Experimental Studies On Effect Of Resveratrol On Migration And Angiogenesis Of Endothelial Progenitor Cells And Regulation Mechanisms Via MiR-138

Deep venous thrombosis(DVT)is a blood clot formation caused by vascular endothelial injury,hemodynamic changes and abnormal hypercoagulability of blood.It is one of the most common clinical diseases.The acute phase of deep venous thrombosis is within two weeks,and the subacute and chronic phases are more than two weeks.Usually acute deep venous thrombosis may progress to femoral bruises,and the following limb thrombosis may lead to pulmonary embolism(PE).Thrombosis may develop into postthrombotic syndrome(PTS),which is characterized by lower extremity swelling,pigmentation,and lower extremity skin ulcers and seriously affects the quality of life of patients.Endothelial progenitor cells(EPCs)are precursors of endothelial cells that are involved in re-endothelialization and neovascularization due to their potential to differentiate into mature endothelial cells.This process plays an important role.Previous experimental studies have found that bone marrow-derived endothelial progenitor cells(BMEPCs)can differentiate into vascular endothelial cells.Transplantation of EPCs can improve the microenvironment in venous thrombosis and promote the thrombosis resolution.At the same time,due to the weaker proliferative capacity and migration homing ability of EPCs transplanted into the body,its therapeutic effect on deep vein thrombosis is limited.Therefore,regulating the biological functions of EPCs and promoting their function in vivo is a hot topic.Resveratrol is originally derived from polyphenolic compounds in plants and can be extracted from plants such as grapes,berries,and peanuts.Previous studies have demonstrated that resveratrol has antibacterial,antifungal,antitumor and anti-inflammatory effects.In France,the incidence of cardiovascular disease is significantly lower than that of other European countries due to daily wine intake.This phenomenon is called "French Paradox".Subsequent studies have further confirmed that resveratrol also has a variety of effects such as protecting the cardiovascular system,regulating blood lipids,and inhibiting platelet aggregation.In addition,many scholars have found that resveratrol also play a significant role in anti-tumor activity.However,resveratrol has no influence on the biological function of EPCs.The influence of resveratrol on migration and angiogenic ability of EPCs has not been reported.MicroRNAs(miRNAs)are a hotspot in genetics research in recent years.They are a class ofRNA sequences of about 22 nucleotides in length.Previous studies have demonstrated that miRNAs can be involved in the regulation of various biological processes such as cell proliferation,differentiation,apoptosis,and development.It was also found to participate in the regulation of EPCs.In this experiment,we will analyze the effects of resveratrol on the biological functions of miRNA-138 as well as EPCs angiogenesis and the recanalization of thrombus.This study will further investigate the mechanism of regulation on EPCs and provide an important theoretical basis for cell therapy research for deep vein thrombosis and other ischemic diseases.Part ?: Isolation,culture and identification of human peripheral blood derivedendothelial progenitor cellsObjective: Collect peripheral blood samples from healthy adults,and purify human peripheral blood-derived endothelial progenitor cells(PBEPCs)by density gradient centrifugation combined with differential adherence speed method.Methods: Peripheral blood mononuclear cells(PBMCs)from adult peripheral blood were isolated by density gradient centrifugation.The obtained cells were induced by differential adherence in EGM-2MV medium containing various growth factors.Proliferation assay was used to detect the proliferation characteristics of EPCs at different time points.Cell surface markers were detected and identified.Dil-Ac-LDL and FITC-UEA-1 were used to identify cell characterizations.Results: After 24 hours culture of PBMCs,a small number of adherent cells were observed.The cells were oval and rod-shaped with different sizes.After one week of culture,the cells formed colonies and the cell morphology showed long fusiform and spindle-shaped as well as irregular and cobblestone shape.After 14-21 days of culture,the number of cells continued to increase.The cell colonies disappeared,and the cell morphology showed a typical cobblestone-like appearance.Double fluorescent staining of Dil-Ac-LDL and FITC-UEA-1 showed that the cells with double positive.Simultaneous detection of CD34,CD31,and VEGFR2 flow cytometry indicated that the cultured cells were in accordance with the characteristics of surface markers of EPCs.Conclusion: Endothelial progenitor cells derived from human peripheral blood by density gradient centrifugation combined with differential adherence method.The obtained cells conform to the morphological characteristics of EPCs.Besides,Dil-Ac-LDL and FITCUEA-1 double fluorescent staining and the cytological test further confirmed that the obtained cells were of high purity and could be used for subsequent experiments.Part ? The effect of resveratrol on the biological function of endothelial progenitor cells and its mechanismsObjective: To investigate the effects of resveratrol on the biological function of human peripheral blood derived endothelial progenitor cells.Methods: 1.EPCs were treated with different concentrations of resveratrol.Cell morphology was observed under light microscope and surface antigen markers were detected by flow cytometry.Cell proliferation assay was also performed to detect the proliferative ability of EPCs.2.Resveratrol at a specific concentration was selected,and the effects of resveratrol on the migration function of EPCs were evaluated by scratch test and invasion test.3.The effects of resveratrol on angiogenesis of EPCs were evaluated by tube-forming test.4.PCR was performed to detect the expression of miR-138 in EPCs after resveratrol treatment.The potential mechanism of resveratrol in regulating the biological function of EPCs was further discussed.The experiment was divided into four groups: A: NC mimics,B.miR-138 mimics,C.NC inhibitor,and D.miR-138 inhibitor.5.Bioinformatics was used to predict potential target genes of miR-138,which was confirmed by experiments such as luciferase reporter gene assay,q RT-PCR and western blots.6.Alter the expression level of target genes in EPCs in order to observe the effects on the biological function of EPCs.Results: 1.Resveratrol promoted the proliferation of EPCs with the increase of concentration in a range.The optimal concentration of resveratrol was 50 ?M.2.Compared with the normal group,the resveratrol group could promote the migration ability of EPCs.The scratch test showed that the migration ability of EPCs in the resveratrol group was higher than that in the control group at 24 and 48 hours.3.Compared with the normal group,the resveratrol can promote the angiogenic ability of EPCs.The angiogenesis experiment showed that the number of EPCs and the length of blood vessels in the resveratrol group were higher than those in the control group.Resveratrol can reduce the expression level of miR-138 in EPCs within 6 hours.5.Resveratrol-pretreated EPCs can change the expression of miR-138 and regulate the biological function of EPCs.Up-regulation of the expression of miR-138 inhibited the angiogenesis and migration of EPCs while down-regulation of the expression of miR-138 improved vascularization and migration capabilities of EPCs.Bioinformatics predicts that focal adhesion kinase(FAK)may be the target gene,and resveratrol regulates the biological functions of EPCs through the miR-138/FAK pathway.Conclusions: 1.Resveratrol can promote the proliferation of EPCs and resveratrol at 50?M can promote the proliferation of EPCs.2.Resveratrol promotes the angiogenesis and migration ability of EPCs.Resveratrol can change the expression level of miR-138 in EPCs and alter the biological function of EPCs by regulating miR-138.4.FAK is a target gene of miR-138,and resveratrol regulates the biological function of endothelial progenitor cells through the miR-138/FAK pathway.Part ? Effect of resveratrol on endothelial progenitor cells for recanalization of venous thrombolysisObjective: To study the effect of resveratrol on the recanalization of venous thrombolysis after regulation of EPCs.Methods: 1.To establish a model of deep vein thrombosis in nude rats.The established models were divided into three groups: group A(10),blank control group,1 ml PBS was injected into the tail vein;group B(10),EPCs group,1 ml of PBS cell suspension containing 1×106 EPCs was injected into the tail vein;group C(10),EPCs/RSV group,1 ml of PBS cell suspension containing 1×106 resveratrol-pretreated EPCs was injected through the tail vein.Thrombus specimens were collected 7 days after surgery,and venous thrombolysis and recanalization were observed by thrombus weighing,HE staining,and digital subtraction angiography.Results: 1.The specimens of thrombus in each experimental group were collected and detected by thrombus weight.The results showed that the weight of thrombus was the lightest in group C,and the weight of thrombus in group B and group C was significantly reduced compared with the blank control group.2.HE staining was compared in each experimental group to show the thrombus recanalization.The results showed that the thrombus in C group had the most new-born blood vessel,and the resolution and recanalization were the best.3.Digital subtraction angiography was investigated to compare the blood flow of inferior vena cava in each experimental group.The results showed that compared with the blank control group(group A),the flow recovery in group B and group C seem better.Conclusion: Resveratrol can regulate the homing ability of EPCs,which participate in the process of thrombus dissolution and recanalization.Finally,the resveratrol-treated EPCs show strong thrombolytic capacity.