Ex vivo gene delivery of ephrin-B2 induces development of functional collateral vessels in a rabbit model of hind limb ischemia.

OBJECTIVE: In this study, we delivered ephrin-B2 to the ischemic hind limb of rabbits using an ex vivo method of gene transfer and evaluated whether the in vivo application of ephrin-B2 contributed to the development of functional collateral vessels. Ephrin-B2 is a transmembrane ligand of several Eph receptors and bidirectional signaling between ephrin-B2 and Eph-B4 is considered to be essential in angiogenesis and the development of arteries and veins. METHOD: The left femoral artery of male Japanese White rabbits was excised to induce limb ischemia, and a primary culture of autofibroblasts was obtained from a skin section. Nineteen days later, the gene expressing ephrin-B2 (ephrin group) or beta-galactosidase gene (control group) was adenovirally transfected to the cultured auto-fibroblasts (5 x 10(6) cells); then 48 hours later, the gene-transduced cells were injected through the left internal iliac artery of the same rabbit. At 28 days after injection, the development of collateral vessels and their function were assessed (control group, n = 12; ephrin group, n = 10). RESULTS: The gene expressing ephrin-B2 was successfully transferred to the rabbit autofibroblasts, and ephrin-B2, expressed on the cell membrane, possessed binding ability with its receptor, Eph-B4. Calf blood pressure ratio (control group: 0.523 +/- 0.047 vs ephrin group: 0.658 +/- 0.049, P < .0001), angiographic score (0.344 +/- 0.091 vs 0.525 +/- 0.109, P = .0006), in vivo blood flow of the left internal iliac artery (rest: 11.963 +/- 2.806 vs 17.202 +/- 3.622 mL/min, P = .0014; maximum: 27.652 +/- 10.377 vs 43.400 +/- 7.108 mL/min, P = .0007), collateral conductance (32.740 +/- 7.408 vs 54.489 +/- 18.809 mL/min/100 mm Hg, P = .0097), and capillary density of the left thigh muscle (118.517 +/- 18.669 vs 167.400 +/- 31.271, P = .0002) showed significant improvement in the ephrin-B2 group compared with controls. CONCLUSION: These findings suggest that auto-fibroblasts expressing ephrin-B2 potentially promote arteriogenesis as well as angiogenesis in the adult vasculature, resulting in the development of functional collateral vessels to an ischemic lesion.

In vivo electroporation enhances plasmid-based gene transfer of basic fibroblast growth factor for the treatment of ischemic limb.

BACKGROUND: Angiogenic therapy for ischemic tissues using angiogenic growth factors has been reported on an experimental and a clinical level. Electroporation enhances the efficiency of plasmid-based gene transfer in a variety of tissues. The purpose of this study was to evaluate the angiogenic effects of plasmid-based gene transfer using basic fibroblast growth factor (bFGF) in combination with electroporation. MATERIALS AND METHODS: The transfection efficiency of in vivo electroporation in rabbit skeletal muscles was evaluated using pCAccluc+ encoding luciferase. To evaluate the angiogenic effects of bFGF gene in ischemic limb, we constructed a plasmid, pCAcchbFGFcs23, containing human bFGF cDNA fused with the secretory signal sequence of interleukin (IL)-2. Then, 500 microg of pCAcchbFGFcs23 or pCAZ3 (control plasmid) was injected into the ischemic thigh muscles in a rabbit model of hind limb ischemia with in vivo electroporation (bFGF-E(+) group and LacZ-E(+) group). Other sets of animals were injected with pCAcchbFGFcs23 (bFGF-E(-) group) or pCAZ3 (LacZ-E(-) group) without electroporation. Then 28 days later, calf blood pressure ratio, angiographic score, in vivo blood flow, and capillary density in the ischemic limb were measured. RESULTS: Gene transfer efficiency increased markedly with the increase in voltage up to 100 V. Regarding angiogenic responses, calf blood pressure ratio, in vivo blood flow, and capillary density only in the bFGF-E(+) group were significantly higher than those in LacZ-E(-) group. Angiographic scores in the bFGF-E(+) and bFGF-E(-) groups were significantly higher than that in the LacZ-E(-) group. CONCLUSION: These data suggest that in vivo electroporation enhances bFGF gene transfer for the treatment of ischemic limb muscles.

Appropriate control of ex vivo gene therapy delivering basic fibroblast growth factor promotes successful and safe development of collateral vessels in rabbit model of hind limb ischemia.

PURPOSE: In our previous study, adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promoted significant collateral vessel development in a rabbit model of hind limb ischemia. The present study examined how to control the efficacy and safety of this gene therapy, and also evaluated the feasibility of repeat application of this procedure. METHODS: Modified hFGF gene with the secretory signal sequence was adenovirally transferred to cultured autologous fibroblasts, and various numbers of the cells (2 x 10(5), 1 x 10(6), 5 x 10(6), or 2.5 x 10(7)) or vehicle was injected through the left internal iliac artery in rabbits in whom the left femoral artery had been excised 21 days previously. Twenty-eight days after cell administration, calf blood pressure ratio, angiographic score, blood flow in the internal iliac artery, and capillary density of muscle tissue were measured to analyze collateral vessel development and tissue perfusion in the ischemic limb. To assess delivery efficiency and viral contamination, the distribution of injected cells and the time course of blood anti-adenovirus antibody titer were examined in rabbits treated with various numbers of gene-transduced cells. In addition, animals received two injections, 21 days apart, of fibroblasts infected with adenovirus vector containing the luciferase gene, and luciferase expression was measured to evaluate whether the present therapy is repeatable. RESULTS: At 28 days after cell administration, significant collateral vessel development without detectable side effects was observed in rabbits who received 5 x 10(6) or 2.5 x 10(7) cells, compared with those who received vehicle, and no significant development was detected in animals with fewer than 5 x 10(6) cells (P <.01 for calf blood pressure ratio and capillary density, P <.05 for angiographic score and maximum blood flow). There was no difference in collateral augmentation between rabbits with 5 x 10(6) and 2.5 x 10(7) cells. However, in animals with 2.5 x 10(7) cells a large number of injected cells accumulated in the lungs, anti-adenovirus antibody titer increased significantly, and calf blood pressure in the left hind limb of two rabbits decreased immediately after injection. Luciferase analysis showed very low gene expression after repeated administration. CONCLUSION: These findings suggest that 5 x 10(6) is a suitable number of cells to induce appropriate collateral vessel development and minimize potential side effects of this procedure. Despite use of ex vivo gene transfer, repeat administration of the cells was not feasible. Clinical relevance Since the present study determined the appropriate conditions for effective and safe stimulation of collateral vessels, the clinical relevance of the ex vivo therapy might be carried forward. However, the findings raised another issue that should be resolved before clinical application; that is, the number of gene-transduced cells able to be injected was strictly limited. To estimate the therapeutic range of cell number in humans, additional experiments using large animals are desirable.

Conduction performance of collateral vessels induced by vascular endothelial growth factor or basic fibroblast growth factor.

OBJECTIVE: In the present study, we delivered vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene to a rabbit model of hind limb ischemia utilizing an ex vivo method of gene transfer, and evaluated the functional performance of the developed collateral vessels. METHOD: The left femoral artery of a male Japanese White rabbit was excised to induce limb ischemia, and a section of skin was resected for culture of auto-fibroblasts. Twenty days later, the VEGF gene, bFGF gene or beta-galactosidase gene (LacZ) was adenovirally transferred to the cultured auto-fibroblasts (5x10(6) cells), and the next day, a pair of specifically infected fibroblasts (total 1x10(7) cells) was injected via the left internal iliac artery of the same rabbit. Pairs of transferred genes into the fibroblasts were as follows: LacZ/LacZ (control group), VEGF/LacZ (VEGF group), bFGF/LacZ (FGF group) and VEGF/bFGF (combination group). Twenty-eight days after cell administration, collateral development and its function were evaluated. RESULTS: Calf blood pressure ratio, resting blood flow of the left iliac artery and capillary density of ischemic muscle showed similar degrees of angiogenic effects in the VEGF and FGF groups, which were significantly greater than those in the control group. On the contrary, angiographic score, collateral conductance and smooth muscle cell (SMC)-positive vessel density in the FGF group were significantly greater than those in the VEGF group. In the combination group, collateral conductance showed synergistic effects, and in vivo blood flow and smooth muscle cell-positive vessel density revealed additive effects of VEGF and bFGF. CONCLUSION: These findings suggested that bFGF-induced collateral development exceeded VEGF-induced collateral development in the induction of arteriogenesis, and that combined gene delivery of VEGF and bFGF produced additive or synergistic effects of collateral development as compared with the effects induced by transfer of each gene alone.

Gene therapy for prostate cancer using the cytosine deaminase/uracil phosphoribosyltransferase suicide system.

BACKGROUND: Cytosine deaminase (CD) activates prodrug 5-FC to 5-FU and is used for suicide gene therapy (the CD/5-FC system). E. coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that directly converts 5-FU into 5-fluorouridine monophosphate and improves the antitumoral effect of 5-FU. This study demonstrates the effectiveness of transduction of the UPRT gene in addition to CD/5-FC cancer suicide gene therapy. METHODS: We investigated a combined suicide gene transduction therapy for human hormone independent prostate cancer cell line DU145 using two separate adenovirus vectors expressing the E. coli CD and E. coli UPRT genes and systemic 5-FC administration (the CD+UPRT/5-FC system). RESULTS: Cells transfected with AdCA-UPRT showed approximately 57 times lower IC50 to 5-FU compared with those transfected with AdCA-LacZ. Furthermore, cells transfected with AdCA-CD and AdCA-UPRT proved to be more sensitive to 5-FC compared with those transfected with AdCA-CD. Intratumoral injection of AdCA-CD and AdCA-UPRT drastically suppressed the growth of tumors which had generated from DU145 cells inoculated into athymic (nude) mice compared with those injected with AdCA-LacZ or AdCA-LacZ and AdCA-CD. CONCLUSIONS: These results suggest that the CD+UPRT/5-FC system could be a powerful factor in human prostate cancer suicide gene therapy.