The Ability Of Endothelial Differentiation Of Periodontal Ligament Stem Cells In Inflammatory Microenvironments And Associated Molecular Mechanisms

Periodontitis is one of the most widespread chronic inflammatory diseases, whichmakes periodontal tissue irreversibly damaged. Inflammation stimulates angiogenesis.There is inflammatory granulation tissue formation in periodontitis. The periodontalligament stem cells (PDLSCs) survive in the periodontal ligament with multidirectionaldifferentiation ability. Recent studys have shown that differentiation of mesenchymal stemcells (MSCs) is regulated by tissue microenvironments. The ability of differentiation ofPDLSCs is affected by inflammatory microenvironments. The finding that inflammationfavors stem cells differentiation into endothelial cells is explained byinflammation-induced production of angiogenic factors. It is not clear whetherinflammatory conditions promote endothelial differentiation of PDLSCs. Stromal-derivedfactor-1α (SDF-1α) and tumor necrosis factor-α (TNF-α) are secrected in periodontitis andplay an important role in the development of periodontitis. CXCR4+MSCs can be inducedto differentiate into endothelial cells by SDF-1α. TNF-α is a key regulator of SDF-1α/CXCR4axis through affecting SDF-1α or CXCR4expression. In this research, itis shown the effect of TNF-α and SDF-1α/CXCR4axis on endothelial differentiation ofperiodontal ligament stem cells in inflammatory microenvironments.Objective: To investigate the ability of endothelial differentiation of PDLSCs ininflammatory microenvironments and associated molecular mechanisms.Methods:1.The stem cells from healthy and inflammatory periodontal ligament tissues(H-PDLSCs and I-PDLSCs) were isolated and examined the ability of colony-forming.Cell-surface-marker characterization of the two kinds of stem cells was checked usingflow cytometry analysis. The osteogenic potential of cells was assayed by alizarin redstaining.2.SDF-1α mRNA and CXCR4mRNA in H-PDLSCs and I-PDLSCs were evaluatedby Real-time PCR. After that two kinds of cells were incubated with SDF-1α, theexpression of endothelial gene in different groups were assayed by Real-time PCR.Matrigel assays analysed the ability of capillary-like structures formation in differentgroups.3.The effet of TNF-α on the proliferative potential of PDLSCs was assessed byCCK-8assays. Real-time PCR analysed the effet of TNF-α on the expression of SDF-1αmRNA and CXCR4mRNA in H-PDLSCs.4.Real-time PCR and Western Blot evaluated the expression of endothelial gene andprotein in only SDF-1α inclubation cells and SDF-1α inclubation cells after TNF-αpretreatment or TNF-α and AMD3100pretreatment. Matrigel assays analysed the abilityof capillary-like structures formation in TNF-α pretreatment group and TNF-α andAMD3100pretreatment groups.Results:1. The two kinds of stem cells both showed a typical fibroblastic spindle shape andwere capable of forming fibroblastic colony-forming units generated from single cells.Thecells from both tissue sources positively expressed CD146, CD29, CD90and CD105,while negatively expressed CD45, CD34, CD31. I-PDLSCs had a lower capacity for osteogenic differentiation compared to H-PDLSCs by alizarin red staining and quantitativeanalysis.2.CXCR4mRNA expression in I–PDLSCs were more than those in H-PDLSCs, butSDF-1α mRNA expression in I-PDLSCs showed less than those in H-PDLSCs. Theendothelial gene expression of CD31and vWF and capillary network in the cellsinclubated with SDF-1α were more than those in the control group. The endothelial geneexpression of CD31and vWF and capillary network in I–PDLSCs inclubated withSDF-1α were more than those in H-PDLSCs inclubated with SDF-1α.3.The cell growth assay revealed that TNF-α at the concentrations of1,10ng/mL hadno effect on cell viability. Nevertheless,100ng/mL TNF-α significantly inhibited cellproliferation.10ng/mL TNF-α inhibited SDF-1α expression of PDLSCs and enhancedCXCR4expression of PDLSCs.4.Real-time PCR and Western Blot displayed that the expression of endothelial geneand protein in SDF-1α inclubation cells after TNF-α pretreatment were more than those inonly SDF-1α inclubation cells. Compared to TNF-α pretreatment group, the expression ofendothelial gene and protein and the ability of capillary-like structures formation weredecreased in TNF-α and AMD3100pretreatment group.Conclusion:The ability of endothelial differentiation of PDLSCs was promoted in inflammatorymicroenvironments. The associated molecular mechanisms was that TNF-α played apositive role in endothelial differentiation of PDLSCs induced by SDF-1α/CXCR4through increasing CXCR4expression in PDLSCs.