Hypoxia Induces The Expression Of RUNX2 Via HIF-1?-mediated VEGF In Periodontal Ligament Stem Cells

Periodontitis is a chronic inflammatory disease that affects the majority of the population in the world.At present,the development of periodontitis could be controlled through scaling,subgingival curettage,or root planning,but the lost alveolar bone could not be restored,hence the problem of loose teeth could not be resolved.Currently,the method of regeneration,e.g.GTR,reaches no ideal effects,where the effects depend on the personal differences.However,it is a promising way to restore the loss of periodontal tissues through the regeneration via PDLSCs.As for periodontitis,the oxygen level of periodontal tissue decreases,and the hypoxia induced by periodontal inflammation affects the ability of PDLSCs.But the effect of hypoxia on PDLSCs in normal periodonta l tissues is rarely studied.In this study,we tried to elucidate the effect and mechanism of hypoxia on the expression of Runx2,a key factor in osteoblast differentiation in PDLSCs.We found that the expression of HIF-1?,VEGF and RUNX2 in PDLSCs increased after hypoxia treatment.In order to further clarify the mechanism of hypoxia on these three factors,we detected the expression of HIF-1?,VEGF and RUNX2 after applying Co Cl2,an agonist of HIF-1?,and YC-1,an antagonist of HIF-1?.In addition,exogenous h VEGF and VEGF siRNA were added and the expression of RUNX2 was detected.The conclusion is that,in the study of periodontal tissue regeneration,hypoxia could mediate the expression of VEGF by HIF-1?,which affects the expression of Runx2.Materials and methodsThe periodontal ligament stem cells were obtained by explant culture mixed with enzymatic digestion,and the cells were further purified by growth pattern observing and single cloning culture.The source was identified by detecting the mesenchymal surface markers by flow cytometry.And the multi-directional differentiation ability was tested through osteogenic inducing,identified by Alizarin red staining,and adipogenic inducing,identified by Oil red O staining.The expressions of HIF-1?,VEGF and RUNX2 in hypoxia(3% O2)were detected by Western blot and immunofluorescence staining.The optimal cell concentration of Co Cl 2 and YC-1 was determined by MTT assay.Co Cl2 and YC-1 were used to promote and inhibit the expression of HIF-1? in normoxia,to determine the effect of HIF-1? on the VEGF and RUNX2 protein expression.After adding exogenous recombinant hVEGF,the expression of RUNX2 was detected by Western blot.With VEGF siRNA interference,the expression of VEGF and RUNX2 in both gene and protein levels was detected by qPCR and Western blot.ResultsStable PDLCs were obtained through explant isolation and culture,growth pattern observation,and cloning and purification.By detecting the expression of mesenchymal surface markers,we found that the positive expressions of these surface markers in PDLSCs,while the hematopoietic stem cell marker was negative.Hence the origin could be further determined.Besides,the Alizarin red staining confirmed red mineralized nodules formation after the osteogenesis inducing,and Oil red O staining confirmed lipid droplets formation after the adipogenic inducing.The expression of HIF-1?,VEGF and RUNX2 was detected by western blot in hypoxia(3% O2),and the results showed that hypoxia promoted the expression of HIF-1?,VEGF and RUNX2 protein(p < 0.05).In consistent with Western blot,cell immunofluorescence staining showed that the expression of HIF-1?,VEGF and RUNX2 was enhanced in hypoxia.The suitable cell concentrations of Co Cl2 and YC-1 were 100 ?mol/L and 10 nmol/L,respectively,by MTT assay.The expression of HIF-1?,VEGF and RUNX2 in CoCl2 group was increased,and the expression of HIF-1?,VEGF and RUNX2 in YC-1 group was lower than that in hypoxia group.After adding exogenous recombinant hVEGF,the expression of RUNX2 protein was found to be increased.And q PCR and Western blot confirmed that the VEGF siRNA was applied successfully,and the RUNX2 express was found to be decreased in the si RNA group.ConclusionIn PDLSCs,hypoxia could mediate the expression of RUNX2 through HIF-1? inducing VEGF.