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 (rhVEGF) on odontoblastic differentiation, in vitro angiogenesis, and expression and activity of lysyl oxidase (LOX) in human dental pulp cells (HDPCs), compared with rhFGF-2. To identify the underlying molecular mechanisms, the study focused on whether LOX was responsible for the actions of rhVEGF.

Methodology

rhVEGF was constructed using the pBAD-HisA plasmid in Escherichia coli. HDPCs were treated with 1- 50 μg/mL rh-VEGF for 14 days. The alkaline phatase (ALP) activity was measured, and the formation of calcified nodules was assessed using alizarin red staining after the induction of odontogenic differentiation of HDPCs. The expression level of of the odontogenic differentiation markers were detected by reverse transcription polymerase chain reaction. Signal pathways were assessed by Western blot and immunocytochemistry. The datas were analysed by ANOVA with Bonferroni's test (α =0.05).

Results

rhVEGF 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 rhFGF-2. rhVEGF significantly increased amine oxidase activity (P < 0.05), and upregulated LOX and LOXL mRNA expression in HDPCs. Additionally, rhVEGF dose-dependently upregulated angiogenic gene mRNAs and capillary tube formation to a greater degree than rhFGF-2. Inhibition of LOX using β-aminopropionitrile (BAPN) and LOX or LOXL gene silencing by RNA interference attenuated rhVEGF-induced growth, ALP activity, mineralization, the expression of marker mRNAs, and in vitro angiogenesis. Furthermore, treatment with rhVEGF resulted in phosphorylation of Akt, ERK, JNK, p38, and activation of NF-κB, which was inhibited by LOX or LOXL silencing and BAPN.

Conclusion

rhVEGF promoted cell growth, odontogenic potential, and in vitro angiogenesis via modulation of LOX expression. These results support the concept that rhVEGF may offer therapeutic benefit in regenerative endodontics.

This article is protected by copyright. All rights reserved.

http://ift.tt/2sgA1I7