Apolipoprotein C-III (APOC3) plays an integral role in the regulation of triglyceride-rich lipoproteins, by inhibiting the clearance of triglycerides carried by VLDL and chylomicron remnants in the blood. Prior work has shown that heterozygous loss of function (LOF) carriers of APOC3 have lower triglyceride levels and a lower risk of coronary artery disease (CAD). The quantitative impact of disease risk remains unknown in complete knockouts (KOs). Additionally, the effects and safety implications of complete APOC3 LOF have not been characterized. APOC3 inhibition is an active therapeutic strategy to lower CAD risk; hence these questions have therapeutic relevance. Among 37,244 unrelated sequenced individuals, including 19,681 cases of myocardial infarction (MI), in the Pakistan Genomic Resource - a biobank with high levels of consanguinity - we identified 207 heterozygous LOF carriers and 14 KOs. As expected, the KOs had undetectable APOC3 levels. We also observed a decrease in plasma triglycerides (P = 9E-85), VLDL-C levels (P = 2E-73), APOE levels (P = 5.4E-9) and an increase in HDL-C levels (P = 1E-34) and APOA1 levels (P = 6E-5) consistent with a gene-dosage effect. We observed a significant decrease in the risk of MI among heterozygous carriers (P = 0.01); however, we did not observe any protection from MI risk in complete KOs. Conversely, we observed a non-significant increase in the risk of MI in complete KOs compared to non-carriers; of the 14 knockouts identified, 9 were found to have MI. The loss of protection against MI could not be explained by the genetic background or by the increase in levels of homozygosity of the KOs. By recalling complete KOs and their family members, we were able to identify and phenotype an additional 33 complete KOs and 152 heterozygotes and assess other safety concerns related to complete APOC3 inhibition (i.e., glucose intolerance, fat content in the liver, etc.). In conclusion, by leveraging a highly consanguineous cohort, we have identified and phenotyped APOC3 KOs that have, hitherto, not been identified elsewhere. We did not observe APOC3 LOF to confer protection in complete KOs and observed other biomarker and phenotypic associations; these findings should inform existing therapeutic programs targeting APOC3.

Objective: The mechanism by which evinacumab, a fully human monoclonal antibody directed against ANGPTL3 (angiopoietin-like 3 protein) lowers plasma LDL (low-density lipoprotein) cholesterol levels in patients with homozygous familial hypercholesterolemia is unknown. We investigated apoB (apolipoprotein B) containing lipoprotein kinetic parameters in patients with homozygous familial hypercholesterolemia, before and after treatment with evinacumab. Approach and Results: Four patients with homozygous familial hypercholesterolemia underwent apoB kinetic analyses in 2 centers as part of a substudy of a trial evaluating the efficacy and safety of evinacumab in patients with homozygous familial hypercholesterolemia. The enrichment of apoB with the stable isotope (5,5,5- 2 H 3 )-Leucine was measured in VLDL (very LDL), IDL (intermediate-density lipoprotein), and LDL at different time points before and after intravenous administration of 15 mg/kg evinacumab. Evinacumab lowered LDL-cholesterol by 59±2% and increased IDL apoB and LDL apoB fractional catabolic rate in all 4 homozygous familial hypercholesterolemia subjects, by 616±504% and 113±14%, respectively. VLDL-apoB production rate decreased in 2 of the 4 subjects. Conclusions: In this small study, ANGPTL3 inhibition with evinacumab is associated with an increase in the fractional catabolic rate of IDL apoB and LDL apoB, suggesting that evinacumab lowers LDL-cholesterol predominantly by increasing apoB-containing lipoprotein clearance from the circulation. Additional studies are needed to unravel which factors are determinants in this biological pathway. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT04722068.

HomeCirculation ResearchVol. 127, No. 8LDL-Cholesterol Reduction by ANGPTL3 Inhibition in Mice Is Dependent on Endothelial Lipase Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBLDL-Cholesterol Reduction by ANGPTL3 Inhibition in Mice Is Dependent on Endothelial Lipase Liya Wu, Mangala M. Soundarapandian, Adam B. Castoreno, John S. Millar, Daniel J. Rader Liya WuLiya Wu Division of Translational Medicine and Human Genetics, Department of Medicine (L.W., J.S.M., D.J.R.), University of Pennsylvania, Philadelphia. , Mangala M. SoundarapandianMangala M. Soundarapandian Alnylam Pharmaceuticals (M.M.S., A.B.C.). , Adam B. CastorenoAdam B. Castoreno Alnylam Pharmaceuticals (M.M.S., A.B.C.). , John S. MillarJohn S. Millar Division of Translational Medicine and Human Genetics, Department of Medicine (L.W., J.S.M., D.J.R.), University of Pennsylvania, Philadelphia. , Daniel J. RaderDaniel J. Rader Correspondence to: Daniel J. Rader, Perelman School of Medicine, University of Pennsylvania, 11-125 Smilow Center for Translational Research, 3400 Civic Center Blvd, Philadelphia, PA 19104. Email E-mail Address: [email protected] https://orcid.org/0000-0002-9245-9876 Division of Translational Medicine and Human Genetics, Department of Medicine (L.W., J.S.M., D.J.R.), University of Pennsylvania, Philadelphia. Departments of Genetics, Medicine, and Pediatrics (D.J.R.), University of Pennsylvania, Philadelphia. Originally published17 Aug 2020https://doi.org/10.1161/CIRCRESAHA.120.317128Circulation Research. 2020;127:1112–1114is related toMeet the First AuthorMeet the First Author, see p 952ANGPTL (Angiopoietin-like)-3, a protein secreted by the liver, is a regulator of lipoprotein metabolism.1 Humans carrying ANGPTL3 loss-of-function mutations have reduced plasma TG (triglyceride), LDL-C (low-density lipoprotein cholesterol), and HDL-C (high-density lipoprotein cholesterol) levels. Antibody-mediated inhibition of ANGPTL-3 reduces TG, LDL-C, and HDL-C in mice,2 monkeys,2 and humans.3 ANGPTL-3 inhibits LPL (lipoprotein lipase) and EL (endothelial lipase). The TG- and HDL-C-lowering effects of ANGPTL-3 inhibition are due to increased LPL and EL activity, respectively2; however, the mechanism for lowering LDL-C remains a mystery. It appears to be independent of the LDLR (LDL receptor), as ANGPTL-3 inhibition reduces LDL-C in Ldlr knockout mice4 and patients with homozygous familial hypercholesterolemia.3We previously reported that hepatic overexpression of EL in hypercholesterolemic mice reduced non-HDL-C, LDL-C, and apoB levels,5 suggesting that increased EL activity as a result of ANGPTL-3 inhibition could contribute to the LDL-C reduction. In this report, we tested the dependence of LDL-C reduction upon Angptl3 silencing on EL by using mice lacking EL (EL-knockout). Because wild-type (WT) and EL-knockout mice have extremely low LDL-C levels, we raised LDL-C by using a siRNA to silence the hepatic Ldlr. Then, we administered an Angptl3 siRNA or a control siRNA and compared the changes in plasma lipid levels in WT and EL-knockout mice.The Figure [A] shows the study design. Two independent experiments were performed and were concordant; results are shown for the first experiment. Hepatic LDLR was undetectable by Western blot (Figure [B]). Angptl3 siRNA robustly reduced hepatic Angptl3 mRNA and plasma ANGPTL-3 levels (Figure [C]). Following Ldlr siRNA injection, all mice had a significant increase in plasma cholesterol and non-HDL-C (Figure [D and E]). Injection of Angptl3 siRNA significantly decreased plasma TG levels to a similar extent in WT and EL-knockout mice but decreased total cholesterol and HDL-C only in WT and not in EL-knockout mice (Figure [D]). Importantly, Angptl3 silencing also significantly reduced plasma non-HDL-C, VLDL-C (very-low-density lipoprotein cholesterol), and LDL-C in WT but not in EL-knockout mice (Figure [E]). These results were confirmed in a separate independent experiment. Our data indicate that EL not only mediates the HDL-C lowering effect of ANGPTL-3 inhibition but also plays a necessary role in mediating the LDLR-independent reduction in LDL-C.Download figureDownload PowerPointFigure. A, Study design: 8- to 10-wk-old wild type (WT; n=10) and EL-KO (endothelial lipase-knockout; n=10) mice on chow diet were used for the study (n=5/treatment group). Fasting bleeds are marked with *. Two independent experiments of this design were performed. The mice and protocol used were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania. B, Hepatic LDLR (low-density lipoprotein receptor) protein: Primary antibodies: anti-LDLR (1:5000, Abcam ab52818), anti-β-Actin (1:10 000 Sigma-Aldrich A5441). C, Hepatic Angptl3 expression: mRNA by quantitative real-time polymerase chain reaction (qRT-PCR) and plasma ANGPTL (angiopoietin-like)-3 levels by ELISA. Data are presented as mean±SEM. Mann-Whitney test was performed to compare between treatment groups. D, Fasting plasma lipids: Measured by autoanalyzer. E, Non-HDL-C (high-density lipoprotein cholesterol), VLDL-C (very-low-density lipoprotein cholesterol), and LDL-C (LDL-cholesterol): Non-HDL-C=TC minus HDL-C. Pooled plasma was fractionated by FPLC and used to determine the % of individually measured non-HDL-C that was VLDL-C and LDL-C. P values (bold) in D and E show overall treatment effect as per repeated-measures 2-way ANOVA. Significant comparisons by Sidak multiple comparisons test between treatment groups are shown at the indicated time points.We previously showed that overexpression of EL in LDLR-knockout mice increased phospholipase activity that accelerated LDL clearance from plasma.5 Thus, increased EL activity in ANGPTL-3 inhibition may accelerate LDL catabolism leading to decreased LDL-C. While kinetic studies in carriers of ANGPTL3 loss-of-function mutations showed increased LDL particle clearance,1 immunologic inhibition of ANGPTL-3 in WT mice did not.4 Alternatively, EL may promote the clearance of LDL precursors, namely VLDL and intermediate-density lipoproteins, leading to reduced LDL production. Although it remains unclear precisely how EL mediates LDL-C lowering in ANGPTL-3 inhibition, our study indicates that EL plays a crucial role in this process. Given the interest in ANGPTL-3 inhibition as a therapeutic approach to reduce LDL-C, this insight into the mechanism of LDL reduction with ANGPTL-3 inhibition is potentially important and suggests that variation in LDL-C reduction with ANGPTL-3 inhibition could be due in part to variation in EL activity.Sources of FundingThis study was supported by the National Institutes of Health grant R01 HL055323. L. Wu was supported by a research fellowship from the German Research Foundation (DFG, WU 939/1-1).DisclosuresD.J. Rader serves on Scientific Advisory Boards for Alnylam, Novartis, Pfizer, and Verve.FootnotesFor Sources of Funding and Disclosures, see page 1113.Correspondence to: Daniel J. Rader, Perelman School of Medicine, University of Pennsylvania, 11-125 Smilow Center for Translational Research, 3400 Civic Center Blvd, Philadelphia, PA 19104. Email [email protected]upenn.eduReferences1. Musunuru K, Pirruccello JP, Do R, Peloso GM, Guiducci C, Sougnez C, Garimella KV, Fisher S, Abreu J, Barry AJ, et al.. Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia.N Engl J Med. 2010; 363:2220–2227. doi: 10.1056/NEJMoa1002926CrossrefMedlineGoogle Scholar2. Gusarova V, Alexa CA, Wang Y, Rafique A, Kim JH, Buckler D, Mintah IJ, Shihanian LM, Cohen JC, Hobbs HH, et al.. ANGPTL3 blockade with a human monoclonal antibody reduces plasma lipids in dyslipidemic mice and monkeys.J Lipid Res. 2015; 56:1308–1317. doi: 10.1194/jlr.M054890CrossrefMedlineGoogle Scholar3. Gaudet D, Gipe DA, Pordy R, Ahmad Z, Cuchel M, Shah PK, Chyu KY, Sasiela WJ, Chan KC, Brisson D, et al.. ANGPTL3 Inhibition in Homozygous Familial Hypercholesterolemia.N Engl J Med. 2017; 377:296–297. doi: 10.1056/NEJMc1705994CrossrefMedlineGoogle Scholar4. Wang Y, Gusarova V, Banfi S, Gromada J, Cohen JC, Hobbs HH. Inactivation of ANGPTL3 reduces hepatic VLDL-triglyceride secretion.J Lipid Res. 2015; 56:1296–1307. doi: 10.1194/jlr.M054882CrossrefMedlineGoogle Scholar5. Broedl UC, Maugeais C, Millar JS, Jin W, Moore RE, Fuki IV, Marchadier D, Glick JM, Rader DJ. Endothelial lipase promotes the catabolism of ApoB-containing lipoproteins.Circ Res. 2004; 94:1554–1561. doi: 10.1161/01.RES.0000130657.00222.39LinkGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited ByReeskamp L, Millar J, Wu L, Jansen H, van Harskamp D, Schierbeek H, Gipe D, Rader D, Dallinga-Thie G, Hovingh G and Cuchel M (2021) ANGPTL3 Inhibition With Evinacumab Results in Faster Clearance of IDL and LDL apoB in Patients With Homozygous Familial Hypercholesterolemia—Brief Report, Arteriosclerosis, Thrombosis, and Vascular Biology, 41:5, (1753-1759), Online publication date: 5-May-2021.Blackburn N, Meikle P, Peralta J, Kumar S, Leandro A, Bellinger M, Giles C, Huynh K, Mahaney M, Göring H, VandeBerg J, Williams-Blangero S, Glahn D, Duggirala R, Blangero J, Michael L and Curran J (2021) Identifying the Lipidomic Effects of a Rare Loss-of-Function Deletion in ANGPTL3, Circulation: Genomic and Precision Medicine, 14:3, Online publication date: 1-Jun-2021.Related articlesMeet the First AuthorCirculation Research. 2020;127:950-952 September 25, 2020Vol 127, Issue 8Article InformationMetrics Download: 572 © 2020 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.120.317128PMID: 32808882 Originally publishedAugust 17, 2020 KeywordsangiopoietinscholesterollipoproteinsmetabolismtriglyceridesPDF download SubjectsTranslational StudiesPhysiologyLipids and CholesterolAnimal Models of Human Disease