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PRECLINCAL RESEARCH

Powerful and controllable angiogenesis by using gene-modified cells expressing human hepatocyte growth factor and thymidine kinase

Yasuyo Hisaka, MS^*, Masaki Ieda, MD, Toshikazu Nakamura, PhD, Ken-ichiro Kosai, MD, PhD, Satoshi Ogawa, MD, PhD and Keiichi Fukuda, MD, PhD^*,*

^* Institute for Advanced Cardiac Therapeutics, Tokyo, Japan
Cardiopulmonary Division, Department of Medicine, Keio University School of Medicine, Tokyo, Japan
Division of Molecular Regenerative Medicine, Course of Advanced Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
Cognitive and Molecular Research Institute of Brain Disease, Kurume University School of Medicine, Fukuoka, Japan

Manuscript received June 28, 2003; revised manuscript received December 10, 2003, accepted January 5, 2004.

^* Reprint requests and correspondence: Dr. Keiichi Fukuda, Institute for Advanced Cardiac Therapeutics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
kfukuda{at}sc.itc.keio.ac.jp

OBJECTIVES: This study investigated the possibility of achieving angiogenesis by using gene-modified cells as a vector.

BACKGROUND: Although gene therapy for peripheral circulation disorders has been studied intensively, the plasmid or viral vectors have been associated with several disadvantages, including unreliable transfection and uncontrollable gene expression.

METHODS: Human hepatocyte growth factor (hHGF) and thymidine kinase (TK) expression plasmids were serially transfected into NIH3T3 cells, and permanent transfectants were selected (NIH3T3 + hHGF + TK). Unilateral hindlimb ischemia was surgically induced in BALB/c nude mice, and cells were transplanted into the thigh muscles. All effects were assessed at four weeks.

RESULTS: The messenger ribonucleic acid expression and protein production of hHGF were confirmed. Assay of growth inhibition by ganciclovir revealed that the 50% (median) inhibitory concentration of50 at first mention (50% "I?" concentration)> NIH3T3 + hHGF + TK was 1,000 times lower than that of NIH3T3 + hHGF. The NIH3T3 + hHGF + TK group had a higher laser Doppler blood perfusion index, higher microvessel density, wider microvessel diameter, and lower rate of hindlimb necrosis, as compared with the plasmid- and adenovirus-mediated hHGF transfection groups or the NIH3T3 group. The newly developed microvessels were accompanied by smooth muscle cells, as well as endothelial cells, indicating that they were on the arteriolar or venular level. Laser Doppler monitoring showed that the rate of blood perfusion could be controlled by oral administration of ganciclovir. The transplanted cells completely disappeared in response to ganciclovir administration for four weeks.

CONCLUSIONS: Gene-modified cell transplantation therapy induced strong angiogenesis and collateral vessel formation that could be controlled externally with ganciclovir.

Abbreviations and Acronyms   DMEM = Dulbecco's modified Eagle's medium   EGFP = enhanced green fluorescent protein   ELISA = enzyme-linked immunosorbent assay   hHGF = human hepatocyte growth factor   IC50 = 50% (median) inhibitory concentration50.>   LDPI = laser Doppler perfusion image   RT-PCR = reverse transcription-polymerase chain reaction   SMA = smooth muscle actin   TK = thymidine kinase   vWF = von Willebrand factor