Adenovirus-Mediated Expression of the Secreted Form of Basic Fibroblast Growth Factor (FGF-2) Induces Cellular Proliferation and Angiogenesis In Vivo

Abstract

Blood supply through collateral arteries is of critical importance in occlusive arterial diseases such as coronary atherosclerosis. Induction of angiogenic growth factor within either the narrowing arteries or jeopardized myocardium may promote angiogenesis in vivo, leading to salvage of ischemic myocardium. We constructed a replication-defective adenovirus (AdCAsFGF-2) coding for human basic fibroblast growth factor (FGF)-2 that is modified, so that its secretion will be facilitated, by tagging a signal sequence derived from FGF-4. A large quantity of FGF-2 was detected in both the cell lysate and culture medium of COS cells infected with AdCAsFGF-2, indicating that FGF-2 was secreted at least partly from the infected cells. The conditioned medium from the infected COS cells stimulated DNA synthesis in and induced cellular proliferation of arterial smooth muscle cells. These effects were eliminated by adenovirus-mediated overexpression of a dominant-negative truncated FGF-receptor type 1. Implantation of a gel of basement membrane proteins containing fibroblasts infected with AdCAsFGF-2 into the ventral subcutaneous space of mice induced extensive cellular proliferation and the formation of functional arterioles. Cells surrounding the vessels were positively immunostained with antibodies recognizing either smooth muscle-specific alpha-actin or factor VIII antigen as a marker for endothelium. These results suggest that AdCAsFGF-2 may be useful for delivering functional FGF-2 into tissues and may lead to therapeutic angiogenesis in vivo.

Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle

“…[4][5][6][7][8][9][10][11] Instead of VEGF, other angiogenic growth factors such FGF, HGF and a transcription factor for angiogenesis, HIF (hypoxiainducible factor), have been considered candidates for therapeutic angiogenesis as gene therapy for the treatment of patients with critical limb ischemia. [12][13][14][15][16][17][18][19][20] Although the feasibility of therapeutic angiogenesis using these angiogenic growth factors has been reported in experimental models and human clinical trials, [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] there are still unresolved problems such as undesirable side-effects. Most clinical trials have employed intramuscular injection of naked plasmid DNA despite its low transfection efficiency.…”

“…[8][9][10][11] In addition to VEGF, the utility of gene transfer of other angiogenic growth factors such as fibroblast growth factor (FGF) or hepatocyte growth factor (HGF) has been reported to stimulate collateral formation. [12][13][14][15][16][17][18][19][20] The feasibility of gene therapy using angiogenic growth factors to treat peripheral arterial disease seems to be superior to recombinant protein therapy for the following reasons: (1) It has the potential to maintain an optimally high and local concentration over time. This issue may be critical in the case of arterial gene therapy.…”

“…Recently, many investigators have focused on the adenoviral gene transfer method. 14,15,17,[20][21][22][23][24][25][26] Although the adenoviral vector is very efficient, 14,15,17,[20][21][22][23][24][25][26] there are some theoretical disadvantages such as strong immunogenicity. 27 In addition to efficiency, safety of the gene transfer method is also an important issue, since infusion of adenovirus has recently been reported to cause deleterious side-effects.…”

Requirement of fibroblast growth factor 10 in development of white adipose tissue

“…After incubation with these reagents, the basal medium was replenished every other day. EFs obtained from E14.5 embryos as described (Miki et al 2001) were induced to differentiate by treating with insulin (10 µg/mL), 250 µM DEX, and 0.5 mM IBMX in the presence of 10% fetal bovine serum for 8 d. The adenovirus vector encoding FGFR-1TR was as described (Ueno et al 1997); that encoding ␤-galactosidase was kindly provided by I. Saito (Tokyo University, Japan). Complementary DNA encoding a constitutively active form of C/EBP␤ (LAP, containing amino acids 21-296) was constructed from the mouse wild-type C/EBP␤ cDNA (kindly provided by S. Akira, Osaka University, Japan); an adenovirus vector containing this cDNA was generated with an adenovirus expression kit (Takara) as described (Sakaue et al 1998).…”

In Vivo Modulation of FGF Biological Activity Alters Cranial Suture Fate

“…15 AdCAsFGF-2 was prepared as previously described. 16 Briefly, a recombinant cDNA for the secreted form of human FGF-2 was constructed by adding the signal sequence of FGF-4 to the 5Ј end of cDNA encoding the full-length human FGF-2. The control adenovirus encoding the Escherichia coli lacZ (␤-galactosidase cDNA) was constructed in a similar manner.…”