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Imaging-defined atherosclerosis represents an intermediate phenotype of atherosclerotic cardiovascular disease (ASCVD). Genome-wide association studies (GWAS) on directly measured coronary plaques using coronary computed tomography angiography (CCTA) are scarce. In the so far largest population-based cohort with CCTA data, we performed a GWAS on coronary plaque burden as determined by the segment involvement score (SIS) in 24,811 European individuals. We identified 20 significant independent genetic markers for SIS, three of which were found in loci not implicated in ASCVD before. Further GWAS on coronary artery calcification showed similar results to that of SIS, whereas a GWAS on ultrasound-assessed carotid plaques identified both shared and non-shared loci with SIS. In two-sample Mendelian randomization studies using SIS-associated markers in UK Biobank and CARDIoGRAMplusC4D, one extra coronary segment with atherosclerosis corresponded to 1.8-fold increased odds of myocardial infarction. This GWAS data can aid future studies of causal pathways in ASCVD.

Despite significant advancements in prevention and treatment, atherosclerotic cardiovascular disease remains a leading cause of mortality and morbidity. Atherosclerosis develops from the accumulation of lipoprotein debris in arterial walls, resulting in plaque buildup that causes arterial narrowing, thickening, or softening and may ultimately trigger thrombosis. Current therapies effectively lower low-density lipoprotein (LDL) levels while insufficiently addressing other atherogenic lipids like very-low-density lipoproteins (VLDL) and chylomicron remnants. Furthermore, the optimal timing for initiating lipid-lowering interventions is debated. Conventional cardiovascular prevention strategies, which base treatment decisions on ten-year risk calculations, may underestimate the cumulative impact of lifelong lipid exposure.

This thesis uses human genetics to explore the lifelong impact of inhibiting specific lipid-lowering drug candidate targets. We examine two key approaches in lipoprotein lowering: activating the rate-limiting enzyme in intravascular triglyceride hydrolysis, lipoprotein lipase (LPL), focusing on its activation through inhibiting the angiopoietin-like (ANGPTL) protein family of regulators; and the reverse cholesterol transport system, reevaluating cholesteryl ester transfer protein (CETP) as a drug target.

Through genetic association studies, Mendelian randomization, genetic mimicry analyses, and meta-analyses of clinical trials, we demonstrate that targeting these proteins may offer protection against atherosclerotic cardiovascular disease. Our findings support the ongoing clinical development of ANGPTL3, ANGPTL4, and CETP inhibitors for cardiovascular prevention while emphasizing the value of human genetics in drug discovery. Lastly, this work improves our understanding of lipid management throughout the lifespan and highlights the potential benefits of early intervention.   

Background: Genetic studies consistently demonstrate that individuals born with reduced Cholesteryl Ester Transfer Protein (CETP) activity experience lower rates of atherosclerotic vascular disease throughout their lives. In contrast, short-term randomized controlled trials of CETP inhibitors have yielded mixed results, with only one of four trials reporting a reduction in clinical events. Several theories have been proposed to explain this discrepancy, but none fully account for the central mechanism of atherosclerosis: the cumulative lifetime exposure to circulating low-density lipoprotein (LDL) particles in the arterial walls.

Objectives: We aimed to reconcile these conflicting findings by examining the relationship between cumulative LDL exposure and atherosclerosis risk across both genetic studies and clinical trials.

Methods: We analyzed 679 carriers of CETP protein-truncating variants (resulting in reduced or non-functional CETP protein) and 505,837 non-carriers in a population with 95,568 atherosclerosis events. Additionally, we assessed treatment effects relative to cumulative LDL reductions in 34 cardiovascular prevention trials involving 328,036 participants and 53,161 events.

Results: Heterozygous CETP protein-truncating variant carrier status reduced atherosclerotic disease risk (odds ratio, 0.70; 95% confidence interval, 0.57– 0.85; P=5×10-4). In clinical trials, we observed a significant interaction between the magnitude and duration of LDL lowering on treatment effects (hazard ratio, 0.69 per 10– mmol/L×years; 95% confidence interval, 0.52–0.90; P=0.007), supporting that reducing cumulative LDL exposure is key to lowering cardiovascular risk. When comparing genetics with trial outcomes, accounting for differences in timing, duration, and follow-up, we observed consistent effects on atherosclerosis-related events per LDL years across genetic and pharmacological CETP inhibition, as well as with statins, ezetimibe, PCSK9 inhibitors, and familial hypercholesterolemia-associated variants (hazard ratio, 0.74 and 0.69 per 10–mmol/L×years, respectively). This suggests that CETP inhibition reduces cardiovascular risk primarily through LDL. Notably, several trials failed to achieve sufficient cumulative LDL reduction to impact clinical events, and this was not unique to CETP inhibitors.

Conclusion: Our findings indicate that future CETP inhibitor trials achieving substantial and sustained LDL reduction will demonstrate efficacy in preventing cardiovascular events. These results highlight the importance of long-term LDL lowering and support further investigation of CETP inhibition as a strategy for cardiovascular prevention.

Aims: APOC3, ANGPTL3, and ANGPTL4 are circulating proteins that are actively pursued as pharmacological targets to treat dyslipidaemia and reduce the risk of atherosclerotic cardiovascular disease. Here, we used human genetic data to compare the predicted therapeutic and adverse effects of APOC3, ANGPTL3, and ANGPTL4 inactivation.

Methods and results: We conducted drug-target Mendelian randomization analyses using variants in proximity to the genes associated with circulating protein levels to compare APOC3, ANGPTL3, and ANGPTL4 as drug targets. We obtained exposure and outcome data from large-scale genome-wide association studies and used generalized least squares to correct for linkage disequilibrium-related correlation. We evaluated five primary cardiometabolic endpoints and screened for potential side effects across 694 disease-related endpoints, 43 clinical laboratory tests, and 11 internal organ MRI measurements. Genetically lowering circulating ANGPTL4 levels reduced the odds of coronary artery disease (CAD) [odds ratio, 0.57 per s.d. protein (95% CI 0.47-0.70)] and Type 2 diabetes (T2D) [odds ratio, 0.73 per s.d. protein (95% CI 0.57-0.94)]. Genetically lowering circulating APOC3 levels also reduced the odds of CAD [odds ratio, 0.90 per s.d. protein (95% CI 0.82-0.99)]. Genetically lowered ANGPTL3 levels via common variants were not associated with CAD. However, meta-analysis of protein-truncating variants revealed that ANGPTL3 inactivation protected against CAD (odds ratio, 0.71 per allele [95%CI, 0.58-0.85]). Analysis of lowered ANGPTL3, ANGPTL4, and APOC3 levels did not identify important safety concerns.

Conclusion: Human genetic evidence suggests that therapies aimed at reducing circulating levels of ANGPTL3, ANGPTL4, and APOC3 reduce the risk of CAD. ANGPTL4 lowering may also reduce the risk of T2D.

Angiopoietin-like proteins, ANGPTL3, ANGPTL4, and ANGPTL8, are involved in regulating plasma lipids. In vitro and animal-based studies point to LPL and endothelial lipase (EL, LIPG) as key targets of ANGPTLs. To examine the ANGPTL mechanisms for plasma lipid modulation in humans, we pursued a genetic mimicry analysis of enhancing or suppressing variants in the LPL, LIPG, lipase C hepatic type (LIPC), ANGPTL3, ANGPTL4, and ANGPTL8 genes using data on 248 metabolic parameters derived from over 110,000 nonfasted individuals in the UK Biobank and validated in over 13,000 overnight fasted individuals from 11 other European populations. ANGPTL4 suppression was highly concordant with LPL enhancement but not HL or EL, suggesting ANGPTL4 impacts plasma metabolic parameters exclusively via LPL. The LPL-independent effects of ANGPTL3 suppression on plasma metabolic parameters showed a striking inverse resemblance with EL suppression, suggesting ANGPTL3 not only targets LPL but also targets EL. Investigation of the impact of the ANGPTL3-ANGPTL8 complex on plasma metabolite traits via the ANGPTL8 R59W substitution as an instrumental variable showed a much higher concordance between R59W and EL activity than between R59W and LPL activity, suggesting the R59W substitution more strongly affects EL inhibition than LPL inhibition. Meanwhile, when using a rare and deleterious protein-truncating ANGPTL8 variant as an instrumental variable, the ANGPTL3-ANGPTL8 complex was very LPL specific. In conclusion, our analysis provides strong human genetic evidence that the ANGPTL3-ANGPTL8 complex regulates plasma metabolic parameters, which is achieved by impacting LPL and EL. By contrast, ANGPTL4 influences plasma metabolic parameters exclusively via LPL.

Patients at a high risk for sudden cardiac death (SCD) without previous history of cardiovascular disease remain a challenge to identify. Atherosclerosis and prothrombotic states involve inflammation and non-cardiac tissue damage that may play active roles in SCD development. Therefore, we hypothesized that circulating proteins implicated in inflammation and tissue damage are linked to the future risk of SCD. We conducted a prospective nested case–control study of SCD cases with verified myocardial infarction (N = 224) and matched controls without myocardial infarction (N = 224), aged 60 ± 10 years time and median time to event was 8 years. Protein concentrations (N = 122) were measured using a proximity extension immunoassay. The analyses revealed 14 proteins significantly associated with an increased risk of SCD, from which two remained significant after adjusting for smoking status, systolic blood pressure, BMI, cholesterol, and glucose levels. We identified leukotriene A4 hydrolase (LTA4H, odds ratio 1.80, corrected confidence interval (CI_corr) 1.02–3.17) and hepatocyte growth factor (HGF; odds ratio 1.81, CI_corr 1.06–3.11) as independent risk markers of SCD. Elevated LTA4H may reflect increased systemic and pulmonary neutrophilic inflammatory processes that can contribute to atherosclerotic plaque instability. Increased HGF levels are linked to obesity-related metabolic disturbances that are more prevalent in SCD cases than the controls.

LPL is a key player in plasma triglyceride metabolism. Consequently, LPL is regulated by several proteins during synthesis, folding, secretion, and transport to its site of action at the luminal side of capillaries, as well as during the catalytic reaction. Some proteins are well known, whereas others have been identified but are still not fully understood. We set out to study the effects of the natural variations in the plasma levels of all known LPL regulators on the activity of purified LPL added to samples of fasted plasma taken from 117 individuals. The enzymatic activity was measured at 25°C using isothermal titration calorimetry. This method allows quantification of the ability of an added fixed amount of exogenous LPL to hydrolyze triglyceride-rich lipoproteins in plasma samples by measuring the heat produced. Our results indicate that, under the conditions used, the normal variation in the endogenous levels of apolipoprotein C1, C2, and C3 or the levels of angiopoietinlike proteins 3, 4, and 8 in the fasted plasma samples had no significant effect on the recorded activity of the added LPL. Instead, the key determinant for the LPL activity was a lipid signature strongly correlated to the average size of the VLDL particles. The signature involved not only several lipoprotein and plasma lipid parameters but also apolipoprotein A5 levels. While the measurements cannot fully represent the action of LPL when attached to the capillary wall, our study provides knowledge on the interindividual variation of LPL lipolysis rates in human plasma.

Increased levels of apolipoprotein CIII (apoCIII), a key regulator of lipid metabolism, result in obesity-related metabolic derangements. We investigated mechanistically whether lowering or preventing high-fat diet (HFD)-induced increase in apoCIII protects against the detrimental metabolic consequences. Mice, first fed HFD for 10 weeks and thereafter also given an antisense (ASO) to lower apoCIII, already showed reduced levels of apoCIII and metabolic improvements after 4 weeks, despite maintained obesity. Prolonged ASO treatment reversed the metabolic phenotype due to increased lipase activity and receptor-mediated hepatic uptake of lipids. Fatty acids were transferred to the ketogenic pathway, and ketones were used in brown adipose tissue (BAT). This resulted in no fat accumulation and preserved morphology and function of liver and BAT. If ASO treatment started simultaneously with the HFD, mice remained lean and metabolically healthy. Thus, lowering apoCIII protects against and reverses the HFD-induced metabolic phenotype by promoting physiological insulin sensitivity.