Introduction: The combined application of cytokines to embryonic fibroblasts and dermal substitute was extensively studied for optimal skin defect coverage. Signals from combined treatment of leukemia inhibitory factor (LIF) and vascular endothelial 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 for 10 days. Results: LIF-transfected cell proliferation was not significantly augmented by any of the various doses of VEGF, while vector-transfected fibroblasts significantly increased with 10 nM and 100 nM of VEGF compared to that with 1 nM of VEGF on days 3 and 5. LIF-transfected cells showed rapid phosphorylation of STAT 3 from 1 minute to 60 minutes after VEGF treatment, while vector-transfected cells failed to induce such phosphorylation after VEGF treatment. Erk MAP kinase phosphorylation was observed from 15 to 60 minutes in LIF-transfected and 10 nM of VEFG and 60 to 120 minutes in LIF-transfected and 100 nM VEFG treatment. In in vivo analyses, LIF-transfected embryonic fibroblasts with 50 µg of VEGF markedly enhanced collagen I expression and CD 34 angiogenic marker on days 7 and 10. Conclusions: LIF transfection induced constitutive STAT signaling and enhanced phosphorylated-Erk MAP kinase with exogenous VEGF. 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 optimal skin collagen production and angiogenesis in the dermal substitute.
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