Doxorubicin Induced The Endothelial Cells Toxicity By Interrupting The Cxosomes Secretion Pathway

Background:Chemotherapy-induced cardiotoxicity has always been a difficult problem to be solved clinically.Limited anthracyclines clinical use,however,is dose-dependent cardiotoxicity with a higher risk of disability and mortality.With the improvement of therapeutic level and treatment methods,the survival rate of patients was greatly increased.However,cardiac toxicity problems such as myocardial dysfunction and heart failure caused by anthracyclines are hidden dangers threatening the health of cancer survivors,and even may occur in patients within a few years to a decade after chemotherapy.Further,the new mechanism should be explored to counter the cardiotoxicity of anthracyclines-induced,and it is necessary for researchers to make continuous progress in cardiac protectionDoxorubicin(DOX)-induced cardiotoxicity is a process involving multiple molecules and factors.Currently,the mechanism of DOX-induced cardiotoxicity is not fully understood.Endothelial cells,located in the inner side of the vascular lumen,constitute a barrier for the communication between the inner and outer of the blood vessels.The integrity of tight junctions and the normal operation of the transport and metabolism processes are important factors for maintaining vascular homeostasis.Endothelial cells are the targets of chemotherapeutic drugs,but the pathophysiologic role and molecular signals of endothelial cells remains elusive in the process of DOX-induced cardiotoxicity.Objective:We wanted to investigate the molecular mechanism and role of exosomes-based secretion pathway in DOX-induced endothelial cytotoxicity and to provide a new notion of strategy for the clinical solution of DOX dose-dependent cardiotoxicity.Methods and results:DOX-induced endothelial cells toxicity was observed by detecting the cells survival rate using CCK-8,the apoptotic cascade related proteins using Western blot,tight junctions protein ZO-1 using immunofluorescence.Further,transcriptome analysis in the endothelial cells revealed that the characteristics genes significantly enriched in transport and metabolism of cell process.Next,we focus on the exosomes-based secretion pathway,playing a vital role in transport and metabolism of cell process and extracting the exosomes from the equal volume of the supernatant cell culture by density gradient ultracentrifugation.The exosomes-based secretion pathway was injuried after DOX-treated revealing by the number of exosomes.To investigate which factors control exosomes secretion,mutivesicular bodies(MVBs)were studied.The results by immunofluorescence show the greatly increased sizes of MVBs and characteristic asymmetrical perinuclear accumulation of CD63-positive organelles in the endothelial cells after DOX-treated.HEK293 CD63-EGFP stable cell lines transfected with Rab5-RFP and the constitutively activated Rab5-Q79L-mCherry mutant plasmids,which facilitates the study of the first steps in MVBs biogenesis.DOX did not affect the sorting of the exosomal protein CD63 into Rab5 endosomes.Bioinformatics analysis suggests that DOX inhibits the Rab27b expression level,one of a small GTPase family,which was responsible for MVBs docking to the cell membrane.Endothelial cells were treated with exosomes inhibitors GW4869,and immunoblotting showed that the aggregation of intracellular proteins increased the apoptotic events.DOX-induced apoptosis in endothelial cells is reserved by melatonin.The results of Western blotting showed that it did not directly affect the protein expression level of Rab27b,but reversed DOX-induced toxic effect in the endothelial cells by reducing the apoptosis cascade.Conclusion:Our studies identify the role of exosomes-based secretion pathway in the DOX-induced cardiotoxicity.DOX inhibits the expression level of Rab27b accounting for MVBs docking at the plasma membrane.The size of MVBs strongly increased in the endothelial cells by DOX-treated,which induced the aggregation of the proteins and accelerated the apoptotic cascade.Melatonin protects the endothelial cells from DOX-induced cardiotoxicity.