The Role Of Platelet-derived Microvesicles In Proliferation And Differentiation Of Endothelial Progenitor Cells During Vascular Intimal Injury

Cardiovascular diseases(CVDs)are serious diseases that endanger human health.Intimal injury is an early stage and cause of many CVDs.After vascular intimal injury,endothelium become dysfunctional,the subendothelial layer,which contains collagen,is exposed,and the vessel will form neointimal in the late stage,resulting in vascular remodeling.Endothelial repair plays an important role in maintaining vascular homeostasis,preventing and reducing the occurrence and development of vascular diseases.It has been reported that endothelial progenitor cells(EPCs)play a crucial role in repairing vascular injury.After intimal injury,EPCs stored in bone marrow were mobilized,entered into peripheral blood,and homed to the site of vascular injury.Then EPCs proliferate and differentiate into endothelial cells(ECs)to participate in intimal repair.At the same time,platelets-derived microvesicles(PMVs)are generated due to the platelets adhering to the collagen fiber layer,resulting in a locally enriched microenvironment.PMVs contain proteins and genetic material,which can participate in regulating cell signaling and functions;however,the effect of PMVs in EPC functions and underlying mechanism are still unknown.This study focused on the role and possible mechanisms of activated PMVs(ac.PMVs)released by platelets upon collagen activation in regulating the proliferation and differentiation of EPCs by transmitting signal molecules.In this study,we established rat carotid artery intimal injury model,and contralateral side was used as control.By using immunohistochemistry and immunofluorescent staining,we detected morphology changes in the damaged blood vessel.The expressions of EC marker von Willebrand Factor(v WF),hematopoietic stem cell marker CD34 and platelet marker CD41 indicated the adhesion and colocalization of PMVs and EPCs at the injury site after vascular injury.In order to explore the regulation mechanism of EPCs in vitro,we used density gradient centrifugation to obtain EPCs from the bone marrow of Sprague Dawley(SD)rats and used differential centrifugation to obtain ac.PMVs from rat abdominal aorta blood.Then ac.PMVs were used to stimulate EPCs for 1 h or 24 h,the results showed that the proliferation ability of EPCs was significantly improved and differentiated to mature ECs.Bioinformatics analysis showed that among the molecules related to the proliferation and differentiation ability of EPCs,the proteins of Smad family played an important role in signal regulation.The protein level of p-Smad3 related to proliferation was significantly increased and the expression of pSmad1/5 related to differentiation was significantly decreased after 1 h stimulation detected by Western blot.Meanwhile,after 24 h stimulation with ac.PMVs,the translocation of Smad3 from cytoplasm to nucleus was incresed and EPC proliferation was enhanced via regulating downstream target genes.Transforming growth factor β1(TGF-β1)is an upstream molecule of Smad3 and plays important role in stem cell growth and proliferation.We found that the content of TGF-β1 was significantly increased in ac.PMVs compared to PMVs which produced from untreated platelets.The stimulation of cells with the recombinant TGF-β1(r-TGF-β1)was consistent with the previous results.In addition,the TGF-β1 pathway specific inhibitor SB431542 and Smad3 phosphorylation inhibitor SIS3 were respectively used to verify the regulation of ac.PMVs on TGF-β1signaling pathway and proliferation.In order to further study the role of TGF-β1 in this process,MEG-01 cell line was applied in the experiment and si RNA interference was used to obtain ac.PMVs in which TGF-β1 was knocked down.The results verified the changes of protein expression level was caused by TGF-β1 that delivered by ac.PMVs.We also performed the in vivo experiments.EPCs were locally incubated after carotid intimal injury,and r-TGF-β1 protein was injected from tail vein for 4 days.Local incubation with EPCs promoted the repair of the vascular intima,and the systemic injection of r-TGF-β1 significantly increased reendothelialization and EPC proliferation in situ.In summary,our data indicated that the exposed collagen at the intimal injury sites stimulated the adherent platelets producing large amount of ac.PMVs,which delivered TGF-β1 to EPCs and activated Smad3 phosphorylation in EPCs,subsequently regulated the m RNA level of TNC,CDKN1 A and CDKN2 A,and enhance the proliferation of EPCs eventually.At the same time,stimulation of ac.PMVs promoted the differentiation of EPCs to endothelial cells by inhibiting the expression level of p-Smad1/5.This study suggests that TGF-β1 delivered by ac.PMVs and Smad protein play an important role in EPCs participating in the repair of intimal injury and provide potential therapeutic targets for the repair of damage in diseases.