HIF1? Up-regulates Runx2 To Promote BMP9-induced Osteogenic Differentiation And Angiogenesis In Mesenchymal Stem Cells

Objective:Mesenchymal Stem Cells(MSCs)play an important role in bone repair after bone fracture.Bone morphogenetic protein 9(BMP9)mediates MSCs during bone regeneration.Tissue in the environment of hypoxia is a kind of key driver of angiogenesis,in which the key molecule hypoxia inducible factor,Hypoxia-inducible factor 1?,(HIF1?)and the Runt-related transcription factor 2(Runx2)may have a cascaded and regulatory relationship,but the two factors how to participate in the regulation of BMP9-induced the MSCs and the specific molecular mechanisms between two factors by which MSCs promote conjugated effects of osteogenesis and angiogenesis still remain unclear.On the basis of these facts,how the regulation mechanism between HIF1?and Runx2 to promote and regulate the effect of BMP9-induced the osteogenic differentiation and angiogenesis of MSCs,which could provide a new idea for clinical treatment of bone defects or bone nonunion.Methods:(1)Mouse embryonic fibroblasts(MEFs)cells were isolated,and the mesenchymal stem cell phenotype was identified by flow cytometry.The osteogenesis,chondrogenic and adipogenic induction medium were used to induce cultivation,the alizarin red,toluidine blue and oil red O staining were used to evaluate the differentiation effect of the MEFs into osteocytes,chondroblasts and adipocytes.By constructing recombinant adenovirus exogenously overexpressing Ad-BMP9,Ad-HIF1?and Ad-Runx2,silencing Ad-simHIF1?and Ad-simRunx2,transfecting and regulating MEFs,the ALP staining was used to analyze the early stage of osteogenic differentiation,the alizarin red staining was used to detect the advanced bone mineralization ability,the quantitative real time polymerase chain reaction(qRT-PCR)and Western blot were used to detect the osteogenic differentiation and angiogenesis related markers.Immunohistochemical staining analysis was performed to detect the angiogenic markers and mutual regulation effect between HIF1?and Runx2 during differentiation process in BMP9-induced MSCs in vitro.(2)The nude mice were used to perform the ectopic ossification experiment,the exogenously overexpressed or silenced HIF1?and Runx2were transfected into BMP9-induced MEFs cells and injected into the nude mice subcutaneously.The bone mass was analyzed by Micro CT,and the growth and number of vascular lumens were detected by H&E staining.The Masson's Trichrome staining was used to observe the trabecular bone distribution,bone tissue sections were detected by immunohistochemical staining for the expression and distribution of osteogenesis and angiopoie-associated markers,to verify the regulation effect between HIF1?and Runx2 to promote osteogenic differentiation and angiogenesis in BMP9-induced MSCs.(3)The plasmid overexpressing BMP9,HIF1?and Runx2 was constructed.The effect of HIF1?and Runx2 on each promoter was explored by using the dual luciferase reporter gene assay.The presence of HIF1?and Runx2 was detected by chromatin immunoprecipitation assay.The relationship between HIF1?and Runx2 on the classical Smads-dependent pathway of BMP and the key factors of non-canonical MAPK pathway were detected by Western Blot,which to explore the possible mechanisms of the regulation effect between HIF1?and Runx2promoted osteogenic differentiation and angiogenesis on BMP9-induced MSCs.Results:(1)The MEFs as a mesenchymal stem cell derived from mouse embryos,the MEFs are pluripotent stem cells which in the possession of self-renewal and replication ability.The MEFs can differentiate into mesodermal tissues such as osteoblasts,chrondroblasts and adipocytes.It has favorable proliferation and cell morphology maintenance ability.(2)After transfection of recombinant adenovirus,exogenous over-expressingAd-BMP9,Ad-HIF1?andAd-Runx2,silencing Ad-simHIF1?and Ad-simRunx2,regulating BMP9-induced MEFs,the fluorescence was observed after transfection of the recombinant adenovirus into MSCs,the expression was sustained and effective.After BMP9transfected C3H10T1/2,iMADs and MEFs with different MSCs,the qRT-PCR results showed the mRNA expression levels of HIF1?and Runx2,which were relatively more persistent in MEFs group among the C3H10T1/2,iMADs and MEFs.(3)HIF1?promotes BMP9-induced osteogenic differentiation of MEFs and accelerates the early stage of osteogenic differentiation,increases bone mineralization and calcium deposition.The mRNA expression of osteogenic markers including ALP,BSP,OCN,OPN,COL-A1 and OSX were detected by qRT-PCR assay.The expression of these factors was up-regulated with the prolongation of induction time(P<0.05),while the exogenous application of silenced HIF1?adenovirus attenuate the effect of the osteogenic differentiation(P<0.05).The Runx2promoted BMP9-induced early stage of osteogenic differentiation of MSCs and maintained advanced bone mineralization and calcium deposition,the qRT-PCR detection of osteogenic markers on the 3~rdd day,the ALP gene expression level was significantly up-regulated(P<0.05),on the 5~thh day,the OCN and COL-A1 expression increased significantly(P<0.05).On the 7~thh day,the mRNA expression levels of OCN,OPN,COL-A1 and OSX in the AdBMP9+Ad-HIF1?group increased significantly(P<0.05)compared with other groups.When the HIF1?and Runx2 genes were silenced by exogenous application,the mRNA expression of the above osteogenic related genes was significantly decreased(P<0.05).Compared with the Ad-BMP9+Ad-simRunx2 group,the Ad-BMP9+Ad-simHIF1?group,on the 7th day,the gene expression levels of osteogenic markers ALP,BSP,OCN,OPN,COL-A1 and OSX were significantly down-regulated(P<0.05).The western blot results showed that the protein expression level of advanced osteogenic indicators OPN and OCN.It was found that on day 7,compared with other groups,the protein expression levels of OPN and OCN were in the Ad-BMP9+Ad-HIF1?group were significantly up-regulated(P<0.05).(4)The HIF1?and Runx2 enhanced BMP9-induced angiogenesis in vitro.The HIF1?promoted angiogenesis after BMP9-induced MEFs.The mRNA expression levels of vascular related markers CD31,VEGF and SLIT3 were significantly increased(P<0.05).Runx2 can significantly increase the mRNA expression of angiogenic factor VEGF after BMP9-induced MEFs(P<0.05).The results of immunehistochemical staining showed that HIF1?and Runx2 could enhance the expression of VEGF in BMP9-induced MEFs,however,after transfected with Ad-simHIF1?and Ad-simRunx2,respectively,the mRNA expression level of angiogenesis related factors were significantly down-regulated.During this process,the Ad-BMP9+Ad-simHIF1?had a more significant inhibitory effect on angiogenesis than in the Ad-BMP9+Ad-simRunx2 group(P<0.05).(5)Exogenous overexpressed or silenced recombinant adenovirus HIF1?and Runx2 were transfected into BMP9-induced MEFs for 24 hours,and then,injected into the nude mice subcutaneously.The Micro CT scan results showed that the bone mineral density,trabecular number,bone volume,trabecularseparationandtrabecularthicknessinthe Ad-BMP9+Ad-HIF1?group were significantly increased compared with other groups(P<0.05).The Masson's Trichrome staining results showed that a large number of trabecular bone formation and trabecular bone network existed in the Ad-BMP9+Ad-HIF1?group.The H&E staining results showed that the number of the intercellular vascular lumen were increased in the Ad-BMP9+Ad-HIF1?group.The immunohistochemical staining results showed that the expression of the angiogenesis related factors VEGF and vWF were increased in the Ad-BMP9+Ad-HIF1?group.However,after HIF1?gene was silenced,it could significantly inhibit BMP9-induced osteogenic differentiation and angiogenic effects of MEFs,and the inhibitory effect was higher than that of silent Runx2 gene.Exogenous overexpression of Runx2 did not significantly enhance osteogenesis in vivo,but the results of Micro CT scan showed that in the Ad-BMP9+Ad-simHIF1?group,the bone mineral density,trabecular number,bone volume,trabecular separation and trabecular thickness were decreased(P<0.05).The Masson's Trichrome staining results showed that the trabecular bone was reduced.The H&E staining results showed that the number of the intercellular vascular lumen were decreased in the Ad-BMP9+Ad-simHIF1?group.The immunohistochemical staining results showed that the angiogenic related factors expression of VEGF and vWF were decreased.(6)HIF1?regulates the initiation and transcription of Runx2 in BMP9-induced MEFs,and the activity of both promoters is detected by dual luciferase gene reporter assay.The results of luciferase gene reporter assay showed that HIF1?can regulate the initiation and activation of Runx2 at the transcriptional level.On the results of the chromatin immunoprecipitation experiments,HIF1?could regulates Runx2 initiation transcription by binding to the-250-700 bp DNA fragment in front of the Runx2 exon in BMP9-induced MEFs.The results of western blot showed that HIF1?can regulate phosphorylation of Smad1/5/8,Erk1/2,p38 and JNK in BMP9-induced MEFs.The level of Runx2 changes to the level of phosphorylation and total protein of Erk1/2.Conclusion:(1)The mouse embryonic fibroblasts are in the possession of the characterization of MSCs,the MEFs have potential to differentiate into osteoblasts,chondroblasts and adipocytes,hence,the MEFs could be selected for seed cells in constructing bone tissue engineering.(2)HIF1?can up-regulate Runx2 to increase the BMP9-induced osteogenic differentiation and angiogenesis of mesenchymal stem cells through direct binding of proteins and chromatin,hence,this experiment could provide a new idea and a new method for clinical treatment of bone defects or bone nonunion.