Delivery of human fibroblast growth factor-1 gene to brain by modified rat brain endothelial cells.
Fibroblast growth factor (FGF) is an endothelial cell mitogen and serves as a mitogen and/or differentiating factor that can be neuroprotective for other cell types within the CNS. We established brain microvascular endothelial cell lines that secrete FGF-1 with the ultimate goal of examining their usefulness as a cellular platform for FGF gene delivery to brain. A chimeric gene consisting of the secretory sequence of FGF-4 linked at the 5' end of human FGF-1 (sp-hst/KS3:FGF-1) was transfected into rat microvascular endothelial cells previously altered to express the lacZ reporter gene (RBEZ), and numerous clones were found to secrete FGF-1 (RBEZ-FGF). Immunoblotting of conditioned medium demonstrated an 18-kDa protein corresponding to FGF-1. Conditioned medium from RBEZ-FGF cells enhanced [3H]thymidine incorporation in BALB/c3T3 fibroblasts by up to sevenfold when compared with conditioned medium of control cell lines, corresponding to as much as 110 ng of active FGF-1/mg of cell protein/24 h. RBEZ-FGF cell lines remained contact-inhibited and proliferated independent of exogenous endothelial mitogens, in contrast to control lines that are mitogen-dependent. Incubation of PC12 cells with RBEZ-FGF cells or their conditioned medium induced neurite outgrowth by PC12 cells. RBEZ-FGF cells survived following implantation to neonatal and adult rat caudate-putamen for at least 21 days based on 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) histochemistry, and FGF-1 gene expression by these cells in vivo was demonstrated by in situ hybridization and reverse transcriptase-PCR. These findings suggest that endothelial cells may be useful for FGF gene delivery to the CNS.
Johnston
P
P
Department of Neurology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.
Nam
M
M
Hossain
M A
MA
Indurti
R R
RR
Mankowski
J L
JL
Wilson
M A
MA
Laterra
J
J
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D017690:Cell Transplantation / Q000379:methods; D002560:Cerebrovascular Circulation; D004247:DNA / Q000096:biosynthesis; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000637:transplantation; D016220:Fibroblast Growth Factor 1 / Q000096:biosynthesis* / Q000235:genetics; D051519:Fibroblast Growth Factor 4; D005346:Fibroblast Growth Factors / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D017403:In Situ Hybridization; D007763:Lac Operon; D008833:Microcirculation; D016501:Neurites / Q000502:physiology; D016716:PC12 Cells; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D011917:Rats, Inbred Lew; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013238:Stereotaxic Techniques; D013936:Thymidine / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; C496445:FGF4 protein, human; C496447:Fgf4 protein, rat; D051519:Fibroblast Growth Factor 4; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D016220:Fibroblast Growth Factor 1; D005346:Fibroblast Growth Factors; D004247:DNA; D013936:Thymidine