Studies Of MiR-150Effects On Endothelial Progenitor Cell Transplantation For Venous Thrombus Recanalization And Resolution

Deep venous thrombosis (DVT) is a common peripheral vascular disease. DVTmay lead to the abnormal swelling and ulceration of lower limb, post-thromboticsyndrome (PTS) or even pulmonary embolism (PE) which can lead to the sudden deathof patients. Currently, the clinical treatment for DVT is using of anticoagulants to reducethe incidence of PE, PTS and recurrence of DVT. However,the usage of anticoagulantsis associated with the high risk of bleeding and wound complications.The discovery of marrow-derived circulating endothelial progenitor cells (EPCs)cause people’s great interest because of their plasticity to differentiate into endothelialcells (ECs) and as a source of paracrine proangiogenic factors. It has been proven thatEPCs are recruited into resolving venous thrombi. Our previous studies also showed thatEPCs significantly improved the microenvironment and promoted the resolution of acutevenous thrombus and the recanalization of chronic thrombus. However, studiesdemonstrated that EPCs recruitment to sites of neovascularition was limited due to theweak viability, migration and homing ability. Therefore, looking for a method toimprove the function of EPCs is being widely discussed.MicroRNAs are a class of~22nucleotides non-coding RNAs that suppress geneexpression at the posttranscriptional level by promoting mRNA degradation or inhibitingmRNA translation. Recent studies have shown that microRNAs are involved in theangiogenesis process. MiR-150, a key regulator for the development of immune cells,was originally detected in mature lymphocyte. It has been proven that miR-150regulatesthe migration of bone marrow stem cells. MiR-150, secreted by monocytes, enhanced the migration of human microvascular endothelial cells. However, the function ofmiR-150in rat EPCs, especially in the ischemia condition such as DVT, remainsunknown.In this study, rat bone marrow was obtained by rinsing bone marrow cavity, thenbone marrow mononuclearcells were isolated by gradient centrifugation using Ficollcentrifugate. mononuclearcells were then cultivated with EGM-2MV mediumcultivation. To investigate the role of miR-150in rat endothelial progenitor cells (EPCs)proliferation and migration, EPCs were transfected with control oligoes and miR-150mimics or inhibitor by electroporation. To further explore the role of miR-150in EPCsunder ischemia condition, EPCs stably expressing miR-150were achieved by lentivirusand delivered into rats with DVT. We demonstrated that ectopic expression of miR-150enhanced rat EPCs motility and tube formation in vitro. MiR-150promoted rat EPCshoming and venous thrombus recanalization and resolution in vivo. Our study revealedthe function of miR-150in EPCs and presents a promising clinical therapy for ischemiadiseases.ChapterⅠ Isolation, culture and identification of endothelialprogenitor cellsObjectives：The goal of this study was to isolate, culture and identify endothelialprogenitor cells from rat bone marrow.Methods：Mononuclear cells (MNCs) from rat bone marrow isolated by densitygradient centrifugation were cultured in EGM-2medium. Morphological changes wereobserved with an inverted microscope. The EPCs were identified by the combinedDil-labeled acetylated low density lipoprotein(Dil-ac-LDL), in vitro tube formationassay, and the cell surface antigen was detected by immunofluorescence technology andflow cytometry.Results：Mononuclear cells isolated from rat bone marrow cultured in EGM-2Medium is round. Cells begin to adherel after48h.The adherent cells are fusiform shape,and cell colony appears. After10~12days culture, cell morphology presentscobblestone appearance. The cells uptake Dil-ac-LDL. Matrigel tubule test show thatcultured cells can form a tubular structure. The resultes of flow cytometry analysis andimmunofluorescence show that the main expression on the surface of cells is VEGFR、vWF; almost no expression of CD133.Conclusions： MNCs isolated from rat bone marrow cultured in EGM-2mediumcan proliferate and differentiate to late EPCs after10~12days culture. Chapter II Studies of miRNA-150effects on endothelialprogenitor cells in vitroObjectives: To investigate the role of miR-150in rat endothelial progenitor cells(EPCs) proliferation,migration and tube formation.Methods: EPCs were transfected with control oligoes and miR-150mimics orinhibitor by electroporation. MTT and flow cytometry analysis was performed toevaluate the growth of EPCs subjecting to miR-150. Cell migration analysis was doneby wound healing and transwell assay. To test whether miR-150affected the angiogenicactivity of EPCs in vitro, we performed matrigel tube formation assay. Bygain-of-function examination, we searched for its putative target genes using onlinesearch tool (TargetScan).Results: The EPCs growth and cell cycle was immue from miR-150application.Both the wound healing and transwell assay showed that miR-150promoted EPCsmigration and miR-150inhibitor inhibited EPCs migration in vitro. Ectopic expressionof miR-150enhanced EPCs tube formation in vitro. C-Myb was predicted to have twoputative miR-150binding sites within its3’UTR.Conclusions: MiR-150had no effect on the growth and cell cycle of EPCs.MiR-150enhanced the migration, tube formation ability of rat EPCs. C-Myb may be amiR-150target gene. Chapter III Construction of the miR-150Lentiviral ExpressionVector and its Expression in endothelial progenitor cellsObjectives: To construct the miR-150lentiviral vector, and to detect itseffectiveness in EPCs．It is the bases of functional study of miR-150effects on ratendothelial progenitor cells (EPCs) in vivo.Methods: MiR-150was amplified from the genomic DNA and inserted intopLVX-IRES-ZsGreenl vector after double digestion to generate pLVX-IRES-ZsGreenl-miR-150．The vector was then confirmed by PCR and DNA sequencing. EPCs wasinfected by the concentrated lentivirus293T produced. MiR-150were assessed byqPCR．Results: The recombinant lentiviral vector was Successfully established andconfirmed by PCR and DNA sequencing. MiR-150was integrated into the genome ofEPCs and miR-150expression was enhanced effectivcly by qPCR．Conclusions: The miR-150lentiviral expression vector was successfullyconstructed, and the miR-150overexpressed in EPCs． Chapter IV Studies of miRNA-150effects on endothelialprogenitor cells in deep venous thrombosisObjectives: To investigate the functional role of miR-150in rat endothelialprogenitor cells (EPCs) and its potential application in deep venous thrombosis.Methods: We ligated the inferior vena cava(IVC) of rats and developed the DVTmodels. EPCs with stable expression of miR-150or empty vector viapLVX-IRES-ZsGreen1lentivirus were transplanted into these DVT models. On the dayof7and14after the transplantation, the rats were sacrificed and the thrombi wereweighed. The homing of EPC was shown by GFP expression and observed using fluorescence microscope. To observe the thrombus organization and recanalization, thesections were performed with HE staining and immunohistochemicalstaining for CD34.To investigate the molecular mechanisms underlying the increased thrombus resolvingability of EPCs overexpressing miR-150, the intrathrombotic expression of MMP-2wasdetected using immunohistochemical staining.Results: Compared with control group, transplanted EPCs were situated around thethrombus in both EPCs/vector and EPCs/miR-150group. The larger number of GFPpositive cells appeared in EPCs/miR-150group at day7and14compared withEPCs/vector group. Compared with control group, significant decrease of thrombusweight was observed in EPCs/vector and EPCs/miR-150groups. The thrombus weightof EPCs/miR-150group was even lower than that of EPCs/vector group. HE stainingand CD34immunohistochemical analysis showed that thrombus organization andrecanalization were better in EPCs/vector and EPCs/miR-150group than control group.Larger and more channels were seen in EPCs/miR-150group in comparison withEPCs/vector group. Compared at the indicated time points, there were moreMMP-2-positive cells in EPCs/vector and EPCs/miR-150group in contrast with controlgroup. Furthermore, there were more MMP-2-positive cells in EPCs/miR-150groupcompared with EPCs/vector group.Conclusions: The exogenous EPCs are recruited into resolving venous thrombi.EPCs transplantation significantly inhibited thrombus formation and improved thrombusorganization and recanalization. MiR-150enhanced the homing, thrombusrecanalization and resolution ability of rat EPCs. Restoring miR-150in EPCs presents apromising clinical therapy for DVT therapy.