Delivery of Human Fibroblast Growth Factor‐1 Gene to Brain by Modified Rat Brain Endothelial Cells

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Abstract
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 β‐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.

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