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Biodegradable gelatin hydrogel potentiates the angiogenic effect of fibroblast growth factor 4 plasmid in rabbit hindlimb ischemia

Hirofumi Kasahara, MD^*, Etsuro Tanaka, MD, PhD, Naoto Fukuyama, MD, PhD, Eriko Sato, MD^*, Hiromi Sakamoto, PhD^||, Yasuhiko Tabata, PhD^¶, Kiyoshi Ando, MD, PhD, Harukazu Iseki, MD, PhD, Yoshiro Shinozaki, BS, Koji Kimura, MD^*, Eriko Kuwabara, MD^*, Shirosaku Koide, MD, PhD^*, Hiroe Nakazawa, MD, PhD and Hidezo Mori, MD, PhD^#,*

^* Cardiovascular Surgery, Tokai University School of Medicine, Isehara, Japan
Physiology, Tokai University School of Medicine, Isehara, Japan
Internal Medicine, Tokai University School of Medicine, Isehara, Japan
Research Center for Genetic Engineering and Cell Transplantation, Tokai University School of Medicine, Isehara, Japan
^|| Genetics Division, National Cancer Center Research Institute, Tokyo, Japan
^¶ Research Center for Biomedical Engineering, Kyoto University, Kyoto, Japan
^# Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Suita, Japan

View larger version (14K): [in a new window]   Figure 1 Decay sequences of radiolabeled deoxyribonucleic acid (DNA), gelatin hydrogel (GHG), and DNA combined with GHG in the hindlimb muscles of mice. Unlabeled GHG impregnated with 125I-labeled DNA (solid line), 125I-labeled GHG (dashed line), and 125I-labeled DNA solution (dotted line) were injected into the hindlimb muscles.

View larger version (41K): [in a new window]   Figure 2 Representative transgene expression demonstrated by reverse-transcription nested polymerase chain reaction (RT-nested PCR). The left adductor muscle of the rabbits was injected with naked fibroblast growth factor 4 (FGF4) gene (lanes 1 to 3), gelatin hydrogel (GHG)-FGF4 (lanes 4 to 6), or GHG-lacZ (lanes 7 and 8). Each sample was obtained from the injection site (lanes 1, 4, and 7) and the adjacent region 10 mm apart from the injection site (lanes 2, 5 and 8) in the left adductor muscle, and from the contralateral adductor muscle (lanes 3 and 6). The RT-nested PCR products from ribonucleic acid of each sample were analyzed on agarose gel; FGF4 expressed Cc1/l6 cells as a positive control (lane 9) and no deoxyribonucleic acid (DNA) template as a negative control (lane 10). A housekeeping beta-actin gene was amplified as a complementary DNA loading control.

View larger version (122K): [in a new window]   Figure 3 Representative gene expression of lacZ in the ischemic adductor muscle in rabbits on day 17. Naked deoxyribonucleic acid (DNA) (A) or gelatin hydrogel-DNA complex (B) was injected into the adductor muscle 10 days after the ischemic insult. X-Gal stain; original magnification x20; bar = 200 µm.

View larger version (148K): [in a new window]   Figure 4 Representative synchrotron radiation microangiograms of the hindlimb ischemia in the rabbits. Synchrotron radiation microangiograms were taken under baseline conditions (A and C) and after repeated adenosine administration (B and D) on day 38. (A and B) Gelatin hydrogel (GHG)-lacZ-treated rabbit; (C and D) GHG-fibroblast growth factor 4-treated rabbit. Arrows indicate the same point in the vessels. Arrowheads reference copper wires with a diameter of 130 µm; bar = 1 mm.