Leukemia inhibitory factor-transfected embryonic fibroblasts and vascular endothelial growth factor successfully improve the skin substitute wound healing by increasing angiogenesis and matrix production.
OBJECTIVE
The combined application of cytokines on embryonic fibroblasts and dermal substitute were studied for optimal skin defect coverage. The mechanism of combined treatment of leukemia inhibitory factor (LIF)-transfected embryonic fibroblasts and vascular endothelial growth factor (VEGF) were elucidated and subsequently the in vivo applications of both were tested in an artificial dermal substitute.


METHODS
Mouse embryonic fibroblast cells, BALB-3T3, were stably transfected with mouse full-length LIF cDNA and added to various doses of VEGF for detection of signaling interaction. LIF-transfected cells and VEGF treatment were tested with pig-tendon derived collagen dermal substitute in the backs of BALB/c male mice up to for 14 days.


RESULTS
LIF-transfected cells as well as vector-transfected fibroblasts significantly proliferated by 1, 10, or 100 ng VEGF on days 3 and 5. Erk mitogen-activated protein (MAP) kinase phosphorylation was observed from 1 to 30 min in LIF-transfected and 10 ng of VEFG, and 1 to 60 min in LIF-transfected and 100 ng VEFG treatments. The cellular fibronectin levels also increased in LIF-transfected cells with 10 and 100 ng VEGF additions. In in vivo analyses, LIF-transfected embryonic fibroblasts with 50 microg of VEGF markedly enhanced collagen I expression and CD34 angiogenic marker on days 7 and 14.


CONCLUSIONS
LIF transfection and VEGF treatment enhanced phosphorylated-Erk-MAP kinase in vitro. In vivo study revealed that the combined application of LIF transfection of embryonic fibroblasts with an angiogenic factor such as VEGF in the template of a dermal substitute induced greater skin collagen production and angiogenesis in the dermal substitute.
Division of Plastic and Reconstructive Surgery, Department of Developmental and Reconstructive Medicine, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto machi, Nagasaki 8528501, Japan. akitas@hf.rim.or.jp
Sadanori
S
Akita
Takahiro
T
Daian
Hiroshi
H
Ishihara
Tohru
T
Fujii
Kozo
K
Akino
D016475:3T3 Cells; D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D015153:Blotting, Western; D049109:Cell Proliferation; D003094:Collagen / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D007150:Immunohistochemistry; D015850:Interleukin-6 / Q000235:genetics*; D053605:Leukemia Inhibitory Factor; D020935:MAP Kinase Signaling System; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009389:Neovascularization, Pathologic; D010766:Phosphorylation; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D012867:Skin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D018952:Antigens, CD34; D018076:DNA, Complementary; D005353:Fibronectins; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D042461:Vascular Endothelial Growth Factor A; D012313:RNA; D003094:Collagen; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3