Welcome to the EAS 2021 Interactive Program
The congress will officially run on EEST time zone (Eastern European Summer Time, Helsinki, CET+1)
274 Presentations
O047 - Functional fine-mapping of coronary artery disease risk variants identified by single-cell profiling of accessible chromatin in human atherosclerotic lesions (ID 195)
Abstract
Background and Aims
Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (CAD). Many of these loci are enriched in cis-regulatory elements (CREs) but not linked to cardiometabolic risk factors nor to candidate causal genes, complicating their functional interpretation.
Methods
We investigated chromatin accessibility profiles of the human atherosclerotic lesions using single nucleus (sn)ATAC-Seq to map cell type-specific patterns of CREs, to understand transcription factors establishing cell identity and to interpret CAD-relevant, non-coding genetic variation.
Results
We identified specific transcription factors associated with macrophage subtypes and the differentiation trajectory of smooth muscle cells transitioning into fibromyocytes. We demonstrate that endothelial and smooth muscle cell enhancers are particularly enriched for blood pressure and CAD associated genetic variants. We prioritized putative target genes and candidate regulatory elements for ~30% of all the CAD loci using single cell co-accessibility and cis-eQTL information. Finally, we performed genome-wide experimental fine-mapping of the CAD GWAS variants using epigenetic molecular QTL analysis in primary human aortic endothelial cells and STARR-Seq massively parallel reporter assay in smooth muscle cells. This analysis identified potential causal SNP(s) and the associated target gene for over 30 CAD loci. We present several examples where the chromatin accessibility and gene expression could be assigned to one cell type predicting the cell type of action for CAD loci.
Conclusions
These findings highlight the potential of applying snATAC-seq to human tissues in revealing relative contributions of distinct cell types to diseases and in identifying genes likely to be influenced by non-coding GWAS variants.
O014 - Highly frequent variants hidden in the KIV-2 region of LPA regulate lipoprotein(a) concentrations and lower coronary artery disease risk (ID 196)
Abstract
Background and Aims
The lipoprotein(a) [Lp(a)] plasma concentration is largely determined by variation in the LPA gene. However, the repetitive KIV-2 domain is difficult to analyze by conventional PCR-based technologies. The KIV-2 variant 4733G>A is a novel and frequent putative splicing modifier but its function, impact on Lp(a) and coronary artery disease (CAD) risk and, importantly, its combined effect with the 4925G>A, another frequent functional KIV-2 variant, have not been characterized.
Methods
We typed the 4733G>A variant in the German Chronic Kidney Disease (GCKD) study (n=4,673) using a novel allele-specific assay. Its function was analyzed by minigene assays. Ethnic frequencies were assessed in 1000Genomes. Proxy SNPs were identified in GCKD and used to analyze the effect of 4733G>A and the combined effect with 4925G>A on CAD in UK Biobank (n=440,234).
Results
The 4733G>A variant has a carrier frequency of 38%, lowers Lp(a) by 14.0 mg/dL ([15.3-12.6], p=4.82e-184) and was the second strongest genetic factor after the apolipoprotein(a) isoforms. It is found in all isoforms and reduces the expression of the mutated allele. Frequencies differ strongly between ethnicities. Compound heterozygous carriers of 4733G>A and 4925G>A have low Lp(a) concentrations (median=7.2 mg/dL) with little variance left (IQR=4.6 mg/dL), independently from the isoform present. 4733G>A alone and compound heterozygosity with 4925G>A are associated with a significantly lower hazard ratio for CAD (0.91 [0.89-0.93], p<0.001 and 0.88 [0.84-0.93], p<0.001).
Conclusions
Hidden functional LPA variants profoundly modulate Lp(a) concentrations and CAD risk. Furthermore, the 4733G>A variant is a novel instrument to dissect the effect of Lp(a) concentrations and apolipoprotein(a) isoforms.
O021 - Genetic and Metabolic Determinants of Plasma Levels of ANGPTL8 (ID 202)
Abstract
Background and Aims
Angiopoietin-like protein (ANGPTL)8 (A8), together with A3 and A4, coordinate changes in triglyceride (TG) delivery to tissues in response to nutritional status. Plasma A8 levels are associated with indices of glucose and TG metabolism, but the causality of these relationships and the contribution of genetic variants to inter-individual differences in A8 levels has not been investigated.
Methods
To address these questions, we developed a sensitive and specific A8 ELISA and measured plasma A8 levels in the Dallas Heart Study (DHS), a large multiethnic population-based cohort.
Results
The distribution of fasting A8 levels was highly skewed to the right with a median (IQR) of 13.3 (6.8–23.1) ng/mL. A8 levels did not differ significantly across age groups or genders. Remarkable differences were found among racial/ethnic groups with Blacks having significantly higher A8 levels than either Hispanics or Whites. A8 levels correlated with BMI, fasting glucose, insulin and TG levels. Exome-wide association study revealed a strong association between the minor T-allele of the A8(R59W) variant with plasma A8 levels. After adjustment for age, sex, race/ethnicity and BMI, the A8(59W) variant explained ~17% of the inter-individual variation in A8 levels. This variant was not associated with any of the metabolic parameters correlated with plasma A8 concentrations despite resulting in a 4-fold increase in A8 levels.
Conclusions
A8 levels are a consequence, not the cause, of the associated metabolic phenotypes.
O015 - Plasma TSH Concentration and Risk of Cardiovascular Disease: A Mendelian Randomization Study of 105,224 Individuals from The General Population. (ID 210)
Abstract
Background and Aims
In observational studies, the association between plasma concentration of thyroid stimulating hormone (TSH) and cardiovascular disease outcomes is conflicting. Using Mendelian randomization, we tested the hypothesis that plasma TSH concentration is associated with risk of ischemic heart disease (IHD), myocardial infarction (MI), and atrial fibrillation (AF).
Methods
In a prospective cohort study of the general population, The Copenhagen General Population Study, including 105,224 individuals, we first tested whether plasma TSH concentration was associated observationally with risk of incident IHD, MI, and AF (=positive control). Subsequently, we tested the genetic association between an allele score weighted on plasma TSH concentrations and risk of the same endpoints.
Results
During a median follow-up of 7 years (0-13 years), 5,425 developed IHD, 2,148 had an incident MI, and 5,105 developed AF. Observationally, using multifactorially adjusted restricted cubic splines, low plasma TSH concentrations (<1.53mIU/L=median) were associated with an increased risk of IHD, MI, and AF. Hazard ratios (HR) increased gradually with lower TSH concentrations up to 1.11(0.99-1.23) for IHD, 1.22(1.03-1.43) for MI, and 1.22(1.10-1.35) for AF, comparing individuals with TSH concentrations in the lowest 5th (≤0.54mIU/L) versus >25th (>1.04mIU/L) percentile (=reference). In genetic analyses, comparing individuals with a gene score ≤5th percentile (who had 16% lower TSH concentrations), versus individuals with a gene score >50th percentile (=reference), HRs were 1.13(1.00-1.28) for IHD, 1.29(1.07-1.55) for MI, and 1.15(1.02-1.31) for AF.
Conclusions
Low plasma TSH concentrations are associated observationally and genetically with an increased risk of IHD, MI, and AF in the general population.
O039 - Comparison Of Two Recent Ceramide-based Coronary Risk Prediction Scores: CERT And CERT-2 (ID 244)
Abstract
Background and Aims
The Coronary Event Risk Test (CERT) is a validated cardiovascular risk predictor that uses circulating ceramide concentrations to allocate patients into one of four risk categories. This test has recently been updated (CERT-2), now additionally including phosphatidylcholine concentrations.
Methods
We investigated the power of CERT and CERT-2 to predict cardiovascular mortality in 999 patients with cardiovascular disease (CVD).
Results
Overall, comparing survival curves (figure) for over 12 years of follow up and the predictive power of survival models using net reclassification improvement (NRI), CERT-2 was the best predictor of cardiovascular mortality, surpassing CERT (NRI=0.456; p=0.01) and also the 2019 ESC-SCORE (NRI=0.163; p=0.04). Patients in the highest risk category of CERT as compared to the lowest category had a HR of 3.63 [2.09-6.30] for cardiovascular death; for CERT-2 the corresponding HR was 6.02 [2.47-14.64]. Among patients with T2DM (n=322), the HR for cardiovascular death was 3.00 [1.44-6.23] using CERT and 7.06 [1.64-30.50] using CERT‑2; the corresponding HRs among non-diabetic subjects were 2.99 [1.20-7.46] and 3.43 [1.03-11.43], respectively.
Conclusions
We conclude that both, CERT and CERT-2 scores are powerful predictors of cardiovascular mortality in CVD patients, especially in those patients with T2D. Performance is even higher with CERT-2.
O071 - Cardiovascular susceptibility loci through the lens of single-cells in plaques: discovery of crucial cell populations and candidate genes for atherosclerosis. (ID 275)
Abstract
Background and Aims
We recently reported on the cellular landscape of advanced carotid atherosclerotic plaques, providing detailed insight on plaque pathology while creating a vast resource for further studies. Large-scale GWAS have identified hundreds of common variants for atherosclerotic disease and cardiovascular risk factors. One of the major challenges in the post-GWAS era remains decoding of susceptibility loci to targets that have the ability to translate into clinical care.
Methods
We developed a framework where we intersected scRNA-seq and GWAS summary statistics to offer a data driven guide towards disease relevant target identification. We performed gene-based association studies using GWAS summary statistics of atherosclerotic disease, cardiometabolic traits and other traits. We calculated a per cell population score to test for enrichment based on expression in individual cells of plaques of associated genes.
Results
We show that loci associated with coronary artery disease have a prominent substrate in plaque SMCs (APOE, KANK2, SORT1), ECs (SLC44A1, ATP2B1) and MCs (APOE, HNRNPUL1), and coronary calcification loci risk loci are present in SMCs and ECs (COL5A1, YWHAE and MORF4L1). Considering plaque SMC-subtypes, risk loci for coronary calcification were enriched in synthetic SMCs. To assess the generalizability of our workflow, we applied our method to scRNAseq-data of liver and show that hepatocyte cell populations are enriched for circulating lipid-associated loci.
Conclusions
We present a data-driven guide to identify putative target genes in known susceptibility loci of cardiovascular traits that can be validated through functional testing in relevant cell types.
O002 - ApoA-I deficiency in apoE-knockout mice induces coronary atherosclerosis and perturbs systemic inflammation (ID 282)
Abstract
Background and Aims
ApoA-I/HDL play a unique role in regulating cell cholesterol homeostasis and in modulating inflammatory response and immune cell activation. In the present study, we investigated the impact of genetic manipulation of apoA-I/HDL levels on lipid deposition in skin and heart vessels in relation to local and systemic immune-inflammatory activation.
Methods
ApoE deficient (EKO) mice, apoE/apoA-I double deficient (DKO) mice, DKO mice overexpressing human apoA-I (DKO/hA-I) and wild-type mice were fed chow diet until 30 weeks of age. Plasma lipids were quantified, atherosclerosis development at the aortic sinus and in coronary arteries was measured, skin ultrastructure was evaluated by electron microscopy. Blood and lymphoid organs were characterized through histological, immunocytofluorimetric and whole transcriptome analyses.
Results
DKO mice were characterized by almost complete HDL deficiency and by plasma total cholesterol levels comparable to those of control mice. Only DKO mice showed xanthoma formation and severe inflammation in the skin-draining lymph nodes, whose transcriptome analysis revealed a dramatic impairment in energy metabolism and fatty acid oxidation pathways. An increased presence of CD4+ T effector memory cells was detected in blood, spleen and in the skin-draining lymph nodes of DKO mice. A worsening of atherosclerosis at the aortic sinus and coronary arteries was also observed in DKO mice vs EKO mice. Human apoA-I overexpression in the DKO background was able to rescue the skin phenotype and to halt atherosclerosis development.
Conclusions
HDL deficiency, in the absence of hyperlipidemia, is associated with severe alterations of skin morphology, aortic and coronary atherosclerosis, local and systemic inflammation.
O011 - Novel method for quantification of lipoprotein(a)-cholesterol: Implications for improving accuracy of LDL-C measurements (ID 310)
Abstract
Background and Aims
Current methods for determining “LDL-C” in clinical practice measure the cholesterol content of both LDL and lipoprotein(a) [Lp(a)-C]. Prior studies suggested Lp(a) mass contains ~30% cholesterol. A high-throughput, sensitive and rapid method to quantitate Lp(a)-C and improve the accuracy of “LDL-C” was developed.
Methods
Lp(a) was isolated on magnetic beads linked to monoclonal antibody LPA4 recognizing apolipoprotein(a) and the cholesterol content measured with standard techniques. This method does not detect cholesterol in plasma samples lacking Lp(a) and is linear up to 747 nM Lp(a). LDL-Ccorr was determined as LDL-C - Lp(a)-C.
Results
Lp(a)-C and LDL-Ccorr were determined in 29 participants from a completed clinical trial of Lp(a) lowering with an antisense oligonucleotide. Baseline Lp(a) ranged from 9.0-822.8 nM, Lp(a)-C ranged from 0.6-35.0 mg/dL and correlated significantly with Lp(a) molar concentration (r=0.76, p<0.001). The percent Lp(a)-C relative to Lp(a) mass varied from 5.8–57.3% and was not affected by Lp(a) lowering. Baseline LDL-Ccorr was significantly lower than “LDL-C” (mean [SD]) 102.2(31.8) versus 119.2(32.4) mg/dL, p<0.001) and did not correlate with Lp(a)-C. Three commercially-available “direct LDL-C” assays could not differentiate LDL-C from Lp(a)-C.
Conclusions
In conclusion, we developed a novel method to quantitate Lp(a)-C that suggests a broader range of Lp(a)-C relative to mass than previously reported. The use of this method to report a more accurate LDL-C will allow a re-assessment of LDL-Ccorr in clinical medicine.
O003 - Identification of Novel Lipid Droplet Factors that Regulate Autophagy and Cholesterol Efflux in Macrophage Foam Cells (ID 335)
Abstract
Background and Aims
Macrophage autophagy is a highly anti-atherogenic process that helps maintain cellular homeostasis. In foam cells, autophagy was demonstrated to contribute to the degradation of lipid droplets (LDs) via a selective form of autophagy called lipophagy. Selective autophagy relies on tags such as ubiquitin and selectivity factors to label specific cargo for degradation. Yet, how LDs are targeted for autophagy remains poorly defined. Our study was aimed at identifying LD factors responsible for lipophagy in macrophage foam cells.
Methods
To identify lipophagy factors in macrophage foam cells in an unbiassed manner, we employed mass spectrometry to qualify the LD proteome. Using siRNA array in combination with high-content microscopy and cholesterol efflux screens, we assessed the functional role of these candidate lipophagy factors.
Results
We confirmed the presence of known LD-associated structural and metabolic proteins in the LD proteome. Additionally, we found the association of several proteins related to the ubiquitination machinery and autophagy, along with other novel factors that could regulate lipophagy. We observed that knocking down several of these genes, including Maplc3b and Tfeb significantly reduced cholesterol efflux, suggesting a role for these proteins in lipophagy-mediated LD catabolism. Furthermore, we identified optineurin as a novel cargo receptor for lipophagy.
Conclusions
Our study is the first to systematically identify several LD-associated proteins of the lipophagy machinery, a finding with important biological and therapeutic implications. Therapeutic targeting of these novel lipophagy factors may represent a means to enhance macrophage lipophagy to promote reverse cholesterol transport for the treatment of heart disease.
O044 - Cytotoxic and inflammatory effects induced by secretome from epicardial adipose tissue of diabetic patients in human cardiomyocytes are partly reverted by HDL and apoJ (ID 422)
Abstract
Background and Aims
Diabetic patients have high incidence of cardiovascular disease, which is associated with increased volume and functional alterations of epicardial adipose tissue (EAT). We aimed to study the inflammatory and cytotoxic effects induced by EAT from diabetic patients on cardiomyocytes, and the counteracting effect of two cardioprotective molecules: HDL and apolipoprotein J (apoJ).
Methods
EAT was obtained after heart surgery from non-diabetic (ND) and diabetic patients, referred as DM-C or DM according to the presence or not of coronary disease. EAT explants were incubated for 24 h, and the secretomes were added to AC16 human cardiomyocytes in the presence or absence of HDL or apoJ. After 24 hours’ incubation, inflammation was assessed by measuring the release of IL6 and MCP1 by ELISA, cytotoxicity was determined by annexin V staining by flow-cytometry, and the expression of selected genes was evaluated by real-time PCR
Results
Secretomes induced the release of MCP1 and IL6 in AC16, being that from DM the major inductor. Secretome from ND had no cytotoxic effect, whereas secretome from diabetic patients induced two-fold the mortality in AC16. The addition of HDL and apoJ inhibited the secretome-induced inflammatory and cytotoxic effects. Secretome from diabetic patients, mainly from DM-C, induced increased expression of genes related to lipid metabolism: DGAT2, PLIN2, PPARα and PPARγ (1.5-2 fold versus ND).
Conclusions
In summary, secretome from EAT of diabetic patients induced increased inflammatory and cytotoxic response in AC16 cardiomyocytes compared with EAT from ND subjects. Both effects were partially inhibited by HDL and apoJ.
O055 - Elevated lipoprotein(a) in mitral and aortic valve calcification and stenosis: The Copenhagen General Population Study (ID 464)
Abstract
Background and Aims
We tested the hypotheses (i) that elevated lipoprotein(a) is causally associated with both mitral and aortic valve calcification, and (ii) that aortic valve calcification mediates the effect of elevated lipoprotein(a) on aortic valve stenosis.
Methods
From the Copenhagen General Population study, we included 12,006 individuals who underwent cardiac computed tomography to measure mitral and aortic valve calcification. Participants had information on plasma lipoprotein(a), LPA kringle IV type 2 number of repeats; and two single nucleotide polymorphisms (LPA rs10455872 and rs3798220); genetic instruments known to be associated with plasma lipoprotein(a). These instruments were used to investigate the genetic association of lipoprotein(a) with mitral and aortic valve calcification to indicate causality.
Results
At age 70-79, 29% and 54% had mitral and aortic valve calcification. For 10-fold higher lipoprotein(a) levels, multifactorially adjusted odds ratios for mitral and aortic valve calcification were 1.26 (95% CI: 1.13-1.41) and 1.65 (1.50-1.81). Correspondingly, for ≤23 versus ≥36 kringle IV type 2 number of repeats the age and sex adjusted odds ratios for mitral and aortic valve calcification were 1.53 (1.18-1.99) and 2.23 (1.81-2.76). For carriers versus non-carriers of LPA rs10455872, odds ratios for mitral and aortic valve calcification were 1.33 (1.13-1.57) and 1.86 (1.64-2.13). For aortic valve stenosis, 20% (95% CI: 12%-28%) of the effect of lipoprotein(a) was mediated through calcification.
Conclusions
Elevated lipoprotein(a) was causally from human genetics and observationally associated with both mitral and aortic valve calcification. Aortic valve calcification mediated 20% of the effect of elevated lipoprotein(a) on aortic valve stenosis.
O051 - The crosstalk between macroautophagy and chaperone-mediated autophagy (CMA) is influenced by the lipid droplet-associated protein perilipin 2 (PLIN2) during lipophagy (ID 467)
Abstract
Background and Aims
PLIN2 is the most prominent lipid droplet (LD)-associated protein in foam cells. PLIN2 stabilizes LDs and LDs protect PLIN2 from rapid degradation. LD-bound PLIN2 degradation is dependent on AMPK-phosphorylation via chaperone-mediated autophagy (CMA). PLIN2 is vital to maintain certain LD size and number. We have recently shown that the Ser251Pro SNP in PLIN2 influences macroautophagy, cholesterol efflux, cholesterol accumulation and is associated with subclinical atherosclerosis development in humans. Since a direct crosstalk between macroautophagy and CMA has been proposed, we aim to investigate whether the Ser251Pro SNP affects CMA and the degradation of PLIN2, thereby influencing lipophagy.
Methods
We utilized stably transfected HEK293 cells carrying either variant of PLIN2 loaded with oleate, followed by treatment with AMPK modulators: we measured PLIN2 and L2A levels by WB and colocalization of Hsc70/L2A by IHC. We used WT, ATG5-/- and L2A-/- MEF cells transiently transfected with PLIN2 variants: we measured LD number by IHC, PLIN2 levels by WB.
Results
Ser251Pro-dependent differences in lipid accumulation observed in WT MEF cells are abolished in both ATG5-/- and L2A-/- MEF cells. Cells carrying the Pro251 allele show lower L2A expression and less Hsc70/L2A colocalization, indicating fewer CMA-active lysosomes. Further, AMPK inhibition in HEK cells induces PLIN2 expression more in Ser251- than in Pro251-carrying cells.
Conclusions
The effect of the Ser251Pro SNP on lipid accumulation is dependent on both macroautophagy and CMA. The presence of the Pro251 allele reduces CMA-activity and modifies PLIN2 degradation via AMPK. Thus, PLIN2 influences lipophagy via crosstalk between macroautophagy and CMA.