| Background:Dental pulp stem cells(DPSCs)are mesenchymal stem cell(MSC)-like populations that can be noninvasively harvested from pulp tissue of extracted human teeth;hence,these cells can be relatively easily obtained and are easy to access for use.Due to their robust selfrenewal capacity and pluripotency,DPSCs have paved the way for not only regenerating tooth related tissues such as dental pulp and the periodontium but also repairing other types of tissue insults such as bone defects,wound defects and central nervous system damage.Besides,at least in certain situations,DPSCs were found to contain a higher percentage of stem/progenitor cells and to exhibit higher proliferation and osteogenic potential than donormatched MSCs derived from bone marrow(BMMSCs).In addition,DPSCs have shown superior resistance to subculture,cryopreservation,and inflammation-induced senescence.Taken together,these findings indicate that DPSCs are highly likely to maintain their multidifferentiation and regenerative potential within an inflamed microenvironment and thus might be suitable for use in regenerative medicine,especially in combating inflammation-induced disease.Although DPSCs can be isolated from extracted wisdom teeth,and sometimes teeth extracted for orthodontic reasons,we still cannot ensure that a patient will have a tooth available for cell isolation when cell therapy is needed.In view of this,scientists have sought to use DPSCs derived from inflamed pulp tissues,as these tissues are more easily accessible in clinics and often can be obtained from teeth with crown fractures,irreversible pulpitis,and periodontitis.Previously,we successfully isolated DPSCs from teeth that were extracted teeth due to severe periodontitis(P-DPSCs);their pluripotential capacity has been demonstrated using in vitro and in vivo models.It was demonstrated that although P-DPSCs suffer relatively long-term stimulation by inflammation,regenerative related properties such as osteoblastic differentiation might increase to some extent,as long as the vitality of the pulp has not been totally damaged,suggesting that these traditionally discarded teeth may be an inexhaustible cell source for research and therapeutic purposes.Although P-DPSCs are becoming relevant in cell production,how to use these cells for therapeutic purposes requires further exploration.Considering that the most direct use of DPSCs is pulp regeneration and that a prerequisite for cells to regenerate functional pulp is their proangiogenic effects for inducing vasculature formation,and H-DPSCs has been proved to promote angiogenesis of endothelial cells(ECs)through its paracrine function,we wonder whether P-DPSCs have the same angiogenic potential as H-DPSCs.Based on current understanding,the beneficial proangiogenic effects of stem cells in cellular therapy are most likely mediated via their paracrine mechanism.It is now well recognized that stem cells can release an abundant mixture of cytokines,growth factors,chemokines,and extracellular vesicles(EVs),all of which have been demonstrated to contain functional elements that are useful for tissue regeneration.In particular,EVs within these secreted elements carry a complex cargo including but not limited to various mRNAs,microRNAs(miRNAs)and a spectrum of anti-apoptotic and proangiogenic factors;these agents are known to be the main mediators contributing to cell paracrine effects.In this context,EVs derived from H-DPSCs(H-EVs)have been demonstrated to trigger regeneration of dental pulp-like tissue and indeed exhibit the capacity to promote angiogenesis in vitro and in vivo.However,it is not clear whether P-DPSCs,especially the EVs secreted by P-DPSCs(P-EVs),have the proangiogenic potential.Therefore,the present study was conducted to explore the proangiogenic function of P-EVs and its underlying mechanisms.Objectives:In this study,we produced CMs using patient-matched H-DPSCs and P-DPSCs(HCM and P-CM),with the aim to investigate the effect of P-CM on EC angiogenesis.Through the patient-matched study,H-EVs and P-EVs were isolated from H-DPSCs and PDPSCs and then were used in parallel to test their proangiogenic effects on ECs.Especially the function of P-EVs.Subsequently,the specific mechanism of P-EVs in regulating angiogenesis of ECs were explored,with the aim to provide theoretical basis for the research and clinical application of P-EVs.Methods:1.Isolation and identification of H-DPSCs and P-DPSCs.In the present study,5 pairs of teeth were successfully obtained from 5 systemically healthy donors(who had(i)at least one tooth with full or partial pulp vitality that was to be extracted due to irreversible periodontitis and(ii)at least one other periodontally healthy tooth that was to be extracted due to nonfunctional or impacted reasons)were used to isolate H-DPSCs and P-DPSCs in parallel.The limiting dilution technique was applied for DPSC purification.DPSCs at passages P3-P5 were characterized by colony-forming assay,proliferation assay,flow cytometry and multiple differentiation assay,and then were used for subsequent experiments.2.To analyze the paracrine effects of DPSCs,ECs were exposed to H-CM and P-CM,then the angiogenic activities related to proliferation,migration,and tube formation were analyzed.More specifically,Cell Counting Kit-8(CCK-8)assay was used to detect the proliferation ability of ECs,transwell and scratch wound healing assays were established to evaluate cell migration potential in response to various CM-based incubations,and Matrigel tube formation assay was conducted to investigate network formation of ECs.Furthermore,in order to clarify the role of EVs in the paracrine function of DPSCs to promote EC angiogenesis,we pretreated H-DPSCs and P-DPSCs with GW4869(a reversible blocker of neutral sphingomyelinase that controls EVs secretion),and then the CMs in terms of H-GW and P-GW were prepared and used for the downstream functional assays.3.Given that EVs played a crucial role in the paracrine function-mediated angiogenesis of DPSCs,H-EVs and P-EVs were then isolated by ultracentrifugation,and detected by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and Western blot,respectively.Similar to CM-based incubation,CCK-8 assay,transwell assay,scratch wound healing assay and Matrigel tube formation assay were established to determine the proliferation,migration,and tube formation abilities of ECs in response to incubation with various EVs.Furthermore,quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR)and Western blot analysis were used to examine the expression levels of angiogenesis-related genes/proteins in ECs in response to EV-based incubation.Finally,a full-thickness skin defect model was applied to test the effects of EVs,whrerein histological analysis(H&E staining)and immunohistochemistry staining for angiogenesis markers(CD31 and VEGF)were used to evaluate wound closure as well as newly formed vessels.4.Circulating specific miRNAs were identified in P-EVs(compared to H-EVs)by Agilent miRNA Microarray and qRT-PCR.Then,cell transfection and RNA interference methods were established to explore the roles of differentially expressed miRNAs(miR378a)in the P-EV-mediated cell angiogenesis.Next,the direct binding affinity between miR-378a and its potential downstream target gene(suppressor of fused,Sufu)was evaluated by luciferase reporter assay,Meanwhile,qRT-PCR and Western blot were performed to validate the relationship between Sufu and miR-378a.Then,the function of Sufu in EC angiogenesis was verified by means of the associated si-RNA.Finally,EVs were separately modified with miR-378a mimic and miR-378a inhibitor(P-EVsmiR-378a mimic and P-EVsmiR-378a inhibitor)using the Exo-Fect Kit,and then added to the EC culture medium.After the treatment,cell proliferation,migration and tube formation on Matrigel assays were carried out to interrogate the EC angiogenesis abilities.In addition,Western blot was employed to measure the protein levels of Hedgehog/Glioma-associated oncogene homologue 1(Glil)signaling pathway and angiogenesis-related protein levels.The angiogenesis abilities of P-EVsmiR-378a mimic treated ECs following addition of GANT61(specifically inhibit Hedgehog/Glil signaling)was evaluated in terms of cell proliferation,migration,tube formation on Matrigel and Western blot(VEGF),with the aim to further validate whether the Hedgehog/Glil signaling was involved in the EC angiogenesis mediated by miR-378a.Results:1、H-DPSCs and P-DPSCs were successfully isolated from pulp tissues of 11 teeth(periodontally healthy teeth,5;periodontitis teeth,6).Almost all of the cells initially grew from pulp tissues after incubation for 5-7 d and exhibited clone-like growth as well as a spindle-shaped appearance.It was apparent that DPSCs in both groups presented colonyforming ability,however,the colony forming ability of H-DPSCs was significantly higher than that of P-DPSCs.In terms of the CCK-8 assay,H-DPSCs displayed a significantly higher proliferative rate than P-DPSCs.Flow cytometry assay was conducted to detect the surface markers of DPSCs.Notably,both P-DPSCs and H-DPSCs were positive for MSC markers,including CD90,CD 105,and CD 146,and negative for hematopoietic cell markers,such as CD31,CD34,and CD45.In addition,both P-DPSCs and H-DPSCs could readily be induced to differentiate into osteogenic,adipogenic and chondrogenic lineages when cultured in their respective differentiation media,as evidenced by Alizarin Red S staining,Oil Red O staining,and Alcian blue staining,respectively.2、It is well recognized that paracrine activity contributes considerably to MSC-based therapy.To analyze the paracrine effects of DPSCs,ECs were exposed to H-CM,P-CM and Control group,then angiogenic activities related to proliferation,migration,and tube formation were analyzed.According to the CCK-8 assay results,both H-CM and P-CM significantly enhanced EC proliferation compared with the Control group,additionally,the proliferation rate was higher in the P-CM group than in the H-CM group.Similar trends were observed in the transwell migration assay and scratch wound healing assay.In addition,tube formation assays revealed that incubation with either P-CM or H-CM could promote angiogenesis when compared with that in the Control group.In addition,ECs incubated with P-CM demonstrated a stronger tube formation ability than those incubated with H-CM in terms of quantitative analyses of the total branching points,total loop numbers,total tube length and covered area.Currently,EVs are considered to be a vital paracrine product of MSCs.If EVs contribute to the proangiogenic effects of DPSCs,then the inhibition of EVs(H-EVs and PEVs)release would be expected to abolish or reduce the proangiogenic function mediated via paracrine activiey of DPSCs(H-CM and P-CM).To verify this hypothesis,we pretreated H-DPSCs and P-DPSCs with GW4869(a reversible blocker of neutral sphingomyelinase that controls EV secretion).Indeed,the increased proliferation trend of ECs exposed to HCM and P-CM was partially inhibited by GW4869 pretreatment,as the CCK-8 assay revealed that both cells exposed to H-GW and P-GW had a lower proliferation rate than cells exposed to H-CM or P-CM,respectively.In addition,the increased migration abilities of ECs stimulated by H-CM and P-CM were also attenuated by GW4869 pretreatment,with fewer migrated cells quantified by transwell migration and larger scratch wounds in the scratch wound assay.Notably,H-GW and I-GW partially reversed the enhanced tube formation trend by H-CM and P-CM,as quantified by the capillary structure formations of ECs.3、Given that EVs played a crucial role in the paracrine function(DPSCs)-mediated angiogenesis,H-EVs and P-EVs were then isolated for further study.As expected,nanoparticles purified from the supernatants were both bilayer membrane vesicles,coinciding with the general characteristics of EVs,and nanoparticle analysis showed that the EV diameters mostly ranged from 50 to 300 nm in both groups.Additionally,Western blot analysis revealed that both H-EVs and P-EVs expressed EV molecular markers,including ALIX,HSP70,CD9,and CD81,further confirming their EV identity.Subsequently,to confirm cellular EV uptake,PKH67-labeled H-EVs and P-EVs were cocultured with ECs.After incubating for 4 h,ECs were observed to stain positive for PKH67 in both groups,confirming EV uptake.ECs were treated with P-EVs,H-EVs,or an equal volume of PBS for a sequence of functional assays,including proliferation,migration and angiogenesis.The results obtained from the CCK-8 assay showed that the proliferation rate of ECs was markedly elevated in response to EV stimulation relative to the Control group,and notably,ECs exhibited a stronger proliferative rate by P-EVs than H-EVs.The migration promoting effect was determined by a scratch wound healing assay and a transwell assay.For the results obtained from transwell assay,there was no difference between the two EV groups,although both of them demonstrated a stronger migration-promoting ability than the Control group.In the scratch wound healing assay,both H-EVs and P-EVs remarkably upregulated the motility of ECs compared with that in the Control group,and P-EVs exhibited a stronger migration ability than H-EVs.Furthermore,we evaluated the effect of EVs on angiogenic activities of ECs.Compared with the Control group,both EV treatments significantly enhanced the angiogenic activities of ECs.Moreover,compared with those treated with H-EVs,ECs treated with P-EVs showed a higher number of capillary-like structures,and quantitative measurements revealed that the total branching points,total loop numbers,and total tube length were all significantly increased,although the covered area was not.qRT-PCR and Western blot analysis were performed to detect the expression levels of angiogenesis-related genes and proteins(VEGF and AngⅡ).The obtained results demonstrated that both groups of EVs exhibited upregulated angiogenesis related gene expression compared with that in the control group;moreover,gene expression in the P-EVs group was higher than that in the H-EVs group.Furthermore,ECs in the P-EVs group secreted more VEGF and AngⅡproteins than ECs in the H-EVs and Control groups.In vivo study,wound closure was markedly accelerated by EVs treatment,illustrated by smaller wound areas measured at days 4,9,and 14 postwounding compared with those in the Control group,and as expected,the rate of wound closure in the P-EVs group was faster than that in the H-EVs group.H&E staining revealed that transplantation with both types of EVs reduced the scar formation of wounds compared with the Control group at day 14 after treatment,and quantitative measurements confirmed that P-EV-treated wounds had a lower level of scar formation than H-EV-treated wounds.Skin images from the undersurface revealed that both EV-treated groups exhibited more newly formed microvessels than Control wounds at day 14 postwounding.Immunofluorescence staining for endothelial markers,including CD31 and VEGF,was also performed to quantify the density of new blood vessels.Representative images of CD31 staining consistently demonstrated that abundant blood vessels appeared in the wounds treated with EVs,whereas the capillary density was significantly lower in the Control group.Quantitative analysis revealed that the proangiogenic effect of P-EVs was stronger than that of H-EVs.Similarly,more extensive blood vessel staining for VEGF was observed in the P-EV group than in the H-EVs and Control groups.4、In the present study,angiogenesis-related miR-378a differentially expressed in PEVs(compared to H-EVs)was identified and its role in P-EV-enhanced cell angiogenesis was confirmed by cell transfection and RNA interference methods.More specifically,overexpression of miR-378a in ECs promotes cell proliferation,migration and angiogenesis.Next,the direct binding affinity between miR-378a and Sufu was evaluated by luciferase reporter assay,wherein Sufu was confirmed as a downstream target gene of miR-378a.Functionally,silencing of Sufu stimulated the proliferation,migration,and tube formation of ECs.A train of assays in terms of proliferation,migration and tube formation on Matrigel were established to interrogate whether P-EV-mediated transmission of miR-378a could exert their pro-angiognic effect on ECs.The proliferation ability of ECs was quantified by CCK-8 assay,which revealed that P-EVsmiR-378a mimic stimulation resulted in a significant increase in EC proliferation,while the effect was inhibited by P-EVsmiR-378a inhibitor treatment.Meanwhile,the results obtained from wound healing and transwell assay demonstrated that P-EVsmiR-378a mimic treatment remarkably upregulated the motility of ECs,while the PEVsmiR-378a inhibitor exerted a contrasting effect Moreover,after the P-EVsmiR-378a mimic treatment,ECs demonstrated a stronger tube formation ability than that in the P-EVsmimicNC group,in terms of quantitative analyses of the total branching points and total tube length.Notably,P-EVsmiR-378 inibitor treatment partially reversed the enhanced tube formation trend by P-EVs stimulation,as quantified by the capillary structure formations of ECs.Subsequently,the Hedgehog/Glil signaling pathway has been evaluated,according to the Western blot analysis,P-EVsmiR-378a mimic initiated elevations in the protein levels of Glil and VEGF,and reductions in the protein level of Sufu,whereas all of which were opposite to that induced by P-EVsmiR-378a inhibitor treatment.Furthermore,we used the Hedgehog/Gli1 signaling inhibitor,GANT61,to specifically inhibit Hedgehog signaling in ECs treated with P-EVsmiR-378a mimic.After treatment with GANT61,the proliferation and migration abilities of ECs were decreased.In addition,GANT61 were demonstrated to impair the pro-angiogenic effect of P-EVsmiR-378a mimic in ECs.Furthermore,the VEGF protein expression level of ECs was down-regulated in GANT61 group when compared to that of the P-EVsmiR-378a mimic group.Conclusion:Both H-CM and P-CM significantly enhanced EC proliferation,migration and angiogenesis,more importantly,compared with H-CM,P-CM had stronger ability to promote EC angiogenesis.Inhibition of EV secretion impaired CM-mediated angiogenesis,and EVs(H-EVs and P-EVs)played a crucial role in the paracrine function(DPSCs)mediated angiogenesis.Both H-EVs and P-EVs could enhance the angiogenesis-related activities of ECs,besides,P-EVs exerted a more robust potential to stimulate cell proliferation,migration and tube formation than H-EVs.miR-378a was largely responsible for the pro-angiogenic potential of P-EVs,which can be delivered to ECs by P-EVs,thereby promoting cell angiogenesis by targeting Sufu to activate the Hedgehog/Gli1 signaling.In this study,H-CM and P-CM were produced using patient-matched DPSCs and were used in parallel to test their proangiogenic effects on ECs.After confirming that P-CM had a stronger function on promoting EC angiogenesis compared with H-CM,and EVs played a crucial role in the paracrine function(DPSCs)-mediated angiogenesis.H-EVs and P-EVs were then isolated and were demonstrated to enhance the angiogenesis-related activities of ECs.Additionally,P-EVs exerted a more robust potential to stimulate EC proliferation,migration,and angiogenesis than H-EVs.More specifically,P-EVs carrying miR-378a promoted the angiogenesis of ECs by down-regulating Sufu to activate the Hedgehog/Gli1 signaling pathway.Our findings provide additional evidence that P-DPSCs derived from periodontitis-compromised teeth represent a potential source of cells for regenerative research and therapeutic use.Particularly,P-EVs may be used to promote new vessel formation in cellular therapy and regenerative medicine. |
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