| Background and Objective Endovascular re-canalization is increasingly being used to restore blood flow to areas of ischemia,tissue loss or gangrene for diabetic patients with peripheral artery disease.However,the early restenosis rate of infrapopliteal occlusions was high,even in lesion treated with drug-eluting balloons.Endothelial cell injury is the central mechanism in the initiation and progression of restenosis.Ongoing deterioration of the endothelial monolayer may induce a continuum of events giving rise to vascular inflammation,lipid deposition,and thrombosis,resulting in neointimal hyperplasia and vessel wall remodeling,which contribute to restenosis.Therefore,an early acceleration of reendothelialization is essential to prevent progression of restenosis.Exogenously infused EPCs could regenerate the endothelium by differentiating into mature endothelial cells and by promoting migration and proliferation of existing endothelial cells.However,recent studies have revealed that the stimulation of resident endothelial cells from adjacent intact endothelium via paracrine mechanisms might be more important for vascular repair than the direct incorporation and expansion of the exogenously transplanted EPCs during tissue repair.Exosomes,small membrane particles(40-100 nm in diameter)originating from multivesicular bodies,are an important component of paracrine secretion and play key roles in the intercellular cross-talk via the horizontal transfer of proteins,lipids and RNAs to target cells.Recent studies have indicated that the exosomes derived from mesenchymal stem cells and cardiac progenitor cells can trigger angiogenesis and restore tissue function,suggesting a significant role of exosomes in the paracrine action of stem or progenitor cells.Therefore,we hypothesized that the protective effects of EPCs in vascular injuries might be partially mediated by their released exosomes.In this study,we isolated and purified exosomes released from human umbilical cord blood-derived EPCs and determined whether these exosomes could accelerate reendothelialization in rat models of balloon-induced vascular injury.Moreover,we studied in vitro the impact of EPCs-secreted exosomes on endothelial cells proliferation,migration,and angiogenesis-related genes expression.Methods 1.Isolation and characterization of EPCs and EPC-Exo1.1 The human umbilical cord blood overlaid on separation medium for density gradient centrifugation.The mononuclear cells were cultured in complete EGM-2 MV media,and plated onto type I collagen precoated flasks.1.2 Immunostaining for v WF,CD34 and CD31.1.3 Flow cytometry analysis for CD31,CD34,CD45,CD133,VEGFR-2,and VE-cadherin.1.4 Assessment of acetylated low density lipoprotein(ac-LDL)uptake and Ulex europaeus agglutinin-1(UEA-l)binding.1.5 EPCs were cultured in serum-free EGM-2 MV media for 48 h.Ultracentrifugation was used to isolate and purify EPC-Exo.1.6 Morphology of EPCs-derived exosomes under a transmission electron microscopy1.7 Western blotting analysis of exosomal surface marker proteins(including CD9,CD63,and CD81).2.In vivo effects of EPCs-derived exosomes in rat models of balloon-induced vascular injury2.1 Establish of vascular injury model.2.2 Evans Blue dye staining of whole-mount en-face carotid arteries after carotid balloon surgery.Non-endothelialized lesions were marked by blue staining,and the reendothelialized area appeared white.Reendothelialization was quantified as white-area/total-area ratio.2.3 Hematoxylin and eosin were performed to measure intimal(between lumen and internal elastic lamina),media(between internal and external elastic laminae)and intima/media.3.In Vitro analyze the mechanism of EPC-Exo in promoting angiogenesis3.1 Fluorescent microscopy analysis of Di O-labeled EPCs-derived exosomes internalization in HMECs.3.2 EPC-Exo effect on HMECs proliferation by Cell Counting Kit-8 assay.3.3 Wound healing assay to test the migration.3.4 EPC-Exo effect on the tube formation of HMECs by metrigel.3.5 Angiogenesis related genes expression level in HUVECs by q RT-PCR.Results 1.Successfully acquaired EPCs and collected EPC-Exo1.1 EPCs-like colonies appeared between 7 and 21 days of culture.Cells within the colonies exhibited the typical cobblestone morphology of endothelial cells1.2 Immunostaining revealed that the adherent cells were positive for v WF,CD31 and CD34.1.3 Flow cytometric analysis showed that the EPCs-like cells were positive for CD31,CD34,CD133,CD144 and CD146,but negative for the hematopoietic cell-specific marker CD45,indicating their EPCs identity.1.4 Acetylated low-density lipoprotein uptaking and ulex europaeus agglutinin binding assay showed that the mononuclear cells were positive for Di I-ac LDL and FITC-UEA-1 stains,and more than 90% cells were double positive.1.5 EPCs-derived exosomes was isolated and purified by ultracentrifugation successfully.1.6 Transmission electron microscopy showed that the majority of EPC-Exo exhibited a cup-or round-shaped morphology,with a size ranging from 30 to 100 nm.1.7 Western blotting analysis of exosomal surface marker proteins(including CD9,CD63,and CD81)in EPCs-derived exosomes.2.The effects of EPCs-derived exosomes on reendothelialization and neointima formation2.1 Evans blue dye was administered premortem at 14,and 21 days after injury.Non-endothelialized lesions were marked by blue staining,whereas the reendothelialized area appeared white.Reendothelialization was quantified as white-area/total-area ratio.At the 14-day time points,the reendothelialization in the exosomes treated arteries was significantly larger than in the control arteries(53.37 ±10.3% vs.87.66 ± 12.3%;n = 8;P < 0.05 for each),while no difference was observed at 21 days(90.65 ± 12.1% vs.92.42 ± 11.6%;n = 8;P < 0.05).2.2 The effect of EPCs-derived exosomes on neointimal thickening was examined by H&E staining.In control rats(n = 7),intimal area/medial area(I/M)ratios increased markedly at day 14(0.587 ± 0.10)and day 21(1.245 ± 0.17).In contrast,I/M ratios of animals treated with EPCs-derived exosomes(n = 7)were 0.282 ± 0.11 at day 14 and 0.885 ± 0.33 at day 21(P < 0.05 vs.control animals).3.EPC-Exo promote endothelial function and angiogenesis in vitro3.1 The green fluorescent lipophilic dye(Di O)labeled exosomes were then incubated with HMECs for 2 hours.Fluorescence microscopy analysis demonstrated that the Di O label was taken up and transferred to the perinuclear region of endothelial cells,demonstrating that the EPCs-derived exosomes entered into the recipient cells,or that these exosomes were internalized by endothelial cells.3.2 CCK-8 cell counting analysis indicated that the exosomes treatment significantly enhanced the proliferation of HMECs.3.3 A scratch wound assay showed that EPC-Exo remarkably enhanced the motility of HMECs,as determined by the increased size of the migration area.3.4 The branch numbers and tube length were measured to assess the ability of HMECs to form tubules.Compared with the control group,all indicators that evaluated the tube formation capability were profoundly increased in HMECs stimulated with EPC-Exo.3.5 q RT-PCR analysis showed various critical pro-angiogenic genes,including endothelial nitric oxide synthase,interleukin-8,angiopoietin-1,and E-selectin,were significantly up-regulated,by more than 4-fold,after stimulation with EPCs-derived exosomes.Vascular endothelial growth factor A,vascular endothelial growth factor receptor-2,hypoxia inducible factor 1 alpha,chemokine(C-X-C motif)ligand-16,and platelet-derived growth factor alpha polypeptide showed >1.5-fold up-regulation in endothelial cells treated with exosomes.In addition,exosomes-stimulated endothelial cells showed profound down-regulation of matrix metalloprotein-9.These data suggested that the EPCs-derived exosomes could modulate the expressions of multiple angiogenesis-related genes.Conclusions Taken together,our results indicated that exosomes are an active component of paracrine secretion by human EPCs and could promote vascular repair in rat modesl of balloon injury by up-regulating endothelial cells function.Further studies are needed to assess the long-term effects of EPCs-derived exosomes-based therapy for vascular diseases,and whether these nanoparticles could be used as a valid and safe therapeutic option for clinical applications. |
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