title: Cholesterol loading reprograms the microRNA-143/145-myocardin axis to convert aortic smooth muscle cells to a dysfunctional macrophage-like phenotype.
abstract: OBJECTIVE
  We previously showed that cholesterol loading in vitro converts mouse aortic vascular smooth muscle cells (VSMC) from a contractile state to one resembling macrophages. In human and mouse atherosclerotic plaques, it has become appreciated that ≈40% of cells classified as macrophages by histological markers may be of VSMC origin. Therefore, we sought to gain insight into the molecular regulation of this clinically relevant process.
  
  
  RESULTS
  VSMC of mouse (or human) origin were incubated with cyclodextrin-cholesterol complexes for 72 hours, at which time the expression at the protein and mRNA levels of contractile-related proteins was reduced and of macrophage markers increased. Concurrent was downregulation of miR-143/145, which positively regulate the master VSMC differentiation transcription factor myocardin. Mechanisms were further probed in mouse VSMC. Maintaining the expression of myocardin or miR-143/145 prevented and reversed phenotypic changes caused by cholesterol loading. Reversal was also seen when cholesterol efflux was stimulated after loading. Notably, despite expression of macrophage markers, bioinformatic analyses showed that cholesterol-loaded cells remained closer to the VSMC state, consistent with impairment in classical macrophage functions of phagocytosis and efferocytosis. In apoE-deficient atherosclerotic plaques, cells positive for VSMC and macrophage markers were found lining the cholesterol-rich necrotic core.
  
  
  CONCLUSIONS
  Cholesterol loading of VSMC converts them to a macrophage-appearing state by downregulating the miR-143/145-myocardin axis. Although these cells would be classified by immunohistochemistry as macrophages in human and mouse plaques, their transcriptome and functional properties imply that their contributions to atherogenesis would not be those of classical macrophages.
authors.affiliation: From the Marc and Ruti Bell Program in Vascular Biology, Division of Cardiology, Department of Medicine, NYU School of Medicine, New York (Y.V., H.N., M.O., N.S., C.P.C., K.J.M., E.A.F.); Center for Cardiovascular Sciences, Albany Medical College, NY (X.L.); Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom (F.O.M.); Department of Biomedical Sciences, Oregon State University, Corvallis (S.A.R.); and Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, NY (J.M.M.).
authors.forename: Yuliya
authors.initials: Y
authors.lastname: Vengrenyuk
authors.affiliation: From the Marc and Ruti Bell Program in Vascular Biology, Division of Cardiology, Department of Medicine, NYU School of Medicine, New York (Y.V., H.N., M.O., N.S., C.P.C., K.J.M., E.A.F.); Center for Cardiovascular Sciences, Albany Medical College, NY (X.L.); Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom (F.O.M.); Department of Biomedical Sciences, Oregon State University, Corvallis (S.A.R.); and Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, NY (J.M.M.).
authors.forename: Hitoo
authors.initials: H
authors.lastname: Nishi
authors.affiliation: From the Marc and Ruti Bell Program in Vascular Biology, Division of Cardiology, Department of Medicine, NYU School of Medicine, New York (Y.V., H.N., M.O., N.S., C.P.C., K.J.M., E.A.F.); Center for Cardiovascular Sciences, Albany Medical College, NY (X.L.); Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom (F.O.M.); Department of Biomedical Sciences, Oregon State University, Corvallis (S.A.R.); and Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, NY (J.M.M.).
authors.forename: Xiaochun
authors.initials: X
authors.lastname: Long
authors.affiliation: From the Marc and Ruti Bell Program in Vascular Biology, Division of Cardiology, Department of Medicine, NYU School of Medicine, New York (Y.V., H.N., M.O., N.S., C.P.C., K.J.M., E.A.F.); Center for Cardiovascular Sciences, Albany Medical College, NY (X.L.); Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom (F.O.M.); Department of Biomedical Sciences, Oregon State University, Corvallis (S.A.R.); and Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, NY (J.M.M.).
authors.forename: Mireille
authors.initials: M
authors.lastname: Ouimet
authors.affiliation: From the Marc and Ruti Bell Program in Vascular Biology, Division of Cardiology, Department of Medicine, NYU School of Medicine, New York (Y.V., H.N., M.O., N.S., C.P.C., K.J.M., E.A.F.); Center for Cardiovascular Sciences, Albany Medical College, NY (X.L.); Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom (F.O.M.); Department of Biomedical Sciences, Oregon State University, Corvallis (S.A.R.); and Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, NY (J.M.M.).
authors.forename: Nazir
authors.initials: N
authors.lastname: Savji
authors.affiliation: From the Marc and Ruti Bell Program in Vascular Biology, Division of Cardiology, Department of Medicine, NYU School of Medicine, New York (Y.V., H.N., M.O., N.S., C.P.C., K.J.M., E.A.F.); Center for Cardiovascular Sciences, Albany Medical College, NY (X.L.); Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom (F.O.M.); Department of Biomedical Sciences, Oregon State University, Corvallis (S.A.R.); and Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, NY (J.M.M.).
authors.forename: Fernando O
authors.initials: FO
authors.lastname: Martinez
authors.affiliation: From the Marc and Ruti Bell Program in Vascular Biology, Division of Cardiology, Department of Medicine, NYU School of Medicine, New York (Y.V., H.N., M.O., N.S., C.P.C., K.J.M., E.A.F.); Center for Cardiovascular Sciences, Albany Medical College, NY (X.L.); Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom (F.O.M.); Department of Biomedical Sciences, Oregon State University, Corvallis (S.A.R.); and Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, NY (J.M.M.).
authors.forename: Courtney P
authors.initials: CP
authors.lastname: Cassella
authors.affiliation: From the Marc and Ruti Bell Program in Vascular Biology, Division of Cardiology, Department of Medicine, NYU School of Medicine, New York (Y.V., H.N., M.O., N.S., C.P.C., K.J.M., E.A.F.); Center for Cardiovascular Sciences, Albany Medical College, NY (X.L.); Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom (F.O.M.); Department of Biomedical Sciences, Oregon State University, Corvallis (S.A.R.); and Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, NY (J.M.M.).
authors.forename: Kathryn J
authors.initials: KJ
authors.lastname: Moore
authors.affiliation: From the Marc and Ruti Bell Program in Vascular Biology, Division of Cardiology, Department of Medicine, NYU School of Medicine, New York (Y.V., H.N., M.O., N.S., C.P.C., K.J.M., E.A.F.); Center for Cardiovascular Sciences, Albany Medical College, NY (X.L.); Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom (F.O.M.); Department of Biomedical Sciences, Oregon State University, Corvallis (S.A.R.); and Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, NY (J.M.M.).
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Ramsey
authors.affiliation: From the Marc and Ruti Bell Program in Vascular Biology, Division of Cardiology, Department of Medicine, NYU School of Medicine, New York (Y.V., H.N., M.O., N.S., C.P.C., K.J.M., E.A.F.); Center for Cardiovascular Sciences, Albany Medical College, NY (X.L.); Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom (F.O.M.); Department of Biomedical Sciences, Oregon State University, Corvallis (S.A.R.); and Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, NY (J.M.M.).
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Miano
authors.affiliation: From the Marc and Ruti Bell Program in Vascular Biology, Division of Cardiology, Department of Medicine, NYU School of Medicine, New York (Y.V., H.N., M.O., N.S., C.P.C., K.J.M., E.A.F.); Center for Cardiovascular Sciences, Albany Medical College, NY (X.L.); Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom (F.O.M.); Department of Biomedical Sciences, Oregon State University, Corvallis (S.A.R.); and Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, NY (J.M.M.). edward.fisher@nyumc.org.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Fisher
mesh_terms: D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism / Q000473:pathology; D001057:Apolipoproteins E / Q000172:deficiency / Q000235:genetics; D050197:Atherosclerosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D001665:Binding Sites; D019070:Cell Lineage; D054338:Cell Transdifferentiation; D002784:Cholesterol / Q000378:metabolism*; D008076:Cholesterol, HDL / Q000378:metabolism; D018920:Coculture Techniques; D004195:Disease Models, Animal; D005487:Foam Cells / Q000378:metabolism* / Q000473:pathology; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D006801:Humans; D019169:Jurkat Cells; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism* / Q000473:pathology; D009336:Necrosis; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010587:Phagocytosis; D010641:Phenotype; D058226:Plaque, Atherosclerotic; D015398:Signal Transduction; D051782:Sterol Regulatory Element Binding Protein 2 / Q000378:metabolism; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000099251:Myocardin
chemical_list: D001057:Apolipoproteins E; D002784:Cholesterol; D008076:Cholesterol, HDL; D035683:MicroRNAs; D009687:Nuclear Proteins; D051782:Sterol Regulatory Element Binding Protein 2; D015534:Trans-Activators; D000099251:Myocardin; C543499:MIRN145a microRNA, mouse; C532884:MIRN143 microRNA, mouse; C497391:Srebf2 protein, mouse
keywords: atherosclerosis; cholesterol; macrophages; plaque, atherosclerotic