| As a widespread chronic inflammatory diseases, periodontitis makes the periodontal tissue damaged, the teeth lost and influences some systemic diseases. However for now the pathogenesis of periodontitis is still not entirely clear. Inflamed tissues can produce angiogenic factors and inflammatory cytokines, which can stimulate the endothelial differentiation of cells and the formation of new vessels. It has been proven that periodontal ligament stem cells(PDLSCs) has the ability to differ into endothelial cells and form vessels, but it is still unclear about the molecular mechanisms of the PDLSCs endothelial differentiation. ERK pathway is one of the key pathways regulates cell physiological and pathological activities, which is widespread in eukaryotic cells and affect cell proliferation, differentiation, variety autophagy and apoptosis. ERK1/2 is a key protein of ERK pathway. Phosphorylationand dephosphorylation of ERK1/2 is the key step of molecular signal transduction from the surface of the receptor to the nucleus.When subjected to growth factor of extracellular matrix, ERK1/2 is activated through a series ofthe enzyme reaction, thereby regulating cellular activity.There has been some studies reported about multi-directional differentiation of PDLSCs under different local microenvironment. However, these studies focused on osteogenic differentiation direction. Other directionand may affect the degree of differentiation is still little studied.In this study, TNF-α analog inflammatory microenvironments to observe the ERK pathway regulating the endothelial differentiation of PDLSCs and to lay the foundations for further exploring the different directions and regulation basis of PDLSCs.Objective:To investigate the effect of extracellular signal-regulated kinase(ERK) signaling pathway on the endothelial differentiation of PDLSCs in normal conditionn and inflammatory microenvironment.1 Human periodontal ligament stem cells(hPDLSCs) is isolated, cultured, and identification in vitro. PDLSCs is induced endothelial differentiation through application of VEGF and FGF. After detecting measures associated with endothelial differentiation, a reliable model of endothelial differentiation is established. In vitro, inflammatory cytokine TNF-α simulates inflammation microenvironment to stimulate PDLSCs. Detecting index associated with endothelial differentiation, then an endothelial differentiation in inflammatory microenvironment model is established.2 According to the established endothelial differentiation model in normal condition and inflammatory microenvironment, PDLSCs is induced endothelial differentiation. The measures about cell proliferation and differentiation are detected after induction. The measures of PDLSCs endothelial differentiation induced in two different microenvironment are compared.3 After inducing PDLSCs as the method of differentiation model, the levels of ERK1/2 phosphorylation in the cell at different duration and methods of inducing are observed.4 Blocking the ERK1/2 phosphorylation of PDLSCs, then cells is induced endothelialdifferentiation in normal condition and inflammatory microenvironment. After detecting measures associated with endothelial differentiation, the differentiations of measures before and after blocking ERK pathway are compared to define the effect of the ERK pathway endothelial to differentiation of PDLSCs.Methods:1 Primary periodontal ligament cells was isolated by enzyme digestion method and single cell cloning. The cells were induced by colony formation assay. The protein of cell surface expression was detected by flow cytometry and multi-directional differentiation.2 PDLSCs was induced endothelial differentiation in normal conditionn and inflammatory microenvironment. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction(qRT-PCR) after induction. The proportion of CD31+and VE-cadherin+cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after induction by counting the number of the total tubes, the total branching points and the total tube length. According to these numbers, PDLSCs endothelial differentiation in normal condition and inflammatory microenvironment was compared.3 With using DMSO as as a negative control, U0126 used as a ERK1/2phosphorylation inhibitor. Protein of PDLSCs induced for 0h, 1h, 3h, 6h, 12 h was extracted. Phosphorylation level of ERK1/2 was detected by western blotting. ERK1/2phosphorylation level of extracting protein was detected by western blot. Protein of PDLSCs which induced in normal condition and inflammatory microenvironment and induction with U0126 for 1h was extracted.4 With adding U0126 blocking phosphorylation of ERK1/2, endothelial differentiation of PDLSCs was induced in normal condition and inflammatory microenvironment.The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction(qRT-PCR) after induction. The proportion of CD31+and VE-cadherin+cells wasidentified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after induction by counting the number of the total tubes, the total branching points and the total tube length. According to these numbers, endothelial differentiation of PDLSCs with blocking and unblocking phosphorylation of ERK1/2 in normal condition and inflammatory microenvironment was compared.Results:1 After primary culture, the cells showed fusiform. The cells were arranged nearly parallel between the long axis, and the major axis of the cell were nearly parallel.Identification of cells found to have the ability to clone formation. Flow cytometry showed high expression of mesenchymal stem cell markers and low expression of hematopoietic stem cell markers on cellular surface.2 Index associated with endothelial differentiation after PDLSCs endothelial differentiation was detected. Real-time PCR and flow cytometry results show that PDLSCs induced differentiation in the inflammatory microenvironment, ve-cadherin and VEGF mRNA transcription level and proportion of CD31+and VE-cadherin+cells were significantly higher than that in normal conditionn. Lumen formation ability in the inflammatory microenvironment was significantly higher than that in normal conditionn.3 Western blotting showed PDLSCs after endothelial differentiation, the phosphorylation level of ERK1/2 increased, which were significantly higher than 1h and3 h before induction. At 1h points after the induction, respectively, ERK1/2phosphorylation of PDLSCs was detected which induced in normal condition and inflammatory microenvironment and with U0126. The p-ERK1/2 levels which induced in the inflammatory microenvironment are higher than in normal conditionn, and U0126 can block ERK1/2 phosphorylation.4 Real-time PCR and flow cytometry results showed that, after adding U0126,m RNA transcription, protein expression levels and angiogenic ability of endothelial PDLSCs were significantly decreased. This explains that blocking ERK1/2phosphorylation process inhibited endothelial differentiation of PDLSCs. The inhibition ofERK1/2 phosphorylation process could not completely inhibit the endothelial differentiation in the inflammatory microenvironment.Conclusions:The endothelial differentiation of PDLSCs induced by VEGF and FGF, is regulated by the ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSCs. The differentiation of PLDSC in the inflammatory microenvironment will promote the endothelial differentiation process. Blocking the ERK pathway may partially inhibit the endothelial differentiation process PDLSCs in inflammatory microenvironment. |
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