title: Coxsackievirus and adenovirus receptor (CAR)-positive immature osteoblasts as targets of adenovirus-mediated gene transfer for fracture healing.
abstract: Adenovirus vectors are expected to be a powerful tool for gene therapy to treat severe fractures. Adenovirus invades cells through binding to the coxsackievirus and adenovirus receptor (CAR) on the cell membrane. CAR expression is low in normal adult animals, but it is induced on regenerating cells in some experimental models. We made a rib fracture model in mice and evaluated the histological changes and CAR mRNA expression by RT-PCR 1, 5, 10, 14, and 21 days after the fracture. CAR mRNA was expressed exclusively in the fractured ribs at each time point, but not in the normal ribs. We detected the CAR protein immunohistochemically in fibroblast-like cells in the fracture callus on days 10 and 14 after fracture. In situ hybridization showed that these fibroblast-like cells expressed mRNA of type I collagen and osteopontin, but not osteocalcin, defining the cells as immature osteoblasts. We then transferred small doses (10(4)-10(8) PFU) of lacZ-expressing adenovirus vector into immature osteoblasts on day 14. beta-galactosidase was detected only on the immature osteoblasts at every dose. Immature osteoblasts play an important role in the matrix replacement step in fracture healing. CAR-mediated gene transfer into immature osteoblasts can be reasonable for adenovirus-mediated treatment of fracture healing.
authors.affiliation: Division of Orthopedic Surgery, Department of Regenerative and Transplant Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Ito
authors.forename: K
authors.initials: K
authors.lastname: Tokunaga
authors.forename: H
authors.initials: H
authors.lastname: Maruyama
authors.forename: H
authors.initials: H
authors.lastname: Kawashima
authors.forename: H
authors.initials: H
authors.lastname: Kitahara
authors.forename: T
authors.initials: T
authors.lastname: Horikoshi
authors.forename: A
authors.initials: A
authors.lastname: Ogose
authors.forename: Y
authors.initials: Y
authors.lastname: Hotta
authors.forename: R
authors.initials: R
authors.lastname: Kuwano
authors.forename: H
authors.initials: H
authors.lastname: Katagiri
authors.forename: N
authors.initials: N
authors.lastname: Endo
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001842:Bone and Bones / Q000378:metabolism / Q000473:pathology; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D017102:Fracture Healing; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000032:analysis; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods; D007763:Lac Operon; D051379:Mice; D023421:Models, Animal; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D011991:Receptors, Virus / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: C568772:CLMP protein, mouse; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D012333:RNA, Messenger; D011991:Receptors, Virus; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)