The Effects And Mechanism Of Exosomes Derived From Krüppel-Like Factor 2 Overexpressed Endothelial Cells On Myocardial Ischemia/Reperfusion Injury

BackgroundAcute myocardial infarction(AMI)usually is the leading cause of death and disability worldwide.Nowadays,the goal of reperfusion therapy with fibrinolytic drugs or primary percutaneous coronary intervention(PCI)is to restore blood flow to ischemic myocardium and reduce infarct size.However,the process of reperfusion could induce further damage known as myocardial reperfusion injury.The ischemia/reperfusion(I/R)process triggers the immune response,particularly the innate response including neutrophils and monocytes,plays a crucial role in both myocardial injury and healing.Accordingly,two distinct waves of monocyte recruitment have been identified in healing myocardial infarct.Early recruitment of pro-inflammatory Ly6Chigh monocytes is mediated through the activation of the monocyte chemoattractant protein-1(MCP-1)/C-C chemokine receptor type 2(CCR2)axis.At a later stage,anti-inflammatory Ly6Clow monocyte subpopulations occur in infarct myocardium and participate in the resolution of the post-infarction inflammatory response.Ly6Chigh monocytes homing is necessary for necrosis cell clearance and tissue repair.Under I/R status these pro-inflammatory monocytes are excessive and thereby constitute a major cause of subsequently myocytes death and myocardium dysfunction.Thus,an optimal,but not excessively level of Ly6Chigh monocytes activation plays an important role in ischemic myocardium repair and better prognosis.Nowadays,clinicians have not achieved effective anti-inflammation therapy to suppress myocardium I/R.Endothelial cells(ECs)have been known as the maj or functional coordinator in the vascular homeostasis,especially in anti-inflammatory and antithrombotic.The inflammatory cells in peripheral blood could be regulated by ECs via paracrine under physiological status.Under pathological stress,ECs would turn toward a proinflammatory phenotype thus take part in inflammatory cell activation and homing.Krüppel-Like Factor 2(KLF2)is identified as a key "molecular switch" to mediate the ECs phenotype to maintain anti-inflammatory phenotype,which is strongly activated by laminar flow through a mechanosensory complex.The previous study illustrated KLF2-transduced ECs could exert its anti-inflammatory effect and mediate monocytes/macrophages(Mo/M?)polarization in atherosclerosis through secreting exosomes.Exosome,which is one of the main media for intercellular communication,has benefit in many aspects including the inherent capacity to pass through biological barriers and escape from phagocytosis by the reticuloendothelial system,and being biocompatible,non-toxic,and immunologically inert.Hence,whether exosomes derived from KLF2-transduced ECs could be used as a potential therapeutic agent to attenuate inflammatory depended myocardium I/R injury need to be explored.Materials and Methods1)Human umbilical vein endothelial cells(HUVECs)were cultured in laminar flow circumstance,and were evaluated in inflammation and KLF2 expression through quantitative reverse transcriptase polymerase chain reaction(qRT-PCR)and Western blot.Then,HUVECs were transduced with a lentiviral vector encoding KLF2 to build KLF2 overexpressed endothelial cells(KLF2-HUVECs).2)Exosomes(KLF2-exo)were isolated from the supernatant of KLF2-HUVECs using gradient centrifugation method,and were identified by western blot,nanoparticle trafficking analysis(NTA),and transmission electron microscopy.3)Mice underwent 45 minutes ischemia,followed by reperfusion,and KLF2-exo was administrated to mice through intravenous injection.The curative effects of KLF2exo on myocardial I/R and cardiac inflammation was evaluated.4)The modifying effects of KLF2-exo on Mo/M? subsets were evaluated in vivo by flow cytometry and immunofluorescence staining analysis.Then,we tested whether systemic depletion of Mo/M? would recapitulate the benefits of KLF2exo therapy by injecting clodronate liposomes into the tail vein of mice.5)The modifying effects of KLF2-exo on Ly6Chigh Mo/M? recruitment,polarization and apoptosis were evaluated by flow cytometry and splenectomy.6)The downstream pathways were explored by qRT-PCR and Western blot,and were further verified in vitro through transwell experiment and cell scratch wound healing assay.7)According to microRNA sequencing results of KLF2-exo and bioinformatics analysis,the microRNA was screened and further verified by miRNA functional experiments.Dual luciferase reporter assay was carried based on the bioinformatics prediction of microRNA target genes.8)We inihited microRNA in KLF2-exo by applying microRNA antagomir to KLF2HUVECs,and tested whether the benefits of KLF2-exo therapy would be recapitulated.Results1)KLF2 was strongly activated by laminar shear stress and conferred its antiinflammatory effects on ECs.We transduced HUVECs with a lentivirus vector encoding KLF2,resulting in a remarkable increase of KLF2 expression,and KLF2HUVECs could mimic the anti-inflammatory phenotype as that in ECs under physiological laminar flow.2)KLF2-exo with lipid bilayer membrane structure,within 30-150nm in diameter expressing specific surface marker were successfully isolated from culture medium using ultracentrifugation method.3)Intravenously injection of exosomes derived from KLF2-transduced HUVECs immediately after reperfusion could improve cardiac function,reduce infarct size,inhibit myocardium fibrosis and ameliorate inflammation.4)KLF2-exo reduced inflammation level after I/R injury by diminishing Ly6Chigh Mo/M? and systemic depletion of Mo/M? obliterated the efficacy of KLF2-exo therapy.5)The protection of KLF2-exo was conserved after splenectomy.KLF2-exo inhibited Ly6Chigh monocytes recruitment from bone marrow through inhibiting CCR2 expression in vivo.6)KLF2-exo restrained RAW264.7 cells migration through inhibiting CCR2 expression in vitro.7)Compared to vec-exo,miRNA24-3p was candidate effector of KLF2-exo mediated Ly6Chigh monocytes recruitment and RAW264.7 cells migration through downregulating CCR2 expression,while inhibiting miRNA24-3p in KLF2-exo blunted those effects.8)miRNA24-3p antagomir abrogated the effect of KLF2-exo on ameliorating myocardial I/R injury in vivo.ConclusionsExosomes derived from KLF2 overexpressed ECs attenuate myocardial I/R injury and reduce cardiac inflammation via restraining the Ly6Chigh monocytes recruitment from the bone marrow.miRNA24-3p packaged in exosomes is involved in the modulation of Ly6Chigh monocytes recruitment by targeting CCR2 expression.