| BackgroundOrthodontic treatment can achieve a healthy occlusal relationship and facial appearance by solving malocclusion.The efficient orthodontic clinical treatment depends on an accurate control of orthodontic tooth movement.Orthodontic tooth movement is a biological consequence of alveolar bone remodeling induced by mechanical force and is controlled by osteoblasts and osteoclasts.Osteoclasts originate from the compressive side of the periodontal ligament by the fusion of pre-osteoclasts recruited during orthodontic tooth movement and decide the efficiency of orthodontic tooth movement.In addition,bone remodeling is also regulated by local microenvironment,in which angiogenesis plays a positive supporting role in the process of osteogenesis and osteoclastogenesis.Angiogenesis refers to the formation of new blood vessels from existing blood vessels,including the proliferation,migration and tube formation of endothelial cells,which can be influenced by hypoxia signals,secreted cytokines,non-coding RNAs and other factors in local tissues.Further,it has been proved that periodontal ligament stem cells,which participate in alveolar bone remodeling,affect the biological activities of endothelial cells.So whether osteoclasts also exert an effect on angiogenesis,so as to regulate alveolar bone remodeling and what is the role of mechanical forces in this intercellular communication need further clarifying.As important mediators of cell-to-cell communication,exosomes transport specific proteins,mRNAs,and microRNA(miRNAs).miRNAs play important role in bone remodeling.Whether and how exosomes and the miRNAs contained participate in bone remodeling through the communication between osteoclasts and endothelial cells remain to be investigated.ObjectivesThe purpose of this study is to explore the effects of exosomes and microRNAs secreted by osteoclasts on the angiogenesis of endothelial cells and the regulated role of mechanical compression;to investigate the mechanism of mir-146a-5p(miR-146a)and adiponectin(ADP)on angiogenesis,and to clarify the effect of miR-146a on orthodontic tooth movement in vivo,so as to provide a theoretical basis for the clinical application of miR-146a in orthodontics and the regulation of angiogenesis.Materials and Methods1.The rat model of tooth movement was established.Osteoclastogenesis and angiogenesis of periodontal ligament were detected 7 and 14 days after orthodontic force loading.2.Osteoclastic differentiation of THP-1 cell line was induced in vitro,the optimal time and value of compression stress to promote osteoclastogenesis were explored.3.The intercellular regulation of osteoclasts on human umbilical vein endothelial cells(HUVECs)was detected through co-culture experiment;Exosomes extracted from compressed osteoclasts(EXOf)and exosomes extracted from static osteoclasts(EXO)were added to HUVECs.The real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR),Western Blot,migration assay,wound-healing cell migration assay and tube network formation assay were used to explore the effects of osteoclasts on endothelial cell proliferation,migration and tube formation.4.High throughput transcriptome sequencing(RNA-seq)was used to detect the changed miRNAs of osteoclasts after compression,and the results were analyzed to find potential regulatory miRNAs and their target genes.The effects of miR-146a on endothelial cell proliferation,migration,tube network formation,and the expression of VEGF,CD31,and ADP were examined by qRT-PCR,Western Blot,migration assay,wound-healing cell migration assay and tube network formation assay.Double luciferase assay was used to verify the relationship between miR-146a and the target gene ADP.5.qRT-PCR was used to verify the different expression of miR-146a in EXO and EXOf;the expression of miR-146a,CD31,VEGF and ADP in HUVECs was verified after adding EXO and EXOf respectively.6.qRT-PCR,Western Blot,migration assay,wound-healing cell migration assay and tube network formation assay were used to explore the effect of ADP expression on endothelial cell proliferation,migration and tube formation.7.Western Blot was used to explore the downstream pathway of miR-146a/ADP after the transfection of miR-146a inhibitor and mimics into HUVECs.8.To observe the effect of miR-146a-5p on tooth movement,appropriate amount of agomiR-146a and antagomiR-146a were injected into periodontal ligament of teeth under orthodontic treatment.Results1.The expression of TRAP and CD31 in the periodontal ligament of the stressed teeth increased on day 7 and day 14 after orthodontic force loading.2.In the process of osteoclastic induction in vitro,the expression of osteoclastic markers increased.After the loading of compression stress during osteoclastogenesis,the increased osteoclasts were showed by TRAP staining.The results of Western Blot and PCR showed that the expression of TRAP increased in compressed osteoclasts,which proved that mechanical compressure promoted the differentiation of osteoclasts.3.The proliferation rate of HUVECs cocultured with osteoclasts increased,and the inhibition of exosome secretion weakened this promotive effect.The proliferation,migration and tube formation of HUVECs were promoted after adding with osteoclast-derived exosomes,and the promotive effect of EXOf was greater than that of EXO.4.67 miRNAs decreased and 170 miRNAs increased in osteoclasts after compression loading.miR-146a inhibitor promoted the proliferation,migration,tube formation and the expression of ADP in HUVECs while miR-146a mimics inhibited the angiogenesis and the expression of ADP in HUVECs.Furthermore,double luciferase assay showed that ADP was the target gene of miR-146a5.The amount of miR-146a in EXOf was less than that in EXO;the expression of miR146a in HUVECs with EXOf was lower than that in HUVECs with EXO.The expression of CD31,VEGF and ADP were increased in HUVECs treated with osteoclastic exosomes,and the effect of EXOf was greater than that of EXO.6.ADP promoted the proliferation,migration and tube formation of HUVECs.The migration and tube formation of HUVECs weakened after the transfection of si-ADP,with the decreased expression of CD31 and VEGF.7.Transfection of miR-146a inhibitor promoted the phosphorylation of ERK pathway and Akt pathway in HUVECs,while miR-146a mimics played the opposite role.The expression of phosphorylated ERK(p-ERK)and phosphorylated Akt(p-Akt)was decreased by si-ADP transfection.It was proved that miR-146a/ADP affected angiogenesis by activating ERK pathway and Akt pathway in HUVECs.8.The tooth movement in rats after the local injection of antagomiR-146a was faster than that in the control group,accompanied by the increase of angiogenesis in periodontal ligament.On the contrary,the tooth movement with the local injection of agomiR-146a was less than that in the control group,accompanied by the decrease of angiogenesis in periodontal ligament.ConclusionCompression stress can promote osteoclastogenesis and reduce the expression of miR146a in osteoclasts.Altered miR-146a promote the expression of ADP in endothelial cells through exosomes.Then the activated ERK and Akt pathways promote angiogenesis of endothelial cells,affecting bone remodeling and tooth movement in vivo. |
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