The Role And Mechanism Of Endothelial Microparticles Induced By Cyclic Stretch In Endothelial Apoptosis And Leukocyte Adhesion

Vascular endothelial cells(ECs),covering the inner surface of blood vessels,serve as a barrier between the blood and the vessel wall,and constantly exposed to mechanical cyclic stretch(CS)caused by rhythmic deformation of the vessel wall following pulsatile blood pressure.In hypertension,elevated blood pressure results in pathological cyclic stretch,stimulates EC apoptosis and causes the dysfunction of ECs.Physiological cyclic stretch inhibits EC apoptosis and inhibits the adhesion of leukocytes to ECs,a crucial step of inflammation,which is beneficial to maintain EC homeostasis.Endothelial microparticles(EMPs)ranging from 0.1 to 1?m in diameter are defined as submicron vesicles,and shed from the surface of activated or apoptotic ECs.EMPs carry different types of molecules both on their surface and in their interior to act as integrated messengers for intercellular communication.Growing evidence has shown that mechanical stimulus can induce EMP release.However,the roles of EMPs induced by CS in EC apoptosis and leukocyte adhesion are still not clear.Using FX-5000T Stretch Unit,ECs were subjected to 5%CS(mimic physiological CS),15%CS(mimic hypertention)respectively,and ECs without mechanical force application were used as static.EMPs were then isolated from the culture supernatant by ultracentrifugation.Using proteomics combined with bioinformatic analysis,the underlying roles of EMPs in EC functions were investigated,and the possible mechanisms of5%CS-induced EMPs in EC-leukocyte adhesion and 15%CS-induced EMPs in EC apoptosis were further demonstrated.Flow cytometry analysis and Nanosight Analyzer revealed that 98.3%of the isolated particles expressed EC marker CD31and ranged from 0.1to 0.6?m in diameter,indicating the efficacy of EMP extraction.Trucount beads and annxin V staining were then used to optimize the amount of isolated EMPs,and cleaved caspase-3 ELISA was used to detected the apoptosis of ECs.The results revealed that 2000 Ann V~+EMPs/?l obviously increased the apoptosis of target ECs.In addition,the adhesion of PKH-26-labeled EMPs to ECs was analyzed at 0 min,30 min,60 min,and 120 min,respectively,and the results showed and the number of EMPs adhered to ECs gradually increased.The fluorescence images were then taken by confocal microscopy and analyzed by Z-stacks,and the results revealed that EMPs were incorporated into ECs.After that,the roles of CS-induced EMPs in EC function were investigated.ECs were incubated with static-or 5%CS-derived EMPs for24 h,and PKH-26 labeled leukocytes were then incubated with ECs for 1h.Fluorescence analysis showed that compared with the static group,the number of leukocytes adhered onto ECs were significantly decreased by5%CS-derived EMPs.To confirm the role of EMPs in cell death and survival,cleaved caspase-3 ELISA was then used to detect EC apoptosis in response to EMPs derived from 5%CS and 15%CS respectively.The results revealed that the apoptosis of ECs was significantly increased by15%CS-derived EMPs compared with 5%CS-derived ones.The circulating MPs generated from the blood of hypertensive and normal rats were intravenous injected into the tail vein of C57BL/6J mice.The result showed that the apoptosis of ECs in vivo were increased with MPs from hypertensive group in comparison with that from normal control.To investigate the underlying mechanisms by which EMPs modulated EC function,the effect of CS on protein composition of EMPs was detected by proteomic approach combined with bioinformatic analysis.Comparative proteomic analysis revealed 373 proteins differentially expressed between static-and 5%CS-derived EMPs,in which 314proteins were uniquely identified in static-derived EMPs,34 proteins uniquely in 5%-CS-derived EMPs,and 25 proteins were detected in both groups but showed obvious difference.Gene Ontology(GO)biological processes analysis showed that these 373 differentially expressed proteins between static and 5%CS groups were involved in cell-cell adhesion,vesicle-mediated transport,and related to cadherin binding involved in cell-cell adhesion.In addition,there were 412 proteins differentially expressed between 5%CS-and 15%CS-derived EMPs,of which 29proteins uniquely expressed in 5%CS-derived EMPs and 362 protein expressed only in 15%CS-derived EMPs,and 21 proteins were detected in both groups but showed significant difference.These 412 differentially expressed proteins between 5%CS and 15%CS group were also analyzed by GO analysis,and the results revealed the extracellular nature of these proteins,which were mainly present in vesicles or extracellular vesicles,and the mainly signaling pathways in which these proteins participated were integrin signaling pathways,angiogenesis pathways,as well as inflammation mediated by chemokine and cytokine signaling pathways.In addition,the molecular function of the differentially expressed proteins was performed by Ingenuity Pathway Analysis(IPA),and the results showed that cell death and survival was ranked first.Based on the comparative proteomic data and bioinformatic analysis,intercellular adhesion molecule 1(ICAM1)was focused to investigate the underlying mechanisms of 5%CS-derived EMPs in EC-leukocyte adhesion.Western blot and flow cytometry analyses confirmed the significant decrease of ICAM1 in 5%-CS-derived EMPs,which subsequently inhibited the phosphorylation of VE-cadherin at Tyr731 in target ECs.Moreover,leukocyte adhesion was obviously decreased when EMPs were pretreated with neutralizing antibody(NAB)of ICAM1.Since Src is a crucial molecule in cell death and survival,the role of EMPs in Src activation at single-cell level was investigated using fluorescence resonance energy transfer(FRET).The results revealed that EMPs derived from both 5%CS and 15%CS induced significant changes in the Src biosensor with gradual increases compared with EMPs derived from the control.Whereas compared with 5%CS group,a significantly weaker response was detected after treatment with 15%CS-derived EMPs.Western blot further confirmed that the phosphorylation of Src(416)in ECs was obviously attenuated by treatment with 15%CS-derived EMPs compared with 5%CS-derived EMPs,but there were no significant effect on the phosphorylation of the inactive residue,Src(527).After pretreatment with Src inhibitor or Src-small interference RNA(si RNA),the apoptosis of ECs significantly increased in both the 5%CS group and the 15%CS group.CD151 was predicted to be the upstream molecule in EMPs which induced Src activation by IPA.Western blot and flow cytometry confirmed the significantly decrease of CD151 in EMPs from 15%CS-group in comparison with 5%CS group.In summary,the research suggested that cyclic stretch changes the protein components of EMPs,which in turn regulate EC functions.Physiological stretch decreases ICAM1 expression in EMPs,which reduces EC-leukocyte adhesion.In addition,EMPs released under pathological cyclic stretch inhibit the phosphorylation of Src(416),which then promote EC apoptosis.This research may provide new biomechanical insights into understanding the underlying mechanism of CS-induced EMPs in intercellular communications and EC homeostasis.