The Mechanism Of RORA Regulating Endothelial Colony-Forming Cells Involved In Distraction Osteogenic Angiogenesis

Objective Distraction osteogenesis(DO)is an endogenous bone tissue engineering technique that induces the formation of new bone by gradually increasing mechanical forces in the gap between the fractures.The regeneration of blood vessels precedes the formation of new bone in time and space and is indispensable in bone repair.A large number of blood vessels and increased blood flow begin to appear in early stretch tissues,but the specific mechanism of rapid angiogenesis in DO is not fully understood.In this study,by analyzing the differential genes in the canine mandibular DO model,the key genes regulating angiogenesis in the process of DO were found,and the potential mechanism of this gene regulating the rapid vasogenesis of DO was studied,providing a theoretical basis for reducing the clinical treatment time.Methods 1.The high-throughput data of early stretch osteogenesis in the research group were re-analyzed to screen out the differentially related genes of angiogenesis;Canine mandibular DO animal model was constructed,and qRTPCR was used to detect DO14 and DO28 tissues.The expression levels of FGF10,TGFBR3,ARNT,XDH,ESM1,CAV2,ENPEP,MEOX2,THBS4,CYP1B1,SEMA3 C,SFRP2,DSP,HPSE and RORA were screened by high-throughput sequencing.Endothelial colony-forming cells from canine venous blood were obtained and identified by cell morphology,cell surface markers,endocytosis test,cell proliferation,migration and tubulogenesis test.Endothelial colonies were constructed to form cell physical hypoxia model,and 12 h normal culture group,24 h normal culture group,12 h hypoxia culture group and 24 h hypoxia culture group were set.Qrt-pcr was used to detect the expression of HIF1-α,VEGFA,FGF10,TGFBR3,ARNT,XDH,ESM1,CAV2,ENPEP,MEOX2,THBS4,CYP1B1,SEMA3 C,SFRP2,DSP,HPSE and RORA in four groups of ECFCs;The key genes that play a role in angiogenesis of Endothelial colony-forming cells(ECFCs)can be obtained by intersection of the two.2.Lentivirus transfection was used to silence and overexpress RORA in ECFCs,and the optimal infection complex number(MOI)of each virus was explored.The transfection efficiency of RORA was detected by QRT-PCR.The effects of silencing and overexpression of RORA on ECFCs angiogenesis were detected by cell proliferation,tubulogenesis and migration experiments.The mRNA relative expression levels of RORA,VEGFA and bFGF in ECFCs were detected by QRTPCR.The protein levels of RORA,VEGFA and bFGF in ECFCs were detected by WB.3.By KEGG signal pathway analysis,the upstream signal regulatory axis of RORA was identified as TGFβ 1-SMADS signal axis.RORA in ECFCs was transfected with lentivirus and treated with TGFβ 1-Sm ADS pathway blocker(E)SIS3.The NC-OE +DMSO group was tested.Smad3 phosphorylated protein levels and Smad3 and TβR-II mRNA and protein levels in ECFCs of NC-OE +(E)SIS3 group,RORA-OE + DMSO group and RORA-OE +(E)SIS3 group;The proliferation,migration and tubeforming abilities of ECFCs in each group were detected by cell proliferation,migration and tubeforming experiments.The mRNA and protein levels of RORA,VEGFA and bFGF were detected by QRTPCR and WB.4.Canine mandibular DO models were constructed and randomly divided into two groups: NC-OE group(ECFCS-Matrigel mixture transfected with empty virus vector by DO space injection)and RORA-OE group(ECFCS-Matrigel mixture transfected with RORA overexpressing lentivirus by DO space injection).In both groups,injection began on day 1 of DO,and samples were collected on day 28 of the fixed period.Then the general view was observed,and micro-CT examination was performed.Finally,immunohistochemical staining,Masson staining and HE staining were performed on the tissues in the DO space.Results 1.The top 15 differentially selected genes related to distraction osteogenesis and angiogenesis were FGF10,TGFBR3,ARNT,XDH,ESM1,CAV2,ENPEP,MEOX2,THBS4,CYP1B1,SEMA3 C,SFRP2,DSP,HPSE and RORA through reanalysis of high-throughput data.Qrt-pcr results showed that FGF10,TGFBR3,ESM1,CAV2,ENPEP,THBS4,CYP1B1,HPSE and RORA genes had statistical significance in DO14 and DO28 tissues among the top 15 genes.Under the inverted microscope,the original adherent cells gradually showed short spindle shape,and then colonies appeared gradually.Flow cytometry showed that CD105 and CD34 were positive,CD45 and CD133 were negative.The results of endocytosis test were also positive.Tube forming experiments show that ECFCs can form tubular structures.Qrt-pcr results showed that the expression levels of HIF-1α and VEGFA in 24 h hypoxia culture group were significantly increased compared with that in 12 h normal culture group,indicating that the hypoxia model was established successfully and the expression level of angiogenic factor was significantly increased under hypoxia environment.At the same time,among the top 15 genes screened,the genes that were significantly increased in 24 h hypoxia culture group compared with 12 h normal culture group were SFRP2,DSP and RORA.In conclusion,the gene with the highest association with the angiogenesis process of ECFCs in distraction osteogenesis is RORA.2.After lentivirus transfection,the expression level of RORA was significantly increased in ECFCs overexpressing RORA,but significantly decreased in ECFCs silencing RORA.The proliferation,migration and tubulogenesis of ECFCs and the expression of VEGFA and bFGF were increased by overexpression of RORA,but these results were reversed after RORA silencing(P<0.05).3.KEGG signaling pathway analysis showed that TGFβ1/Smads was upstream signal regulatory axis of RORA.Compared with ror A-OE +DMSO and NCOE+DMSO groups,TGFβ 1-Sm ADS pathway blockers down-regulated the phosphorylated protein levels of Smad3 and mRNA and protein levels of Smad3 and TβR-II in RORA-OE+(E)SIS3 groups and NC-OE+(E)SIS3 groups.After treatment with(E)SIS3,the in vitro angiogenesis of ECFCs in NC-OE+(E)SIS3group was significantly decreased,and the expression levels of RORA,VEGFA and bFGF were the lowest,which were lower than those in RORA-OE+(E)SIS3group(P<0.05).4.In general,it was found that the gap at both ends of the stretch gap in ror A-OE group was not clear,and the new callus in the stretch gap was basically bone connection,while the boundary at both ends of the stretch gap in NC-OE group was still vaguely visible,and there were still fibrous tissue bands in the gap.Compared with the NC-OE group,the RORA-OE group had stronger stretch gap blocking.Micro-ct results showed that the mean trabecular number(Tb.n),bone mineral density(BMD),mean trabecular thickness(TB.TH)and bone volume fraction(BV/TV)in DO space in RORA-OE group were significantly higher than those in NC-OE group(P<0.05).The mean trabecular space(TB.SP)was lower than that in NC-OE group(P<0.05).The results of HE staining,Masson staining and immunohistochemical staining showed that more bone trabeculae were formed and more mature in the RORA-OE group than in the NC-OE group.Conclusions 1.RORA is a key differential gene in the angiogenesis of DO tissue and ECFCs and regulates the biological function of ECFCs.2.In ECFCs,TGFβ-SMADS signaling axis can target RORA and up-regulate VEGFA and bFGF expression levels,promoting the angiogenesis ability of ECFCs.