Lysyl oxidase‐mediated VEGF‐induced differentiation and angiogenesis in human dental pulp cells

Abstract Aim To investigate the effects of recombinant human vascular endothelial growth factor (rh VEGF ) on odontoblastic differentiation, in vitro angiogenesis, and expression and activity of lysyl oxidase ( LOX ) in human dental pulp cells ( HDPC s), compared with rh FGF ‐2. To identify the underlying molecular mechanisms, the study focused on whether LOX was responsible for the actions of rh VEGF . Methodology Recombinant human vascular endothelial growth factor (rh VEGF ) was constructed using the pBAD ‐HisA plasmid in Escherichia coli . HDPC s were treated with 1–50 μg mL −1 rh VEGF for 14 days. Alkaline phosphatase ( ALP ) activity was measured, and the formation of calcified nodules was assessed using alizarin red staining after the induction of odontogenic differentiation of HDPC s. The expression level of the odontogenic differentiation markers was detected by reverse transcription polymerase chain reaction. Signal pathways were assessed by Western blot and immunocytochemistry. The data were analysed by anova with Bonferroni's test (α = 0.05). Results Recombinant human vascular endothelial growth factor significantly increased cell growth ( P < 0.05), ALP activity ( P < 0.05) and mineralization nodule formation and upregulated the mRNA expression levels of the osteogenic/odontogenic markers that were lower with rh FGF ‐2. rh VEGF significantly increased amine oxidase activity ( P < 0.05) and upregulated LOX and LOXL mRNA expression in HDPC s. Additionally, rh VEGF dose‐dependently upregulated angiogenic gene mRNA s and capillary tube formation to a greater degree than rh FGF ‐2. Inhibition of LOX using β‐aminopropionitrile ( BAPN ) and LOX or LOXL gene silencing by RNA interference attenuated rh VEGF ‐induced growth, ALP activity, mineralization, the expression of marker mRNA s and in vitro angiogenesis. Furthermore, treatment with rh VEGF resulted in phosphorylation of Akt, ERK , JNK and p38, and activation of NF ‐κB, which was inhibited by LOX or LOXL silencing and BAPN. Conclusion Recombinant human vascular endothelial growth factor promoted cell growth, odontogenic potential and in vitro angiogenesis via modulation of LOX expression. These results support the concept that rh VEGF may offer therapeutic benefits in regenerative endodontics.