title: Induction of sustained hypercholesterolemia by single adeno-associated virus-mediated gene transfer of mutant hPCSK9.
abstract: OBJECTIVE
  Patients with mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have hypercholesterolemia and are at high risk of adverse cardiovascular events. We aimed to stably express the pathological human D374Y gain-of-function mutant form of PCSK9 (PCSK9(DY)) in adult wild-type mice to generate a hyperlipidemic and proatherogenic animal model, achieved with a single systemic injection with adeno-associated virus (AAV).
  
  
  RESULTS
  We constructed an AAV-based vector to support targeted transfer of the PCSK9(DY) gene to liver. After injection with 3.5×10(10) viral particles, mice in the C57BL/6J, 129/SvPasCrlf, or FVB/NCrl backgrounds developed long-term hyperlipidemia with a strong increase in serum low-density lipoprotein. Macroscopic and histological analysis showed atherosclerotic lesions in the aortas of AAV-PCSK9(DY) mice fed a high-fat-diet. Advanced lesions in these high-fat-diet-fed mice also showed evidence of macrophage infiltration and fibrous cap formation. Hepatic AAV-PCSK9(DY) infection did not result in liver damage or signs of immunologic response. We further tested the use of AAV-PCSK9(DY) to study potential genetic interaction with the ApoE gene. Histological analysis of ApoE(-/-) AAV-PCSK9(DY) mice showed a synergistic response to ApoE deficiency, with aortic lesions twice as extensive in ApoE(-/-) AAV-PCSK9(DY)-transexpressing mice as in ApoE(-/-) AAV-Luc controls without altering serum cholesterol levels.
  
  
  CONCLUSIONS
  Single intravenous AAV-PCSK9(DY) injection is a fast, easy, and cost-effective approach, resulting in rapid and long-term sustained hyperlipidemia and atherosclerosis. We demonstrate as a proof of concept the synergy between PCSK9(DY) gain-of-function and ApoE deficiency. This methodology could allow testing of the genetic interaction of several mutations without the need for complex and time-consuming backcrosses.
authors.affiliation: From the Cardiovascular Development and Repair Department (M.R.-M., F.M.C., J.A.B.), and Epidemiology, Atherothrombosis and Imaging Department (D.S.-R., J.G.-P., V.F., B.I.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Seville, Spain (S.L., R.A., F.J.G.M.); The Zena and Michael a Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (V.F.); and Cardiovascular Institute, Hospital Clínico San Carlos, Madrid, Spain (B.I.).
authors.forename: Marta
authors.initials: M
authors.lastname: Roche-Molina
authors.affiliation: From the Cardiovascular Development and Repair Department (M.R.-M., F.M.C., J.A.B.), and Epidemiology, Atherothrombosis and Imaging Department (D.S.-R., J.G.-P., V.F., B.I.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Seville, Spain (S.L., R.A., F.J.G.M.); The Zena and Michael a Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (V.F.); and Cardiovascular Institute, Hospital Clínico San Carlos, Madrid, Spain (B.I.).
authors.forename: David
authors.initials: D
authors.lastname: Sanz-Rosa
authors.affiliation: From the Cardiovascular Development and Repair Department (M.R.-M., F.M.C., J.A.B.), and Epidemiology, Atherothrombosis and Imaging Department (D.S.-R., J.G.-P., V.F., B.I.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Seville, Spain (S.L., R.A., F.J.G.M.); The Zena and Michael a Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (V.F.); and Cardiovascular Institute, Hospital Clínico San Carlos, Madrid, Spain (B.I.).
authors.forename: Francisco M
authors.initials: FM
authors.lastname: Cruz
authors.affiliation: From the Cardiovascular Development and Repair Department (M.R.-M., F.M.C., J.A.B.), and Epidemiology, Atherothrombosis and Imaging Department (D.S.-R., J.G.-P., V.F., B.I.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Seville, Spain (S.L., R.A., F.J.G.M.); The Zena and Michael a Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (V.F.); and Cardiovascular Institute, Hospital Clínico San Carlos, Madrid, Spain (B.I.).
authors.forename: Jaime
authors.initials: J
authors.lastname: García-Prieto
authors.affiliation: From the Cardiovascular Development and Repair Department (M.R.-M., F.M.C., J.A.B.), and Epidemiology, Atherothrombosis and Imaging Department (D.S.-R., J.G.-P., V.F., B.I.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Seville, Spain (S.L., R.A., F.J.G.M.); The Zena and Michael a Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (V.F.); and Cardiovascular Institute, Hospital Clínico San Carlos, Madrid, Spain (B.I.).
authors.forename: Sergio
authors.initials: S
authors.lastname: López
authors.affiliation: From the Cardiovascular Development and Repair Department (M.R.-M., F.M.C., J.A.B.), and Epidemiology, Atherothrombosis and Imaging Department (D.S.-R., J.G.-P., V.F., B.I.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Seville, Spain (S.L., R.A., F.J.G.M.); The Zena and Michael a Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (V.F.); and Cardiovascular Institute, Hospital Clínico San Carlos, Madrid, Spain (B.I.).
authors.forename: Rocío
authors.initials: R
authors.lastname: Abia
authors.affiliation: From the Cardiovascular Development and Repair Department (M.R.-M., F.M.C., J.A.B.), and Epidemiology, Atherothrombosis and Imaging Department (D.S.-R., J.G.-P., V.F., B.I.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Seville, Spain (S.L., R.A., F.J.G.M.); The Zena and Michael a Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (V.F.); and Cardiovascular Institute, Hospital Clínico San Carlos, Madrid, Spain (B.I.).
authors.forename: Francisco J G
authors.initials: FJ
authors.lastname: Muriana
authors.affiliation: From the Cardiovascular Development and Repair Department (M.R.-M., F.M.C., J.A.B.), and Epidemiology, Atherothrombosis and Imaging Department (D.S.-R., J.G.-P., V.F., B.I.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Seville, Spain (S.L., R.A., F.J.G.M.); The Zena and Michael a Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (V.F.); and Cardiovascular Institute, Hospital Clínico San Carlos, Madrid, Spain (B.I.).
authors.forename: Valentín
authors.initials: V
authors.lastname: Fuster
authors.affiliation: From the Cardiovascular Development and Repair Department (M.R.-M., F.M.C., J.A.B.), and Epidemiology, Atherothrombosis and Imaging Department (D.S.-R., J.G.-P., V.F., B.I.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Seville, Spain (S.L., R.A., F.J.G.M.); The Zena and Michael a Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (V.F.); and Cardiovascular Institute, Hospital Clínico San Carlos, Madrid, Spain (B.I.).
authors.forename: Borja
authors.initials: B
authors.lastname: Ibáñez
authors.affiliation: From the Cardiovascular Development and Repair Department (M.R.-M., F.M.C., J.A.B.), and Epidemiology, Atherothrombosis and Imaging Department (D.S.-R., J.G.-P., V.F., B.I.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), Seville, Spain (S.L., R.A., F.J.G.M.); The Zena and Michael a Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (V.F.); and Cardiovascular Institute, Hospital Clínico San Carlos, Madrid, Spain (B.I.). jabernal@cnic.es.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Bernal
mesh_terms: D000818:Animals; D001011:Aorta / Q000201:enzymology / Q000473:pathology; D001018:Aortic Diseases / Q000097:blood / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D001057:Apolipoproteins E / Q000172:deficiency / Q000235:genetics; D050197:Atherosclerosis / Q000097:blood / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D015415:Biomarkers / Q000097:blood; D002784:Cholesterol / Q000097:blood*; D000229:Dependovirus / Q000235:genetics*; D059305:Diet, High-Fat; D004195:Disease Models, Animal; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D006937:Hypercholesterolemia / Q000097:blood / Q000201:enzymology* / Q000235:genetics*; D008297:Male; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D058226:Plaque, Atherosclerotic; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000235:genetics* / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors
chemical_list: D001057:Apolipoproteins E; D015415:Biomarkers; D002784:Cholesterol; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases
keywords: PCSK9; atherosclerosis; hypercholesterolemia