| Objective: Exosomes that are generated through two consecutive invaginations of plasma membrane are nanosized membranous vesicles ranging from 40 to 120 nm in diameter and play important roles in cell-cell communication and exchange of signalmolecules.Unlike traditional gene delivery vectors(liposome,plasmids,viruses,etc.)that bear many defects such as toxicity,off-target phenomenon,low delivery efficiency,quickly clearance from body,short biological half-life,undesired inflammatory and immune response,and tumorigenecity,exosomes possess many characteristics such as natural,good biological distribution profile and compatibility,stability,biological barrier permeability,negligible immunogenicity,and capacity of carrying single or a combination of therapeutic agents.Naturally occurring exosomes,when given systemically,are usually trapped in liver,kidney,lungs,spleen and other organs,and were soonremoved.Exosomes displaying a target molecule on their surface usually have an increased half-life and a shortened period to deliver the therapeutic cargos in targeted organs.During exosome genesis,they selectively enrich signal molecules that reflect the nature and the pathophysiological states of the donor cells,which have been widely used in the diagnosis,treatment,and prevention of many diseases including cardiovascular disease.The aims of our study was to test whether the plasmid(p LVX-IRES-Zs Green1-m LAMP2b-CSTSMLKAC)expressing a homing peptide targeting hypoxia/reoxygenation injured cardiomyocytes can be effectively displayed on the surface of exosomes,and whether the engineered exosomes can be specifically internalized into the injured cardiomyocytes,thus providing a novel vehicle for targeting therapeutics to the ischemia injured hearttissue.Methods: HEK293 T cells were transfected with either the said plasmid or a plasmid expressing FLAG tag,respectively,using Lipofectamine 2000.After 48 hours exosomes-free media were harvested,from which exosomes were isolated using ultracentrifugation.Electron microscope,NTA,and Western blot were employed to characterize the exosomes,and immuno-coprecipitation was used to prove that FLAG tag can be displayed on the surface of exosomes.Neonatal rat cardiomyocytes(CMs)were isolated using a two-step enzyme digestion method and cultured as described.H/R-CMs was induced by incubation of cardiomyocytes with 250 ?M cobalt chloride for 12 hours,followed by 2 hours of incubation with normal complete medium.H/R-CMs model was verified by q PCR to detect the expression of hypoxia induced factor-1A and by LDH kit to determine the LDH activity in medium.The engineered exosomes were labeled by Cell Tracker TM CM-Di I(in red),which were added to normal CMs and H/R-CMs.After incubation,cells were observed under microscopy to evaluate the internalization of the labeled exosomes.Results:(1)Exosomes are spherical or cup-shaped with an average diameter close to 100 nm by electron microscopy,positive for the well-known exosome markers CD9 and CD63,and negative for endoplasmic reticulum proteins,calnexin,GRP9 by Western blot.Nano Sight tracking analysis further confirmed that most exosomes have a diameter ranging from 50-100 nm;(2)FLAG tag was successfully displayed on the surface of exosomes,whichwas confirmed by immunprecipitated with anti-FLAG antibody and immunobloted with anti-CD63 antibody recognizing CD63,a marker of exosomes.(3)CMs stimulated by cobalt chloride had a high level of HIF-1A m RNA expression compared to that of normal CMs(p < 0.01);The LDH activity was significantly increased in the medium of H/R-CMs than the normal CMs(P < 0.01);(4)Immunofluorescence microscopy showedthat particulate immunofluorence were observed in the cytosol of most H/R-CMs,but not in the normal CMs.Conclusions: Peptide can be successfully displayed on the surface of exosomes,and a peptide homing to H/R-CMs,when displayed on the surface of exosomes,facilitates the internalization of the engineered exosomes into H/R-CMs,but not the CMs under normoxia.Objective: To investigate the role and mechanism of Ecrg4 in cardiac ischemia/reperfusion injury.Methods: Myocardial ischemia-reperfusion injury models were established by ligation of left anterior descending coronary artery in vivo and in vitro by isolation of neonatal rat myocardial cells subjected to cobalt chloride treatment respectively.Real-time quantitative PCR(q PCR),Western blot and immunohistochemistry were used to detect the expression of Ecrg4 after myocardial ischemia /reperfusion injury.Luciferase activity was used to detect ECRG4 promoter's reaction to hypoxia.The expression of inflammation related genes(IL1,CD44,NF-KB)and apoptosis-related genes(Bcl2 and Bax)in hypoxia / reoxygenation cardiomyocytes were detected by q PCR.Flow cytometry was used to detect the apoptosis rate of cardiomyocytes after overexpression of ECRG4 in cardiomyocytes.Results: 1.Ecrg4 expression was rapidlydownregulated after ischemia/reperfusion injury by q PCR,Western blot and immunohistochemistry(p<0.01);2.There exists a classical hypoxia res ponse element(HRE)from-304 to-300 of ECRG4 promoter,which was highly sensitive to cobalt chloride-induced hypoxia.Mutation of the HRE significantly reduced the sensitivity of the promoter to cobalt chloride-induced hypoxia and hypoxia-inducible factor(HIF-1A)overexpression;3.Overexpression of ECRG4 down-regulated the expressi on of inflammatory related genes(IL1,CD44 and NF-KB),up-regulated the expression of anti-apoptotic gene Bcl2,and down-regulated the apoptosis-inducing gene Bax(p<0.05);4.Flow cytometry showed that the apoptosis rate of cardiomyocytes was significantly reduced after overexpression of Ecrg4 on cardiomyocytes subjected to hypoxia-reoxygenation.Conclusion: 1.Ecrg4 plays a role in cardiac reactivity to hypoxia;2.Ecrg4 may protect cardiac IRI through suppression of cardiomyocyte inflammatory response and apoptosis. |
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