Methods: Cells were stimulated with EMD, transforming growth factor-β (TGF-β), or fibroblast growth factor 2 (FGF-2), with or without antibodies to TGF-β or FGF-2. The levels of VEGF in the culture media were measured using an ELISA. Quantitative real-time PCR was carried out to investigate the mRNA expression profiling in the gingival fibroblasts. We examined the effects of SB203580 [a p38 mitogen-activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal-regulated kinase (ERK) inhibitor], SP600125 [a c-Jun N-terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor] on EMD-induced VEGF production.
Results: EMD stimulated the production of VEGF in a dose- and time-dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF-β or FGF-2 significantly decreased EMD-induced VEGF production, and the addition of exogenous TGF-β and FGF-2 stimulated VEGF production. We observed that EMD stimulated a significant increase in FGF-2 mRNA expression in the gingival fibroblasts compared with control. Furthermore, EMD-stimulated cells produced significantly higher levels of FGF-2 protein, compared to control cells. EMD-induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002.
Conclusions: Our results suggest that EMD stimulates VEGF production partially via TGF-β and FGF-2 in human gingival fibroblasts and that EMD-induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. EMD-induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.