Study On The Effect And Mechanism Of Hypoxic Preconditioning Bone Marrow Mesenchymal Stem Cell Derived Exosomes On Multi-Territory Perforator Flap Survival

Chapter 1:IntroductionFlaps serve as a reconstructive measure to repair extensive soft tissue defects caused by tumors or trauma.Among them,the multi-territory perforator flaps,with their unique tissue structures and clinical application advantages,have become one of the essential techniques in microsurgery.However,the survival and success of such flaps are influenced by a series of biological and clinical factors.Necrosis of the flap is a severe complication in perforator flap transplantation surgeries,potentially leading to transplant failure,infection,additional surgeries,and a decrease in the patient’s quality of life.The survival of the transplanted flap largely depends on the efficacy of angiogenesis,which,in turn,is influenced by various factors such as ischemia-reperfusion injury and patient age.During the process of flap necrosis,inflammatory factors,apoptosis,oxidative stress,and endothelial cells play crucial roles.However,current therapies,including melatonin,febuxostat,and other drugs,as well as cell-based treatment methods,have certain limitations and their clinical efficacy is not optimal.Autophagy-related signaling pathways have been proven to play a critical role in angiogenesis and ischemia-reperfusion,but the process of autophagy is highly complex,and the varying effects of autophagy in different organs are not clear.This study attempts to investigate the role of autophagy in perforator flap transplantation and seeks a new therapeutic method that can alleviate the impact of inflammatory factors,cell apoptosis,and oxidative stress in flaps,thereby further promoting flap angiogenesis and alleviating ischemia-reperfusion injury.Exosomes derived from bone marrow mesenchymal stem cells（BMSC-exos）have been shown to promote angiogenesis and alleviate ischemia-reperfusion injury.Considering that BMSCs adapt to the hypoxic environment in the body over the long term and can activate autophagy in response to hypoxia,this study hypothesizes that exosomes derived from hypoxia-preconditioned BMSCs（Hypo-BMSC-exos）possess stronger biological activity than BMSC-exos.The bioactive components carried by these exosomes may regulate autophagy,alleviate inflammation,apoptosis,and reactive oxygen species（ROS）production,thereby further promoting flap angiogenesis,reducing ischemia-reperfusion injury（IR）,and promoting the survival of multi-territory perforator flaps.Chapter 2:Preparation and biological activity identification of Hypo-BMSC-exosObjective:Hypoxic preconditioning of BMSC enhances the biological activity of exosomes.Methods:Rat BMSCs were cultured to the third generation and divided into the hypoxic preconditioning group and the regular culture group.The hypoxic preconditioning group was subjected to hypoxic preconditioning for 24 hours,while the regular culture group was cultured under normal oxygen conditions for 24 hours.Hypo-BMSC-exos and BMSC-exos were extracted from the supernatant of both groups.The morphology,diameter,protein concentration,and exosomal surface markers of the two exosomes were identified.Hypo-BMSC-exos and BMSC-exos were labeled with the fluorescent dye DIO and co-cultured with human umbilical vein endothelial cells（HUVECs）,and the process of exosome phagocytosis by endothelial cells was observed under a fluorescence microscope.Additionally,the two types of DIO-labeled exosomes were co-cultured with HUVECs and rat bone marrow endothelial progenitor cells,with cell nuclei stained with the fluorescent dye DAPI.Subsequently,flow cytometry was used to detect the phagocytosis rate of the two types of exosomes.The two types of exosomes were co-cultured with HUVECs,and the impact and optimal intervention dose of Hypo-BMSC-exos on cell viability,toxicity,proliferation,and apoptosis were evaluated through cell viability and cytotoxicity tests（Calcein/PI）,CCK-8 proliferation experiments,EdU proliferation experiments,and flow cytometry cell count.The migration and angiogenic ability were then evaluated by scratch assays,Transwell assays,and tube formation assays.Results:Successful extraction of Hypo-BMSC-exos and BMSC-exos was confirmed,with typical exosomal morphology observed under a transmission electron microscope.Nanoparticle tracking analysis（NTA）indicated no significant difference in the particle diameter of the two types of exosomes.BCA protein quantification results suggested that hypoxic preconditioning promoted the secretion of BMSC exosomes.Western Blot（WB）results showed that Hypo-BMSC-exos and BMSC-exos both expressed exosome surface molecular markers,but Hypo-BMSC-exos had higher expression of certain markers.Fluorescence microscope results indicated that Hypo-BMSC-exos were more readily phagocytosed by HUVECs compared to BMSC-exos.Flow cytometry results showed that Hypo-BMSC-exos were more readily phagocytosed by HUVECs and rat bone marrow endothelial progenitor cells.CCK-8 proliferation experiments indicated that Hypo-BMSC-exos enhanced endothelial cell proliferation more effectively than BMSC-exos,and different concentrations of Hypo-BMSC-exos and BMSC-exos showed varied effects on endothelial cell proliferation,with 50μg/ml being the optimal intervention concentration for Hypo-BMSC-exos.Cell viability and cytotoxicity test results suggested that even with an eight-fold increase in the optimal intervention concentration of Hypo-BMSC-exos co-cultured with endothelial cells,almost no apoptotic cells were observed.EdU proliferation experiment and flow cytometry cell count results indicated that Hypo-BMSC-exos promoted cell proliferation.Scratch,migration,and tube formation assay results indicated that Hypo-BMSC-exos promoted HUVEC migration and angiogenesis in vitro,showing superior effectiveness compared to BMSC-exos.Conclusion:Hypoxic preconditioning promotes the secretion of BMSC exosomes,and Hypo-BMSC-exos are more readily absorbed by endothelial cells than BMSC-exos.Hypo-BMSC-exos exhibit stronger biological activity,promoting angiogenesis in vitro.Chapter 3:The effect and mechanism of Hypo-BMSC-exos on the survival of multi-territory perforator flapsObjective:The effect and mechanism of Hypo-BMSC-exos on the survival of multi-territory perforator flaps.Methods:18 C57BL/6 mice were purchased and a model of multi-territory perforator flaps on the backs of mice was established.The mice were randomly divided into 3 groups:the Hypo-BMSC-exos group,BMSC-exos group,and PBS group,each with 6 mice.For each group,3mice were injected with DIR-labeled Hypo-BMSC-exos,BMSC-exos,and PBS respectively via tail vein.In vivo imaging was performed daily for 4^thdays post-surgery to evaluate the distribution and metabolic rate of exosomes in flaps across all groups.On the fourth day,organs such as the heart,liver,spleen,kidneys,lungs,and back flap of the mice were dissected and imaged to assess the fluorescence intensity of exosomes in different organs.The remaining 3 mice in each group were injected with Hypo-BMSC-exos,BMSC-exos,and PBS respectively,and the survival area and the number of vessels in the Choke Vessel region of the flap were evaluated on the 7^th day post-surgery.Subsequently,flap tissue samples from each group were collected,and tissue necrosis was evaluated using HE staining.The expression levels of 4 inflammatory factors（TNF-α,IL-1,MCP-1,and IL-6）in flap tissues were detected by ELISA.The number of CD31-positive vessels in the flap tissue was counted through IHC staining,the total amount of ROS in the flap tissue was evaluated by immunofluorescence staining,and the number of TUNEL-positive cells in the flap tissue was counted.Results:In the animal experiments,both Hypo-BMSC-exos and BMSC-exos enhanced the survival of the transverse island skin flap model in mice,compared to the control group.The flap survival area was 92.7%in the Hypo-BMSC-exos group,82.3%in the BMSC-exos group,and 71%in the control group,showing statistical significance（n=3,*P<0.05,***P<0.001）.An increase in the number of blood vessels was observed in the Choke Vessel region in the Hypo-BMSC-exos group（n=3,***P<0.001,****P<0.0001）.The in vivo imaging results indicated that Hypo-BMSC-exos demonstrated a higher biological activity than BMSC-exos in mice and concentrated in the distal ischemic hypoxic tissue of the flap.Histological evaluation of the flap tissue showed that,compared with the BMSC-exos group and the control group,the Hypo-BMSC-exos group had more CD31-positive vessels,simultaneously reducing the expression of inflammatory factors in the flap,decreasing the total amount of ROS,and lessening cell apoptosis（n=3,*P<0.05,**P<0.01,***P<0.001,****P<0.0001）.Conclusion:Hypo-BMSC-exos can promote the survival of multi-territory perforator flaps on the backs of mice.The mechanism is associated with promoting angiogenesis,reducing the release of inflammatory factors,alleviating oxidative stress,and exhibiting anti-apoptotic effects.Chapter 4:Hypo-BMSC-exos Alleviates Ischemia-Reperfusion Injury in Rat Multi-Territory Perforator Flaps through miR-421-3p Targeting mTOR to Activate AutophagyObjective:The effects and mechanisms of Hypo-BMSC-exos on the survival of ischemia-reperfusion multi-territory perforator flaps in rats.Methods:6 Sprague-Dawley rats were randomly divided into Hypo-BMSC-exos group and BMSC-exos group,and a model of ischemia-reperfusion multi-territory perforator flaps was established.DIR-labelled Hypo-BMSC-exos and BMSC-exos were injected locally immediately after surgery,and the distribution and metabolism of both exosomes in the flap were observed 24h later using in vivo imaging.Additionally,18 Sprague-Dawley rats were randomly divided into Sham group,Hypo-BMSC-exos group,BMSC-exos group,Ischemia-reperfusion group,Hypo-BMSC-exos+3MA group,and Hypo-BMSC-exos+PBS group,with 3 rats in each group.After establishing the model of abdominal multi-territory perforator flap ischemia-reperfusion in rats,the vascular pedicle was sutured without interruption in the sham surgery group,while in the remaining groups,the vascular pedicle was sutured after6 hours of interruption.The survival of the flap was assessed daily after surgery,and on the 7th postoperative day,the blood flow perfusion and skin temperature of the flaps in each group were evaluated using indocyanine green and infrared detectors.At the same time,flap tissue samples from each group were collected,and HE staining was used to evaluate the degree of necrosis and inflammatory infiltration in the flaps of each group.The expression levels of inflammatory factors TNF-α,IL-1,MCP-1,and IL-6in the flaps of each group were evaluated by qPCR.The number of CD31-positive vessels in the flaps of each group was assessed by IHC,and the total amount of ROS in the flaps of each group was evaluated by immunofluorescence.The number of TUNEL-positive cells in the flaps of each group was evaluated by TUNEL assay.The expression of proteins such as FUNDC1,LC3,VEGFA,BAX,BCL-2,and GAPDH in the flaps of each group was evaluated by WB.Co-expression of FUNDC1/LC3 in the flaps of each group was assessed by immunofluorescence.The expression of mTOR and FUNDC1 in the flaps of each group was assessed by immunohistochemistry.Endothelial cells were divided into Hypo-BMSC-exos+OGD/ROG group,BMSC-exos+OGD/ROG group,PBS+OGD/ROG group,PBS control group,and Hypo-BMSC-exos+3-MA+OGD/ROG group,and the migration and angiogenic abilities of endothelial cells were evaluated by Transwell and tube formation assays.Differential sequencing was carried out on Hypo-BMSC-exos and BMSC-exos.The data obtained were quality-controlled and bioinformatically analyzed to identify enriched pathways and potential target sites for differential miRNA.Endothelial cells were co-cultured with m Cherry-GFP-LC3 adenovirus and transfected.They were then divided into Hypo-BMSC-exos+OGD/ROG group,BMSC-exos+OGD/ROG group,PBS+OGD/ROG group,and PBS control group.Autophagy activation in each group was assessed and autophagic flux was calculated using fluorescence microscopy.The expression of several miRNAs,including miR-421-3p,in Hypo-BMSC-exos and BMSC-exos was validated by qPCR.Endothelial cells were then divided into Hypo-BMSC-exos+OGD/ROG group,BMSC-exos+OGD/ROG group,and PBS+OGD/ROG group,and the expression of miR-421-3p in the cells was detected by qPCR.Further,endothelial cells were transfected with miR-421-3p Inhibitor,Inhibitor NC,and transfection reagents,and the expression of miR-421-3p in endothelial cells was detected by qPCR.Finally,endothelial cells were divided into Hypo-BMSC-exos+NC+OGD/ROGgroup,Hypo-BMSC-exos+miR-421-3p Inhibitor+OGR/ROG group,NC+OGD/ROG group,and miR-421-3p Inhibitor+ORG/ROG group.The expression of mTOR,ULK1,FUNDC1and downstream related proteins were validated by WB.Results:In vivo imaging results showed that Hypo-BMSC-exos demonstrated better biological activity in the rat multi-territory perforator flap ischemia-reperfusion model than BMSC-exos.The survival area of the flap,indocyanine green angiography,and thermal imaging results showed that Hypo-BMSC-exos promoted the survival of rat ischemia-reperfusion multi-territory perforator flaps compared with BMSC-exos and PBS（（n=3,*P<0.05,**P<0.01,***P<0.001,****P<0.0001））.Histological results of rat ischemia-reperfusion perforator flaps showed that Hypo-BMSC-exos reduced the release of inflammatory factors,cell apoptosis,and ROS production compared with BMSC-exos and PBS.Transwell and tube formation experiment results showed that Hypo-BMSC-exos alleviated endothelial cell OGD/ROG damage compared with BMSC-exos and PBS.High-throughput sequencing results showed that compared with BMSC-exos,56 miRNAs were upregulated and 44 miRNAs were downregulated in Hypo-BMSC-exos.qPCR results confirmed that miR-421-3p and other miRNAs were highly expressed in Hypo-BMSC-exos.Bioinformatics analysis results showed that the mTOR and autophagy-related mechanisms were highly enriched.m Cherry-GFP-LC3 transfection of endothelial cells experiment results confirmed that Hypo-BMSC-exos activated autophagy and increased autophagic flux in endothelial cell OGD/ROG model compared with BMSC-exos and PBS.WB,immunofluorescence colocalization results showed that compared with BMSC-exos and ischemia-reperfusion group,Hypo-BMSC-exos group increased the expression of autophagy-related proteins FUNDC1 and LC3in the flap,thereby activating autophagy.After autophagy was inhibited by3-MA,the biological effects of Hypo-BMSC-exos in vitro and in vivo disappeared.Using public databases and bioinformatics analysis techniques to further screen micro RNA and potential target genes,bioinformatics prediction results showed that Hypo-BMSC-exos activated autophagy by miR-421-3p targeting mTOR.A large number of experimental results such as immunohistochemistry,WB,qPCR,miR-421-3p Inhibitor,and cell transfection confirmed that Hypo-BMSC-exos carried a large amount of miRNA-421-3p to target and regulate mTOR,thereby activating autophagy.Conclusion:Hypo-BMSC-exos promoted the survival of rat multi-territory perforator flaps under ischemia-reperfusion.The mechanism is related to the miR-421-3p carried by Hypo-BMSC-exos targeting and regulating mTOR to activate autophagy and protect vascular endothelial cells.The study includes 51 figures,4 tables,and 155 references.