Circulating Exosomes-mediated THBS1 Protein Regulates Angiogenesis Of Canine Mandibular Distraction Osteogenesis Through PI3K/Akt/ERK Signaling Pathway

Objective1.We analyze the differential protein expression profiles of circulating exosomes in canine mandibular distraction osteogenesis(DO)and bone fracture(BF)models,to identify the key protein that regulate angiogenesis in the process of distraction osteogenesis.2.To explore the possible mechanism of the key protein regulating the rapid angiogenesis and osteogenesis of distraction osteogenesis.3.To further clarify the biological mechanism of distraction osteogenesis and shorten the treatment period of distraction osteogenesis.Methods1.Experimental study on the angiogenesis characteristics of distraction osteogenesis.Twenty-four healthy dogs were divided into two groups,and animal models of canine mandibular distraction osteogenesis(DO)and bone fracture(BF)were constructed respectively.After the operation,the animal specimens were taken in 14,21,28,and 42 days.We detected the new bone formation by Micro-CT.HE staining was used to observe the blood vessels and new bone formation in the new tissues.Immunohistochemistry is used to detect the protein expression of endothelial cell marker CD31 and osteoblast marker OCN,and the microvessel density was detected.The m RNA expression of angiogenic factor VEGF was detected by qRT-PCR.2.Proteomic and angiogenic functional properties of circulating exosomes in DO.Both DO group and BF group animals were harvested peripheral blood at preoperation and post-operative 7,8,14,and 21 days.Peripheral blood exosomes were extracted and identified.Exosomes were used for DIA proteomics detection and bioinformatics analysis.The angiogenesis-related proteins were selected for Western blot verification.The effect of DO serum exosomes on the angiogenesis function of HUVEC was detected by CCK8,transwell,tube formation experiment,qRT-PCR,and western blot.3.In vitro study on the effect of THBS1 mediated by ECFCs-Exos on angiogenesis.We collected peripheral blood from adult dogs,to isolated and cultured endothelial colony forming cells(ECFCs).The characteristics of ECFCs were identified.Silencing or overexpression of THBS1 in ECFCs by lentiviral transfection.Western blot and qRT-PCR were used to detect the transfection efficiency of ECFCs,and the effect of THBS1 on the biological properties of ECFCs were detected.Exosomes secreted by lentivirus transfected ECFCs were extracted and identified.Co-culture the ECFCs-Exos that silence or overexpress THBS1 with HUVEC.After that,CCK8,transwell,tube formation,qRT-PCR,and Western blot were used to detect the effects on endothelial cell proliferation,migration and angiogenesis.Meanwhile,explore the mechanism of regulating angiogenesis through PI3K/Akt/ERK signaling pathway.4.In vivo study on the effect of THBS1 mediated by ECFCs-Exos on DO angiogenesis.Canine mandibular DO model was established,and in vivo tracer experiment was performed by intravenous injection of ECFCS-Exos labeled with p KH26 fluorescence.27 healthy dogs were taken to construct the animal models of mandibular distraction osteogenesis,and THBS1-silent ECFCs exosomes were injected intravenously(DO + sh THBS1-Exos group)during distraction period,and normal saline injection group(DO + NS group)and negative virus exosomes injection group(DO + Con1-Exos group)animal models were also constructed.After the operation,the animal specimens were taken in 14,21,and 42 days.We observed the tissue healing of the mandibular distraction gap in general,and detected the new bone formation by Micro-CT.QRT-PCR was used to detected the expression of angiogenesis-related factors VEGF 、 b FGF 、 VE-cadherin m RNA in tissues.HE staining and Masson staining were used to observe the blood vessels and new bone formation in new tissues.Immunohistochemistry was used to detecte the expression of CD31,VEGF,OCN protein,and the microvessel density was detected.Results1.The characteristics of angiogenesis and new bone formation during distraction osteogenesis.The results of HE staining and immunohistochemistry both showed that the angiogenesis of the DO group was more abundant than that of the BF group.At 21 days postoperatively,the microvessel density was the highest(P<0.05).QRT-PCR results showed that the highest m RNA expression of VEGF in the distraction gap 21 days after surgery,which was significantly higher than that in the BF group(P<0.05).New bone formation was discovered in the early consolidation period(7 days).Forty-two days of post-operation,the quantity and quality of trabecular bone formation in DO group were better than those in BF group.Micro-CT showed that 28 days and 42 days after surgery,more bone callus was formed in the distraction area than in the BF group.The bone parameters of DO group such as BMD,BV/TV,Tb.Th,Tb.N were higher than BF group,while Tb.Sp was less than BF group(P<0.05).2.Proteomic and angiogenic functional properties of circulating exosomes in DO.The serum exosomes were successfully extracted,and their morphology was cup holder-like,with a particle size of about 50-150 nm,and expressed exosome markers such as CD63,CD81,and TSG101.DIA proteomics detected 681 proteins,and,there were 43 differential proteins that we believed to be involved in DO angiogenesis.After bioinformatics analysis,the angiogenesis-related proteins THBS1,FN1,and possible regulatory signaling pathways PI3K-Akt signaling pathway were screened out.Western blot verified the expression changes of THBS1,FN1,Tln1,and the results were basically consistent with the results of DIA protein quantitative experiments.In vitro experiments confirmed that DO serum exosomes can significantly promote the endothelial cell proliferation,migration,tube formation and the expression of angiogenesis-related factors VEGF,b FGF,and CD31(P<0.05).3.ECFCS-Exos mediated THBS1 inhibits the angiogenesis of endothelial cells.ECFCs were successfully isolated,cultured and identified.After ECFCs were transfected with THBS1-silencing lentivirus,the ability of proliferation,migration,tube formation and the expression ability of VEGF and b FGF were increased,and the results were reversed after transfection with THBS1-overexpression lentivirus.We successfully extracted exosomes secreted by ECFCs that silenced or overexpressed THBS1.After HUVEC treatmented by those exosomes,we found that the exosomes extracted from THBS1-silencing ECFCs could promote the HUVEC ability of proliferation,migration,tube formation and expression of VEGF,b FGF,meanwhile Promotes the protein phosphorylation of PI3 K,AKT1 and ERK.The experimental results of exosomes overexpressing THBS1 were opposite.4.ECFCs-Exo promote the angiogenesis and new bone formation of distraction osteogenesis by down-regulating THBS1.The result of laser scanning confocal microscope observation showed that the red fluorescently labeled ECFCs-Exo could be gathered in the distraction zone.The results of HE staining and Masson staining showed that in the early consolidation period(14days and 21 days of post-operation),the DO + sh THBS1-Exos group had more angiogenesis in the distraction space,and earlier osteoid deposition than other two groups.Forty-two days of post-operation,the DO + sh THBS1-Exos group produced more trabecular bones in the distraction space,and the new bone was more mature than those in other two groups.The results of immunohistochemistry showed that the expression of CD31,VEGF,OCN proteins,and the microvessel density in the DO + sh THBS1-Exos group were higher in the early consolidation period than control groups(P<0.05).QRTPCR results showed that the m RNA expressions of VEGF,b FGF and VEcadherin in the distraction zone of the DO + sh THBS1-Exos group were higher than those of the control groups.Micro-CT showed that the DO + sh THBS1-Exos group formed more bone callus than control group,and the BMD,BV/TV,Tb.Th,Tb.N of new bone were higher than that of the control groups(P<0.05).Conclusions1.Distraction osteogenesis is a process accompanied by abundant angiogenesis,and the peak of angiogenesis in distraction osteogenesis occurs 7 days after DO consolidation.2.DO serum exosome proteins may play a key role in the regulation of distraction osteogenic angiogenesis and promote the proliferation,migration and angiogenesis of endothelial cells.3.ECFCs-Exos mediated THBS1 to regulate endothelial cell function through activation of PI3K/ Akt /ERK signaling pathway.4.Angiogenesis and osteogenesis were accelerated by downregulating the expressions of THBS1 on ECFCs-Exos during DO in vivo.