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Zebrafish models for large-scale genetic screens in dyslipidemia and atherosclerosis: Validation and application
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. (Marcel den Hoed)ORCID iD: 0000-0002-5664-6711
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Hundreds of loci have been robustly associated with circulating lipids, atherosclerosis and coronary artery disease; but for most loci the causal genes and mechanisms remain uncharacterized. The overall aim of my thesis is to develop and validate novel in vivo model systems that are suitable for high-throughput, image-based genetic screens in coronary artery disease and related traits, and use these model systems to systematically characterize positional candidate genes.

In Study I, I developed an experimental pipeline to validate the suitability of zebrafish larvae as a model system for systematic, large-scale characterization of drugs and genes associated with dyslipidemia and atherosclerosis. Using this pipeline, I showed that five days of overfeeding and cholesterol supplementation have independent pro-atherogenic effects in zebrafish larvae, which could be diminished by concomitant treatment with atorvastatin and ezetimibe. CRISPR-Cas9-induced mutations in orthologues of proof-of-concept genes resulted in higher LDL cholesterol levels (apoea), and more early stage atherosclerosis (apobb.1). Finally, the pipeline helped me to identify putative causal genes for circulating lipids and early-stage atherosclerosis (LPAR2 and GATAD2A).

In Study II, I characterized cardiometabolic traits in apoc2 mutant zebrafish larvae and found that, similar to humans, larvae with two non-functional apoc2 alleles have higher whole-body levels of triglycerides and total cholesterol, and more vascular lipid deposition than larvae without mutations in apoc2. Interestingly, apoc2 mutant larvae also had lower glucose levels after adjusting for triglyceride levels, suggesting that therapeutic stimulation of apoc2 to prevent hypertriglyceridemia may result in hyperglycemia. Still, zebrafish larvae with mutations in apoc2 can be a useful model to identify and characterize additional causal genes for triglyceride metabolism.

In Study III, I examined the effects of mutations in pcsk9 on atherosclerosis and diabetes-related traits in nearly 5,000 zebrafish larvae. Similar to the loss-of-function mutations in PCSK9 in humans, larvae with mutations in pcsk9 had lower LDLc levels and were protected from early-stage atherosclerosis. Interestingly, mutations in pcsk9 also resulted in fewer pancreatic β-cells in 10 days old larvae, which suggests the higher risk of diabetes in humans with mutations in PCSK9 may result from a direct effect on the beta cell.

Based on these large-scale proof-of-concept studies, my thesis confirms that zebrafish larvae can be used for large-scale, systematic genetic screens in dyslipidemia and early-stage atherosclerosis.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 46
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1619
Keywords [en]
zebrafish, dyslipidemia, atherosclerosis, genetic screens, high-cholesterol diet, APOE, APOB, LDLR, APOC2, PCSK9, LPAR2, GATAD2A
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-397715ISBN: 978-91-513-0822-7 (print)OAI: oai:DiVA.org:uu-397715DiVA, id: diva2:1372477
Public defence
2020-01-16, Humanistiska teatern, Engelska Parken, Thunbergsv. 3H, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-12-20 Created: 2019-11-24 Last updated: 2020-01-14
List of papers
1. Zebrafish larvae as a model system for systematic characterization of drugs and genes in dyslipidemia and atherosclerosis
Open this publication in new window or tab >>Zebrafish larvae as a model system for systematic characterization of drugs and genes in dyslipidemia and atherosclerosis
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(English)In: Article in journal (Refereed) Submitted
Abstract [en]

Background: Hundreds of loci have been robustly associated with circulating lipids, atherosclerosis and coronary artery disease; but for most loci the causal genes and mechanisms remain uncharacterized.

Methods: We developed a semi-automated experimental pipeline for systematic, quantitative, large-scale characterization of mechanisms, drugs and genes associated with dyslipidemia and atherosclerosis in a zebrafish model system. We validated our pipeline using a dietary (n>2000), drug treatment (n>1000), and genetic intervention (n=384).

Results: Our results show that five days of overfeeding and cholesterol supplementation had independent pro-atherogenic effects, which could be diminished by concomitant treatment with atorvastatin and ezetimibe. CRISPR-Cas9-induced mutations in orthologues of proof-of-concept genes resulted in higher LDL cholesterol levels (apoea), and more early stage atherosclerosis (apobb.1).

Conclusions: In summary, our pipeline facilitates systematic, in vivo characterization of drugs and candidate genes to increase our understanding of disease etiology, and can likely help identify novel targets for therapeutic intervention.

National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-378939 (URN)
Available from: 2019-03-11 Created: 2019-03-11 Last updated: 2019-11-24
2. Apoc2 mutant zebrafish: a model for hypertriglyceridemia and early-stage atherosclerosis
Open this publication in new window or tab >>Apoc2 mutant zebrafish: a model for hypertriglyceridemia and early-stage atherosclerosis
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Zebrafish larvae in a hypertriglyceridemic background can be useful to identify and characterize causal genes for triglyceride metabolism. A previous, small-scale study suggested that apolipoprotein C-II (apoc2)-mutant zebrafish larvae can be used to model hypertriglyceridemia-induced atherosclerosis. We aimed to replicate these findings in a large-scale study and asses if APOC-II may represent a useful therapeutic target. We generated apoc2 mutant zebrafish using CRISPR-Cas9 and examined cardiometabolomic traits in their offspring (F1 generation). Systematic characterization of 384 larvae using our image and assay-based, high-throughput pipeline showed that compound heterozygous larvae for loss of function mutations in apoc2 (n=35) have higher whole-body levels of triglycerides (0.71±0.16 SD), HDL cholesterol (0.32±0.15 SD) and total cholesterol (0.37±0.18 SD), and a trend for lower whole-body glucose levels (0.23±0.14 SD) compared with larvae without mutations in apoc2 (n=174). Such larvae also tended to have more vascular lipid deposition, however this effect did not reach significance (P=0.12). Interestingly, the trends for lower whole-body glucose levels and more vascular lipid deposition in larvae with anticipated loss of functional apoc2 reached significance when larvae (n=3812) from other screens, in which apoc2 was not experimentally perturbed were included as additional wildtype controls. Thus, our large-scale study confirms the role of apoc2 in hypertriglyceridemia and early-stage atherosclerosis. While apoc2 mutant zebrafish model can be used as a genetic background to identify and characterize causal genes for triglyceride metabolism, independent and opposite effects on triglycerides and glucose suggest that APOC-II is likely not a suitable target for prevention and treatment of coronary artery disease.

Keywords
Apolipoprotein C-II, APOC2, Hypertriglyceridemia, Dyslipidemia, Atherosclerosis, Zebrafish, CRISPR
National Category
Medical Genetics
Research subject
Medical Genetics; Genetics
Identifiers
urn:nbn:se:uu:diva-396067 (URN)
Available from: 2019-11-19 Created: 2019-11-19 Last updated: 2019-11-24
3. Image-based, in vivo characterization of cardiometabolic consequences of mutations in pcsk9
Open this publication in new window or tab >>Image-based, in vivo characterization of cardiometabolic consequences of mutations in pcsk9
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Based on the association of loss-of-function mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) with low plasma LDL cholesterol levels, inhibition of the PCSK9 protein using monoclonal antibodies have emerged as an effective treatment option to lower LDL cholesterol levels and reduce the risk of coronary artery disease. Despite these beneficial effects, PCSK9 inhibitors may increase the risk of diabetes. In this study, we mimicked the mechanistic action of PCSK9 inhibitors in humans by inducing mutations in pcsk9 in zebrafish and examining their effects on dyslipidemia, early-stage atherosclerosis and diabetes-related traits in data from nearly 5000 zebrafish larvae. At 10 days of age, larvae with mutations in pcsk9 were characterized by lower whole-body LDL cholesterol levels (beta±SE -0.056±0.025 SD units) and protection against early-stage atherosclerosis, with less vascular lipid deposition (-0.133±0.035 SD) and less co-localization of macrophages with lipids (-0.086±0.032 SD). Mutant larvae also had fewer pancreatic β-cells (-0.153±0.055 SD). Thus, our findings in pcsk9 mutant larvae are in line with results from people carrying loss-of-function PCSK9 mutations, and are also in line with the effects of PCSK9 inhibitors in humans. Further, our results suggest that mutations in pcsk9 may increase the risk of diabetes through a direct effect on pancreatic β-cells.

Keywords
Proprotein convertase subtilisin/kexin type 9, PCSK9, LDL cholesterol, Diabetes, Zebrafish, CRISPR
National Category
Medical Genetics
Research subject
Medical Genetics; Genetics
Identifiers
urn:nbn:se:uu:diva-396068 (URN)
Available from: 2019-11-19 Created: 2019-11-19 Last updated: 2019-11-24

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