Background and Aims

Cardiovascular disease (CVD) is the leading cause of death worldwide and is often related to high plasma concentrations of low-density lipoprotein cholesterol (LDL-c). One of the most frequent dyslipidaemias is familial hypercholesterolemia (FH), which is mostly caused by mutations in the LDL receptor (LDLr). Although an early identification is essential to reduce premature mortality, only the 10 % of FH patients are properly diagnosed.

In vitro characterization of variants is time consuming and expensive, so computational predictors of pathogenicity of mutations are under constant development. The aim of our work was to create a machine learning-based model that can predict the pathogenicity of LDLr missense variants, the most common ones, on an easy and reliable way.

Methods

Using more than 700 LDLr missense variants characterized on ClinVar database and Excel solver Evolutionary algorithm, we created a Machine Learning model that predicts the pathogenicity of a variant based on seven characteristics of the mutated amino acid: Conservation of the residue, original and substituting amino acid, hydrophobicity, size, charge and affected domain.

We used a part of the dataset as training group for the obtention of pathogenicity frequency distribution and other essential parameters. Then, the model was tested with the other part of the dataset, the validation group.

Results

MLb-LDLr shows a sensitivity of 92.5% and a specificity of 91.5%, matching or even surpassing other predictor software such as PolyPhen-2 or SIFT.

Conclusions

With an accuracy higher than 90%, we conclude that in silico predictions are a reliable source of information about LDLr variant pathogenicity.

Background and Aims

Mendelian randomisation as well as epidemiology studies have established an association between elevated lipoprotein(a) [Lp(a)] levels and increased cardiovascular risk. Besides the pro-atherogenic properties of the LDL- like moiety, Lp(a) further contributes to the disease pathology by carrying pro-inflammatory oxidized phospholipids (OxPLs). While blocking these OxPL epitopes profoundly reduces the monocytic inflammatory response, a residual inflammatory risk remains.

Methods

To determine the contributing factors resulting in this residual inflammation, we performed lipidomics on complete plasma from healthy individuals with either elevated [median 87 mg/dL (218 nmol/L); N=12]or low [median 7 mg/dL (18 nmol/L); N=13] levels of Lp(a).

Results

Using this unbiased lipidomics approach, we discovered a distinct “lipidome” in individuals with elevated Lp(a) levels as displayed by an upregulation of several diacylglycerol species (DAGs) and lysophosphatic acid (LPA). Further fractionation and purification of different lipoprotein fractions showed that these DAGs are preferentially carried by Lp(a). Next, we functionally assessed if these upregulated DAGs are able to elicit an inflammatory response in monocytes. Upon ex vivo DAG stimulation, monocytes elicited a dose dependent increase in IL-8, IL-6 and IL-1β secretion. Functionally, our preliminary data demonstrated this coincided with increased transendothelial migration.

Conclusions

By using transwell migration assays with fluorescently labelled monocytes, combined with sprouting assays and in-depth phosphorylation analysis, we aim to further characterise the pathways effected by DAGs and LPA to further unravel the atherogenicity of Lp(a) and will be available at the EAS 2021.

Background and Aims

Elevated Lp(a) serum levels are associated with increased risk for atherosclerotic coronary artery disease and stroke. Here, we analyzed the effect of plasma Lp(a) levels on caspase-1 and the pro-inflammatory cytokines it processes. We further directly compare the molecular composition of Lp(a) and LDL isolated from the same donors as well as their pro-atherogenic and pro-inflammatory potential.

Methods

Human plasmas with varying Lp(a) levels and isolated Lp(a) and LDL were incubated with THP-1 macrophages for three hours and caspase-1 activation and the release of pro-inflammatory cytokines IL-1β, IL-1α, and IL-18 was measured. Molecular composition of Lp(a) and LDL isolated from the same donors was determined by lipidomics and proteomics approaches.

Results

Plasma Lp(a) levels ranging from 1.7 to 165.3 mg/dL correlated significantly with caspase-1 activity (r = 0.496), IL-18 (r = 0.496), and IL-1α (r = 0.447) in macrophages. IL-1β secretion correlated significantly with plasma triglycerides, and not with Lp(a) levels.

Lipidomics comparison of LDL and Lp(a) indicated that Lp(a) was significantly depleted of poly-unsaturated fatty acids in all lipid classes. Proteomics analyses revealed that Lp(a) is enriched in inflammation-associated proteins. Lp(a) from induced robust and dose-dependent caspase-1 activation and release of IL-1β and IL-18 compared to a mild induction upon incubation with LDL from the same donors.

Conclusions

Our data show that plasma Lp(a) levels directly correlate with inflammasome activation in macrophages, isolated Lp(a) induces stronger dose-dependent caspase-1 activation than LDL, and LDL and Lp(a) have clear structural differences additionally to apo(a).

Background and Aims

Background:Lipoprotein(a) [Lp(a)] is a recognized cardiovascular risk factor. Lp(a) concentration in heterozygous familial hypercholesterolemia (heFH) is not well established. Whether the genetic defect responsible for heFH plays a role in determining Lp(a) concentration is unknown.

Aims:To study Lp(a) concentration in subjects genetically diagnosed with heFH and to assess the influence of the genetic defect responsible for heFH on its concentration.

Methods

Methods: Cross-sectional study, performed in a lipid clinic in Spain. We studied 511 heFH adults according to the responsible gene (LDLR, APOB, APOE and PCSK9). We selected 443 subjects LDLR, 27 subjects APOB, 37 subjects carriers of the p.(Leu167del) mutation in APOE, and 4 subjects PCSK9.

Results

Results:

Lipid levels differed across gene groups after adjusting for age, sex, and BMI.Lp(a) concentration differed among subjects with LDLR, APOB, and APOE mutation(p <0.001).Median Lp(a) concentration was greatest in APOB-dependent FH 36.5 mg/dL(IQR 22.0,60.8),intermediate in LDLR-dependent FH,21.7 mg/dL (IQR 17.9, 26.4)(and independent on the affected LDL receptor protein domain) and lowest in carriers of the p.(Leu167del) mutation in APOE, 7.9 mg/dL (IQR 4.9,12.7).Lp(a) geometric means, adjusted for age, sex, and BMI differed significantly. The geometric mean of LPA KIV-2 repeats did not differ among the FH gene subgroups and the estimations and differences for Lp(a) remained unchanged after adjustment for the number of KIV-2 repeats.

Conclusions

Conclusions: The concentration of Lp(a) in heFH is depending on the responsible gene. Lp(a) concentration was gratest in APOB- dependent FH. In LDLR-dependent, FH Lp(a) levels are not different depending on the affected protein domain.

Background and Aims

Elevated lipoprotein(a) [Lp(a)] levels have been demonstrated to be a causal risk factor for the development of cardiovascular disease. While previous studies have shown the atherogenicity of Lp(a) on monocyte activation and migration as well by activation of the vessel wall, the atherosclerotic plaque phenotype of individuals with elevated Lp(a) levels have not been studied up till now.

Methods

Therefore, we measured Lp(a) levels in 1506 subjects of the Athero-Express Biobank located at the University Medical Centre Utrecht, The Netherlands. Athero-Express started in 2002 and is a prospective ongoing biobank study that includes all patients undergoing carotid or iliofemoral endarterectomy in two referral hospitals in the Netherlands. From these 1506 subjects we compared the plaques from patients with extremely high (>195 mg/dl; N=57) and low (<7 mg/dl; N=106) Lp(a) levels.

Results

Analysis showed that plaques from patients with elevated Lp(a) levels demonstrated a 33% increase in blood vessel area per plaque, indicating an increased plaque angiogenesis. Intraplaque neovascularization has been shown to drive the progression of atherosclerosis and enhance plaque instability leading to increased cardiovascular risk.

Conclusions

So far, we have shown that human arterial endothelial cells stimulated with Lp(a) show enhanced collagen degradation capacity, probably due to increased migratory capacity and matrix metalloproteinase (MMP) activity. By using whole plaque RNA sequencing, combined with immunohistochemistry and ex-vivo validation studies (data available at the EAS 2021), we aim to further characterise the plaque phenotype in patients with elevated levels of Lp(a) and thereby provide more insight in the atherogenic potential of Lp(a).

Background and Aims

In observational studies, dietary intake, as dietary components or supplements, and blood concentrations of vitamin E, C, lycopene and carotenoids were associated with a lower risk of ischemic stroke. However, these studies were prone to residual confounding and reverse causation, and thereby limit the ability for causal inference. We investigated the associations between genetically-determined antioxidant concentrations and ischemic stroke using Mendelian Randomization.

Methods

For each circulating antioxidant (vitamin E, C, lycopene, β-carotene and retinol), which were assessed as absolute levels and/or metabolites, single-nucleotide polymorphisms (SNPs) were retrieved from earlier genomics studies and used as genetic instrument variables. We obtained summary statistics for gene-stroke associations from three European-ancestry cohorts (cases/controls): MEGASTROKE (67 162/454 450), UK Biobank (2404/368771) and FinnGen study (4026/90211). MR analyses were performed on each exposure per outcome database using inverse-variance weighted analyses, and subsequently meta-analyzed.

Results

In a combined sample of 986 964 individuals (73 592 cases), none of the genetically-determined absolute antioxidants or antioxidant metabolite concentrations were causally associated with the risk of ischemic stroke. For absolute antioxidants, the odds ratios (95% CI) ranged between 1.00 (95% CI: 0.99 to 1.01) for vitamin C and 1.06 (95% CI: 0.74 to 1.52) for retinol. For metabolites, they ranged between 1.00 (95% CI: 0.98 to 1.03) for vitamin C and 1.17 (95% CI: 0.92 to 1.49) for vitamin E.

Conclusions

Our study did not provide evidence supporting a causal association between dietary-derived antioxidant levels and ischemic stroke. Therefore, taking antioxidant supplementation seems unlikely to be of clinical benefit to prevent ischemic stroke.

Background and Aims

DOT1L is the only histone methyltransferase for H3K79 and has recently emerged as a central player in the immune system.

Methods

Here we investigate the role of DOT1L in macrophages by application of a selective DOT1L inhibitor on either mouse or human macrophages and using myeloid-specific Dot1l deleted mice. Furthermore, we investigated myeloid Dot1l in vivo in a mouse model for atherosclerosis including scRNAseq on atherosclerotic plaques.

Results

Using RNA-seq and in vitro assays, we found that Dot1l represses macrophage activation and impacts cellular lipid metabolism. ChIP-seq for H3K79me revealed that DOT1L regulates H3K79 methylation of RXRα in macrophages, leading to reduced RXRα mRNA and protein expression, both upon Dot1l deletion as well as DOT1L inhibition. Given the established role for RXRα in controlling macrophage activation and lipid homeostasis, the phenotype we observe can thus be at least partly explained by impaired RXRα signaling. Moreover, we could induce a similar suppressed lipid phenotype using an RXRα antagonist in wild-type macrophages.

In vivo, we confirmed that myeloid Dot1l deletion increases the activation of plaque macrophages. Moreover, although plaque area was not affected, Dot1l deficiency led to enhanced necrosis, indicating potential plaque destabilization. Apart from these direct effects, we found that myeloid Dot1l also affects other immune cell subsets illustrated by reduced production of IgG and IgM antibodies targeting atherosclerosis-related antigens.

Conclusions

Our data show that myeloid DOT1L is a critical regulator of macrophage inflammatory responses and lipid homeostasis and that it impacts in vivo immune responses and atherosclerosis development.

Background and Aims

Background and Aims: Evolocumab and alirocumab (mAbs anti-PCSK9) block the function of PCSK9 but determine a significant increase of its plasma levels, an effect that can be determined by 1) an increased synthesis or 2) reduced clearance by the liver.

Methods

Methods: We have measured total PCSK9 plasma levels, by ELISA assay, in hypercholesterolemic patients at baseline and under treatment with mAbs anti-PCSK9. In vitro human hepatocarcinoma cell line (Huh7) was incubated with evolocumab or alirocumab and total PCSK9 and LDLR were determined.

Results

Results: Baseline plasma levels of PCSK9 (n=26 patients) was 458.8±297.4 ng/ml and increased to 1533.3±333.5 ng/ml in response to mAbs anti-PCSK9. In a second cohort of patients (n=30) we observed levels of PCSK9 equal to 1702.0±164.9 ng/ml which declined to 1360.0±344.8 ng/ml after 2 weeks post-injection.

Huh7 were incubated with simvastatin (5 µM), evolocumab (10 µg/ml) and alirocumab (10 µg/ml) for 4, 24 and 48 h. Simvastatin induced both the LDLR and PCSK9 in a time-dependent manner with maximal effect at 48 h (2.15 and 10.8-fold for LDLR and PCSK9, respectively). mAbs anti-PCSK9 induced the LDLR already after 4 h incubation (+46% and +43% for evolocumab and alirocumab, respectively). On the contrary, evolocumab and alirocumab strongly reduced both intracellular and extracellular PCSK9 at 48 h, as determined by western blot and ELISA assays (0.29 and 0.16 vs basal).

Conclusions

Conclusions: Our in vitro results suggest that alirocumab and evolocumab increase PCSK9 plasma levels by reducing its hepatic clearance.

Background and Aims

Apolipoprotein C-III (apoC-III) is a key regulator in triglyceride metabolism and correlates positively with hypertriglyceridemia and incidences of CVD. Recent studies also identified apoC-III as an inducer of sterile inflammation by activating the inflammasome, another CVD risk factor. It remains unclear if therapeutic apoC-III lowering can reduce CVD risk, nor is it clear if this is depended on lipid-lowering or anti-inflammatory properties or both.

Methods

In this study, we determined the impact of interventional apoC-III lowering on atherogenesis using an apoC-III antisense oligonucleotides (ASOs) in hypertriglyceridemic mouse models where the intervention results in triglyceride-lowering (Apoe^-/-Ndst1^f/fAlb-Cre⁺, Ldlr^-/-Ndst1^f/fAlb-Cre⁺) or not (Ldlr^-/-Lrp1^f/fAlb-Cre⁺).

Results

ApoC-III ASO treatment did not alter atherosclerotic lesion volume in the murine models mice simultaneously fed a Western diet. However, when triglyceride-lowering was obtained, apoC-III ASO treatment significantly attenuated advanced and unstable plaque development. In contrast, no improvement in lesion composition and hyperlipidemia was observed in apoC-III ASO-treated Ldlr^-/-Lrp1^f/fAlb-Cre⁺ mice. To mimic an intreventional setting we tested the impact of therapeutic apoC-III lowering in combination with a switch to a lipid-poor chow diet intervention after 12-week Western diet feeding. We observed that apoC-III ASO treatment significantly reduced atherosclerotic lesion size progression when an additive triglyceride-lowering was achieved. No differences in lesion development were observed in similarly treated Ldlr^-/-Lrp1^f/fAlb-Cre⁺ mice wherein no additive triglyceride-lowering was achieved by apoC-III ASO intervention.

Conclusions

Our data highlight that the impact of apoC-III on atherogenesis depends on its lipid-modifying properties. Hence, our findings suggest that interventional apoC-III lowering in hypertriglyceridemia patients can prevent plaques rupture and reduce CVD-associated mortality.

Background and Aims

Macrophages are key regulators of inflammatory responses in atherosclerosis. Since epigenetic processes are important in controlling macrophage function, interfering with epigenetic pathways might be a novel approach to combat atherosclerosis. Histone H3K27 trimethylation is a repressive histone mark catalyzed by the Polycomb Repressive Complex 2 (PRC2) with EZH2 as the catalytic subunit. We previously showed that myeloid deletion of Kdm6b, an enzyme that in contrast to PRC2 removes repressive H3K27me3 marks, results in advanced atherosclerosis. Because of its opposing function, we here studied macrophage EZH2 and JARID2, both part of the PRC2 complex, in macrophage activation and atherosclerosis.

Methods

Myeloid-specific Ezh2 (Ezh2^del) and Jarid2 (Jarid2^del) deficient mice strains were generated (LysM-Cre+ x Ezh2^fl/fl or Jarid2^fl/fl) and bone marrow from Ezh2^del or Ezh2^wt mice was transplanted to Ldlr^-/- mice which were fed a high fat diet for 9 weeks to study atherosclerosis.

Results

Atherosclerotic lesion size was significantly decreased in Ezh2^del transplanted mice compared to control. The percentage of macrophages in the lesions was similar. However neutrophil numbers were lower in Ezh2^del transplanted mice. Correspondingly, the migratory capacity of neutrophils was decreased. Moreover, peritoneal Ezh2^del foam cells showed a reduction in the inflammatory response with reduced production of IL-6, IL-12 and NO. Currently, we are investigating the effects of JARID2 as important co-factor for the PRC2 complex on macrophage inflammatory responses by use of the Jarid2^del mouse strain and siRNA’s against JARID2 in human macrophages.

Conclusions

Myeloid Ezh2 deficiency impairs neutrophil migration and reduces macrophage foam cell inflammatory responses, both contributing to reduced atherosclerosis.

Background and Aims

Vascular smooth muscle cells (VSMC) are not terminally differentiated and undergo complex phenotypic changes during atherosclerosis. However, the complex heterogeneity of VSMC and how VSMC de-differentiation affects human carotid artery disease (CAD) risk has not been clearly established. The aim of the present study is to comprehensively characterized the transcriptomic profile of phenotypically modulated VSMC and to identify mediators of VSMC transition to osteogenic like cells with likely detrimental role for atherosclerosis plaque stability.

Methods

Single-cell RNA sequencing of CD45 negative cells derived from Apoe^-/- mice on a normal (NCD) or high cholesterol diet (HCD) was performed. To confirm the identified marker mediator of VSMC transition to osteogenic like cells, immunofluorescence staining of aortic roots of VSMC lineage tracing Apoe^-/- Myh11-CreERT2, ROSA26 STOP-flox eYFP^+/+ mice fed an NCD or HCD and of internal carotid plaque specimens from symptomatic and asymptomatic patients with CAD was used. VSMC osteogenic switch was quantified upon GDF10 and oxLDL stimulation by flow cytometry.

Results

Disease-relevant gene signature of VSMC macrophagic calcific phenotype, VSMC mesenchymal chondrogenic phenotype, VSMC inflammatory and fibro phenotype and VSMC inflammatory phenotype was revealed. The osteoblast factor GDF10 was highly expressed in phenotypically modified VSMC clusters, in parallel GDF10 stimulation in combination with oxLDL triggered VSMC osteogenic switch in vitro. Moreover, higher GDF10 expression in phenotypically modified VSMC in human atherosclerotic plaques was associated with an increased risk of plaque rupture in CAD patients.

Conclusions

Osteoblast factor GDF10 is associated with VSMC transition to osteogenic like cells with likely detrimental role in atherosclerosis plaque stability.

Background and Aims

Aortic stenosis is most common valvular heart disease in the Western world. Lipoprotein(a) [Lp(a)] is an independent risk factor of atherosclerotic cardiovascular diseases (ASCVD) and calcific aortic valve stenosis (CAVS).

To assess the role of Lp(a) as well as autoantibodies to Lp(a) in CAVS in patients with and without ASCVD.

Methods

The study included 250 patients (mean age 69±3, males 42%) that were divided into 3 groups. There were 2 groups of patients with CAVS depending on presence (group 1) or absence of ASCVD (group 2). Control group included the patients without ASCVD or CAVS. Lipids, Lp(a), autoantibodies to Lp(a) and Cu²⁺oxidized Lp(a) [autoAbs to oxLp(a)] were measured in serum for all the patients.

Results

Indicators of aortic stenosis severity in group 2 were significant higher than in group 1: median [25;75%] of maximum jet velocity 4.65 [3.77;5.28] vs 3.85 [2.6;4.5] m/s (p<0.05), mean gradient of pressure 52.9 [40.25;69.50] vs 41.0 [24.0;55.0] mmHg (p<0.05). Lp(a) level in group 1 was 22.6 [6.5;51.1] vs group 2 (14.0 [5.9;48.3]) and group 3 (12.1[4.9;25.1]) mg/dl, (p<0.05 for both). Level of IgM autoAbs to oxLp(a) in group 1 was (8.6 [7.3;10.5]) vs group 2 (9.4 [7.7;11.2]) and group 3 (11.6 [9.4;14.6] lab.units), p<0.001. According to multifactors logistic regression analysis concentration of Lp(a), level of IgM autoAbs to oxLp(a) (p<0.05 for both) and age (p<0.001) were independent predictors of CAVS.

Conclusions

Concentration of Lp(a) associated with CAVS in patients with ASCVD. The level of IgM autoAbs to oxLp(a) are associated with CAVS regardless ASCVD.