| Background and objectivesAngiogenesis,developing new capillaries from the preexisting blood vessels,is recognized as a pivotal process with a sufficient supply of oxygen and nutrition and removal of metabolic products to meet the needs of local metabolism.This process plays an important role in promoting wound healing,the treatment of ischemic diseases and the anti-tumors treatment by targeting tumor angiogenesis.At present,many studies have found that the process of angiogenesis can be regulated by the application of various exogenous angiogenesis related factors.The wide applications of these factors are limited by some disadvantages,such as drug toxicity,drug resistance and poor performance stability.Therefore,it remains to be solved that how to regulate the angiogenesis safely and efficiently in the clinical treatment.Mesenchymal stem/stromal cells(MSCs),as the ideal seed cells for tissue regeneration and tumor biological therapy,have been reported to play the positive therapeutic effects through extracellular vesicle-mediated paracrine mechanism.Exosomes,as a type of extracellular vesicles,have attracted extensive attention due to the superior biological properties and important roles in intercellular communication and drug delivery.Presently,the roles of exosomes derived from MSCs on the regulation of angiogenesis are diverse and controversial.Exosomes derived from MSCs obtained from different tissues have played diverse roles on angiogenesis.Even,the exosomes from the same type of MSCs,which are cultured in different microenvironments,such as normoxia or hypoxia may play different roles on angiogenesis.SHED cells,first reported in the year of 2003,are one type of immature MSCs with a stronger proliferative ability and capability for osteogenic and odontogenic differentiation and a more prominent and versatile differentiation ability.At the onset of physiological root resorption,the blood vessels in the dental pulp tissue are reduced and oxygen availability is decreased.Thus,we used hypoxic culture condition(2%O2)in vitro to mimic the physiological cellular microenvironment in vivo.Therefore,the aim of our study was to explore the roles of exosomes derived from SHED cultured at normaxic and hypoxic culture conditions on angiogenesis and the underlying molecular mechanism in order to develop a novel therapeutic approach for regulating the angiogenesis.Methods1.Comparison of exosomes derived from SHED cututured at normaxia and HypoxiaFirstly,we isolated and cultured SHED from the dental tissues,and used flow cytometry to analyze specific MSCs surface markers.The osteogenic differentiation potential of SHED cells was verified by using osteogenesis induction assay.Exosomes from SHED cututured at normaxia(Norm-exos)were characterized using transmission electron microscope,nanoparticle tracking analysis and Western blot.Then the morphologies and mean diameters of Norm-exos and exosomes from SHED cututured at Hypoxia(Hypo-exos)were compared by transmission electron microscopy and nanoparticle tracking analysis.Furthermore,the concentration of exosomes from hypoxic and normoxic conditions were measured.Nextly,we added those exosomes labeled with the PHK67 fluorescent dye to HUVECs.2.The effects of Norm-exos and Hypo-exos on angiogenesis in vitroCCK-8 assay and Ki67 immunofluorescent staining were performed to assess the effect of Norm-exos and Hypo-exos on the growth of endothelial cells.Moreover,the roles of these exosomes on the migration ability of endothelial cells were validated by conducting Scratch wound healing assays and Transwell migration assays.Additionally,the effects on angiogenesis ability of endothelial cells were assessed by performing tube formation assays in vitro.Meanwhile,the proangiogenic factors were verified by Western blot assays.3.The effects of Norm-exos and Hypo-exos on angiogenesis in vivoChick chorioallantoic membrane(CAM)assays were used to evaluate the anti-angiogenesis effects of Norm-exos in vivo.Norm-exos or PBS was injected into in middle of each CAM of chicken eggs of 7～8 days.After incubation for 48 h,the CAM assays were cut out and the pictures were taken.In addition,CAL27 cells were subcutaneously injected subcutaneously to establish the xenogeneic oral squamous cell carcinoma transplantation(OSCC)model.We performed multi-point intratumoral injections near the base of the tumor four times at three day intervals and the tumor volume was calculated.Tumors were isolated for photography and immunohistochemical staining for CD31 was performed.Besides,the matrigel plug assays were performed to examine the angiogenesis ability of endothelial cells in vivo.Matrigel containing Hypo-exos or Norm-exos were mixed with HUVECs and then were subcutaneously injected.At 10 days after injection,the mice were sacrificed and the immunofluorescence staining for VEGF and immunohistochemical staining for CD31 were performed.4.Molecular mechanism of the effects of Norm-exos and Hypo-exos on angiogenesisTo further characterize the miRNA cargoes of Norm-exos and Hypo-exos,miRNAs were sequenced in three independent experiments.Primarily,the top 15 highly expressed miRNAs were targeted according to their expression level and we used qRT-PCR assays to verify the expression levels of miR-100-5p and miR-1246 in Norm-exos.Additionally,the expression profiles of miRNAs between Hypo-exos and Norm-exos were compared and the targeted miRNAs were validated by using qRT-PCR.Next,the assays of vascular tube formation,qRT-PCR and Western blot were performed to explore the molecular signaling pathways.Results1.Comparison of exosomes derived from SHED cututured at normaxia and HypoxiaSHED are long spindle shaped.Flow cytometric assays showed that SHED cells expressed high levels of the MSCs markers,but did not express any endothelial cell markers.SHED cells presented osteogenic differentiation potential.It was also validated that the extracellular vesicles derived from SHED cultured at normaxia were exosomes.Besides,similar morphologies with a typical cup shape were observed in Hypo-exos and Norm-exos.Nanoparticle tracking analysis showed that the mean diameters of Hypo-exos were significantly larger than those of Norm-exos.Furthermore,we found that the exposure of SHED to hypoxia significantly increased the concentration of exosomes and the PHK67-labeled Hypo-exos and Norm-exos were efficiently taken up by HUVECs.2.The effects of Norm-exos and Hypo-exos on angiogenesis in vitroThe assays in vitro showed that Norm-exos significantly suppress the growth and migration ability compared with PBS.Meanwhile,we found that Hypo-exos significantly enhance the growth and migration of endothelial cells in vitro compared with Norm-exos.Additionally,it was revealed that Norm-exos inhibited the tube formation of endothelial cells in vitro associated with the downregulation of VEGF,MMP-9 and ANGPT1.Furthermore,we found that Hypo-exos enhance the tube formation of endothelial cells,which was associated with the upregulation of VEGF.3.The effects of Norm-exos and Hypo-exos on angiogenesis in vivoThe CAM assays showed that the number of formed micro-vascular was significantly decreased in the Norm-exos treated group compared with PBS group.In addition,after performing the multi-point intratumoral injections of Norm-exos,we found that the tumor volumes were significantly smaller in Norm-exos treated tumors compared with PBS treated groups.Besides,OSCCs injected with Norm-exos had a lower percentage of CD31 positive cells than the PBS treated group.In addition,matrigel plug assays showed that a redder appearance was observed in plugs containing Hypo-exos than in plugs containing Norm-exos.Besides,a higher expression of VEGF and a higher number lumenal structures stained by CD31 were displayed in the Hypo-exos group.4.Molecular mechanism of the effects of Norm-exos and Hypo-exos on angiogenesisAccording to the Norm-exos miRNA sequencing,we targeted miR-100-5p and miR-1246 from the top 15 highly expressed miRNAs.QRT-PCR analysis confirmed that both miR-100-5p and miR-1246 are enriched in Norm-exos compared with SHED,Besides,it was shown that let-7f-5p and miR-210-3p significantly upregulated in Hypo-exos.Finally,the results of tube formation,qRT-PCR and Western blot indicated that both miR-100-5p and miR-1246 are enriched in Norm-exos and can inhibit angiogenesis of endothelial cells by indirectly targeting VEGF expression,through the miR-100-5p/mTOR/HIF-1a and the miR-1246/ACE pathway.Beside it was also demonstrated that let-7f-5p and miR-210-3p rich in Hypo-exos regulate angiogenesis via the let-7f-5p/AGO1/VEGF and miR-210-3p/EphrinA3 signal pathways.Conclusions1.The effects of Norm-exos and Hypo-exos on angiogenesis are different.Norm-exos suppress angiogenesis compared with PBS.Compared with Norm-exos,Hypo-exos enhance angiogenesis in vitro and in vivo.2.The normaxia and hypoxia affected the expression levels of different miRNAs to regulate angiogenesis.3.MiR-100-5p and miR-1246 are enriched in Norm-exos and can inhibit angiogenesis of endothelial cells by indirectly down-regulated VEGF expression,through the miR-100-5plmTOR/HIF-1a and the miR-1246/ACE pathway.4.Hypo-exos enhanced the angiogenesis by the transfer of let-7f-5p and miR-210-3p through the let-7f-5p/AGO1/VEGF and the miR-210-3p/EphrinA3 signal pathways. |
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