Visualizing Intracellular Autophagy Flux During Endothelial Injury With A Small Molecular Fluorescent Probe

Objective:Autophagy is a homeostatic cellular process that plays a role in the metabolic adaptation,clearance of damaged organelles and serves to control protein quality.To assess the translational relevance in treating autophagy-related diseases,it is vital to have a reliable assay for the real-time and specific tracing of the disturbed autophagy flux under pathological contexts of vascular diseases.To elucidate the detailed molecular mechanisms of autophagy and its role in various pathophysiological processes,we have developed a small-molecule probe(AFG-1).METHOD:The photophysical properties of AFG-1 was studied by using chemical methods,including the sensitivity and specificity.Immunofluorescence(IF)was used to evaluate the changes of autophagy machinery protein.Real-time changes of autophagy flux in endothelial cells following starvation were recorded by time-lapse recording.RESULT:Chemical analysis showed that AFG-1 itself in PBS(pH 7.4,100 mM)was almost non-fluorescent.The fluorescent intensity enhancement of 15-fold could be observed in the presence of peroxynitrite or acidify the solution to PH 4.0,indicating that the fluorescence of AFG-1 could indeed be activated by a two-step stimulation of sequential peroxynitrite/oxidation and acid protonation.Sensitivity and specificity of AFG-1 in detection of autophagy were further verified.A time dependent accumulation of AFG-1 fluorescence was observed in EA.hy926 endothelial cells over 1 to 12 h following nutrient deprivation.And the sensitivity could be further enhanced following treatment by Bafilomycin A1,3-Methyladenine or si-ATG 5.To further confirm that AFG-1 is a sensitive probe for peroxynitrite,endothelial cells were treated with uric acid or FeTTPs to deplete peroxynitrite.In these treated cells,starvation induced much less AFG-1 fluorescence.In addition,AFG-1 fluorescence was colocalized with the RFP-LC3 staining in EA.hy926 endothelial cells indicating that AFG-1 is a sensitive probe for autophagy.Time-lapse recording demonstrated that AFG-1 could monitor dynamic change of autophagy flux following starvation in endothelial cells.CONCLUSION:A small-molecule synthetic fluorescent probe was reported for the specific visualization of autophagy flux.The probe can only fluoresce first by peroxynitrite stimulation and then by protonation under acid context,two sequential features characterizing autophagy.More importantly,the probe AFG-1 can visualize the dynamic changes of autophagy flux in a temporal and spatial manner with high-resolution images during endothelial injury.