune response throughout the healing process. Unexpectedly, by day 28 post MI when a stable scar had formed, resident and recruited macrophage population transcriptomes converted onto a common, pro-inflammatory steady state phenotype, that differed from their originally divergent gene expression profiles in the healthy heart by over one thousand genes. In the remote zone, however, resident macrophages maintained cellular homeostasis function, unlike recruited macrophages that retained an inflammatory program. Following TAC surgery, transcriptional profiles remained differentially regulated at all time points tested, where recruited subset induced immune response and resident macrophages upregulated tissue remodeling genes. Conclusion Ischemic and non-ischemic cardiac injuries induce a transient peak of monocyte recruitment and macrophage differentiation in and around cardiac lesions which lead to a permanent integration of recruited monocyte-derived macrophages into the pool of tissue resident macrophages – albeit at different paces. The type of injury and macrophage localization within the changing tissue microenvironment determine partial or complete override of ontogenic cell programs.

Modulating sialylation as therapeutic approach for preventing age-associated cardiac dysfunction

Abstract Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): Ministry of Health Voltage-gated sodium channel Nav1.5 plays a pivotal role in the proper functioning of cardiac muscle cells. Sialylation, a post-translational modification involving the addition of negatively charged sialic acid moieties, is essential for Nav1.5 functionality. Altered sialylation has been observed in various cardiovascular diseases, including Chagas disease and congenital disorders of glycosylation affecting the heart. Furthermore, sialylation status has been shown to change during human adult life, impacting brain and muscle structural properties. Cardiac arrhythmias increase in prevalence with age, contributing to higher morbidity and mortality in older people. Using an aging mouse model, we observed a decline in cardiac sialic acid levels and NaV1.5 associated with the aging process. In compelling in vitro experiments on HEK293 cells and induced pluripotent stem cell-derived cardiomyocytes, diminished external sialic acid induces Nav1.5 channel internalization, activating endocytic pathways and leading to proteasome-mediated degradation. Intriguingly, proteomic analysis revealed that introducing a specific sialic acid precursor increased membrane protein trafficking, restoring Nav1.5 protein levels and the sodium current. Electrophysiological experiments demonstrated functional recovery and highlighted the rapid response, observable within just 2 hours of sialic acid stimulation. In summary, this study provides novel insights into the complex relationship between sialic acid and NaV1.5 channel trafficking, offering new perspectives and promising therapeutic strategies to prevent cardiac dysfunction in elderly.Modulating sialylation on NaV1.5 channelOur project goal

Metabolic plasma signatures in thoracic aortic aneurysm associated to tricuspid and bicuspid aortic valves

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III with co-funding from the ERDF and Comunidad de Madrid Background/Introduction Thoracic aortic aneurysm (TAA) develops asymptomatic with life-threatening consequences and its diagnosis is usually fortuitous. TAA is mostly idiopathic, although its prevalence is significantly higher in subjects with bicuspid aortic valve (BAV) for unknown reasons. We hypothesize that it is essential to investigate idiopathic TAA by differentiating between BAV and tricuspid aortic valve (TAV) patients for personalized diagnosis and treatment. Purpose We aimed to identify plasma metabolic profiles of aortic dilatation in idiopathic TAA, differentiating between subjects with BAV or TAV. Methods Plasma samples from 41 idiopathic TAA patients and 57 subjects without aortic dilatation (control, C) were collected and classified according to aortic valve type (BAV or TAV). Full metabolomics coverage was carried out by 1H RMN and LC-MS/MS untargeted analysis, including reverse phase and hydrophilic interaction chromatography and positive and negative electrospray ionization modes. Significant variation in metabolite levels were considered if FDR-adjusted p-value <0.05. Results Our analyses highlighted a metabolic signature specific for BAV and TAV subjects. BAV patients presented a marked glycerophospholipid metabolism alteration involving different lysophospholipids (LPAs, LPCs and LPEs) and phospholipids (PCs, PEs, PAs and PIs). Particularly, LPCs and LPEs were increased in BAV-TAA vs BAV-C, being an indicator of stimulation of glucose utilization through glycolysis and suppression of fatty acid biosynthesis. In contrast, carnitine-related metabolism was the most altered pathway observed in TAV subjects. In particular, we detected an increment in plasma acylcarnitines in TAV-TAA compared to TAV-C suggesting an altered β-oxidation of fatty acids and mitochondrial overload. Conclusions Idiopathic TAA development is differentially reflected in plasma from BAV and TAV patients, supporting an individualized diagnosis valve-dependent and suggesting also differential therapeutic targets.