title: Potential beneficial effects of ixmyelocel-T in the treatment of atherosclerotic diseases.
abstract: BACKGROUND
  Advanced atherosclerotic lesions are characterized by lipid accumulation, inflammation, and defective efferocytosis. An ideal therapy should address all aspects of this multifactorial disease. Ixmyelocel-T therapy, an expanded autologous multicellular therapy showing clinical promise in the treatment of diseases associated with advanced atherosclerosis, includes a novel population of M2-like macrophages. Here, we examine the macrophages of ixmyelocel-T and determine their ability to influx modified cholesterol in an atheroprotective manner, maintaining cholesterol homeostasis and preventing cellular dysfunction and death, ultimately promoting reverse cholesterol efflux.
  
  
  METHODS
  Approximately 50 ml of whole bone marrow was obtained from healthy donors and shipped overnight. Bone marrow mononuclear cells (BMMNCs) were produced by using density gradient separation and cultured for approximately 12 days to generate ixmyelocel-T. CD14+ cells were isolated from ixmyelocel-T via positive selection for analysis. Ixmyelocel-T and human leukemia monocyte (THP-1) cells were loaded with acetylated low-density lipoprotein (Ac-LDL) for analysis. Flow cytometry and immunofluorescence were used to examine Ac-LDL uptake, expression of cytokines was analyzed by enzyme-linked immunofluorescence assay (ELISA), and quantitative real-time PCR was used to analyze expression of cholesterol-transport genes. Both the in vitro cholesterol efflux assay and in vivo reverse cholesterol transport assay were used to examine cholesterol transport.
  
  
  RESULTS
  Ixmyelocel-T macrophages take up acetylated low-density lipoprotein and express the scavenger receptors CD36 and scavenger receptor-B1 (SR-B1). Ixmyelocel-T did not become apoptotic or proinflammatory after lipid loading. The cholesterol transporter genes ABAC1 and ABCG1 were both statistically significantly upregulated when ixmyelocel-T macrophages were loaded with cholesterol. Ixmyelocel-T also exhibited enhanced apolipoprotein A-I (ApoAI)-mediated cholesterol efflux. In addition, in vivo reverse cholesterol-transport assay demonstrated that ixmyelocel-T was able to efflux cholesterol in this model.
  
  
  CONCLUSIONS
  Ixmyelocel-T macrophages influx modified cholesterol, remained anti-inflammatory in the face of lipid loading and inflammatory challenge, and displayed enhanced cholesterol efflux capabilities. These combined features suggest that this autologous multicellular therapy may exert beneficial effects in atherosclerotic diseases.
authors.forename: Kelly J
authors.initials: KJ
authors.lastname: Ledford
authors.forename: Nikki
authors.initials: N
authors.lastname: Murphy
authors.forename: Frank
authors.initials: F
authors.lastname: Zeigler
authors.forename: Ronnda L
authors.initials: RL
authors.lastname: Bartel
mesh_terms: D000070998:ATP Binding Cassette Transporter, Subfamily G, Member 1; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D016632:Apolipoprotein A-I / Q000378:metabolism; D050197:Atherosclerosis / Q000378:metabolism / Q000473:pathology / Q000601:surgery; D001854:Bone Marrow Cells / Q000166:cytology; D018955:CD36 Antigens / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D006801:Humans; D008077:Lipoproteins, LDL / Q000633:toxicity; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism* / Q000637:transplantation; D051122:Scavenger Receptors, Class B / Q000378:metabolism
chemical_list: C419353:ABCG1 protein, human; D000070998:ATP Binding Cassette Transporter, Subfamily G, Member 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D018955:CD36 Antigens; D016207:Cytokines; D008077:Lipoproteins, LDL; C495124:SCARB1 protein, human; D051122:Scavenger Receptors, Class B; C032629:acetyl-LDL; D002784:Cholesterol