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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

View larger version (27K): [in a new window]   Figure 1 (A) Expression of human hepatocyte growth factor (hHGF) messenger ribonucleic acid in the hHGF-transfected NIH3T3 cells. The primer set of reverse transcription-polymerase chain reaction specifically detects hHGF but not mouse HGF. pUC-SR/hHGF plasmid and mouse liver were used as a positive and negative control, respectively. M = the X174-HaeIII digest. (B) Production of hHGF protein. This ELISA system specifically detects only hHGF because of the lack of cross-reactivity by the antibodies. Data are expressed as hHGF concentrations adjusted for cell number. Both NIH3T3 + hHGF and NIH3T3 + hHGF + thymidine kinase (TK) groups expressed hHGF messenger ribonucleic acid and produced hHGF protein (n = 5).

View larger version (19K): [in a new window]   Figure 2 (A) Influence of hHGF and/or TK genes on cell growth in vitro. The growth rate of the hHGF-transfected NIH3T3 cells was slightly higher than that of the nontransfected cells, but TK had no effect on cell growth. (circles = NIH3T3; diamonds = NIH3T3 + hHGF; squares = NIH3T3 + hHGF + TK) (n = 3). (B) Growth-inhibitory effect of ganciclovir. The IC50 of ganciclovir for the NIH3T3 + hHGF + TK group (solid bars) was 1,000 times lower than that for the NIH3T3 + hHGF group (open bars) (n = 5). (C) Apoptotic cells stained with annexin V-EGFP at the cell membrane after exposure to ganciclovir. Abbreviations as in Figure 1.

View larger version (25K): [in a new window]   Figure 3 Frequency of necrosis in the ischemic hindlimbs. Severe hindlimb necrosis was significantly reduced in the NIH3T3 + hHGF + TK group. Open areas = negative necrosis; lined areas = necrosis on toes; solid areas = necrosis on foot. Abbreviations as in Figure 1.

View larger version (50K): [in a new window]   Figure 4 (A, panels a to d) Immunohistochemical staining for von Willebrand factor in the triceps muscle of the left calf revealed the presence of numerous vessels. Vessels were larger and more numerous in the NIH3T3 + hHGF + TK group (panels c and d) than in the saline (panel a) and NIH3T3 groups (panel b). Scale bars = 100 µm. (B) The number of vessels was determined by observation of 20 random fields from 10 mice (2 fields per mouse; *p < 0.01). (C) Distribution of the minimum diameters of the von Willebrand factor-positive vessels (n = 25; *p < 0.0001). Abbreviations as in Figure 1.

View larger version (47K): [in a new window]   Figure 5 (A, panels a to c) Three consecutive frozen sections of NIH3T3 + hHGF + TK transplanted muscle. (panel a) Immunohistochemical staining for vWF and (panel b) -smooth muscle actin (SMA) and (panel c) elastica van Gieson staining. Scale bars = 100 µm. (B) Maturation of vessels was compared by using three consecutive frozen sections. Most of the von Willebrand factor (vWF)-positive vessels in NIH3T3 + hHGF + TK transplanted mice also stained with -SMA (n = 20; *p < 0.05, p < 0.001, p < 0.01). Abbreviations as in Figure 1.

View larger version (71K): [in a new window]   Figure 6 (A) Representative laser Doppler perfusion images. (B) Quantitative analysis of the rate of blood perfusion of the ischemic/nonischemic limb. Panel a = Control mouse on postoperative day 1; panels b to f = four weeks after treatment (panel b = saline injection; panel c = NIH3T3 transplantation [107 cells]; panel d = NIH3T3 + hHGF + TK transplantation [104 cells]; panel e = NIH3T3 + hHGF + TK transplantation [107 cells]; panel f = beginning two weeks after transplantation of NIH3T3 + hHGF + TK (107 cells), ganciclovir (50 mg/kg/day) was administered orally for four weeks. Oral ganciclovir administration adjusted the blood perfusion rate of the ischemic limb to the same level as that of the nonischemic limb (eight mice/group). *p < 0.01, p < 0.001 vs. saline, p < 0.05, ¶p < 0.01 versus NIH3T3. Abbreviations as in Figure 1.

View larger version (75K): [in a new window]   Figure 7 (A) Immunohistochemical staining for hHGF in transplanted NIH3T3 cells (panel a) and NIH3T3 + hHGF + TK cells (panel b) in the skeletal muscle. Scale bars = 50 µm. (B) The NIH3T3 + hHGF + TK (107) cells were transplanted, and two weeks later, various concentrations of ganciclovir were administered for another four weeks. (C) Hematoxylin-eosin staining. (Panels a to c) The natural history of the transplanted NIH3T3 + hHGF + TK (107) cells is shown. (Panels d to f) Beginning two weeks after transplantation, ganciclovir (50 mg/kg/day) was administered orally for two to four weeks. The cells had completely disappeared after four weeks of ganciclovir treatment. Arrows indicate the microvessels. Scale bars = 100 µm. Abbreviations as in Figure 1.