Imaging Withaferin A‐Vimentin Interactions in Live Cells and Fibrotic Tissues

Abstract

The objective of this study was to illuminate the targeting of vimentin by the anti‐fibrotic natural product withaferin A (WFA). Here we have developed a novel WFA‐BODIPY‐FL imaging reagent (WFA‐Verde) to probe the functions of type III intermediate filament (IF) protein vimentin, a prominent biomarker of fibrotic cells and invasive cancer cells. WFA‐Verde exhibits high cell permeability demonstrating rapid entry into cultures of baby hamster kidney‐1 (BHK‐1) cells and rabbit corneal fibroblasts (RCFs). The staining pattern of WFA‐Verde in RCFs cells treated for 5 min and then fixed showed diffuse cytoplasmic fluorescence, which over time showed temporal incorporation of the probe into the filamentous vimentin forms by 30–60 min post treatment. Since WFA targets soluble vimentin and causes increased phosphorylation at serine 38 residue, we investigated also the staining characteristics of RCFs treated with drug‐like higher concentrations of WFA‐Verde. Subconfluent RCFs treated for 1 h, washed and left in culture medium showed recovery of cell shape and cytoskeleton by 18 hours. Cells stained for phosphorylated serine 38 vimentin showed extensive overlap with BODIPY‐labeled fluorescence that clearly illuminated individual filaments throughout the cell cytoplasm. Next, to investigate early kinetics of polymerization of soluble vimentin into filamentous cytoskeleton we employed a cell spreading assay. BHK‐1 cells were trypsinized, plated for 1 h and treated with 250 nM WFA‐Verde for 5 min, washed and immediately imaged every 5 seconds for 10–12 mins. In these live‐cell imaging experiments we found the bulk of soluble vimentin labeled was perinuclear and newly formed filaments precursors (represented by dots, squiggles and looping structures) showed dynamic movements similar to that reported with hybrid cDNA constructs containing green fluorescent protein‐vimentin in transfection studies. Dots‐like structures moved bilaterally and some dots moved at speeds of ~0.5 micrometers/sec, which is in the range previously reported. Since WFA exhibits potent ant‐fibrotic activity by targeting type III IFs, we next investigated whether WFA‐Verde is also incorporated into fibrotic tissues in vivo. Employing a model of ocular alkali‐injury that results in corneal fibrosis due to abundant overexpression of vimentin in myofibroblasts, we investigated the in vivo delivery of WFA‐Verde on corneal staining in eyes of uninjured mice and from those injured for 14 days. The fluorescent reagent within 1 hour of delivery achieved tissue permeabilization and accumulated abundantly in corneal myofibroblasts that overexpress vimentin as corroborated by staining overlap with anti‐vimentin antibody as well as anti‐phosphorylated serine 38 vimentin antibody. The uninjured eye sections due to very low expression of vimentin did not show comparable strong WFA‐Verde fluorescence as that observed with injured eyes. Dose response studies and time course investigations are currently being conducted to optimize target labeling. Thus, in conclusion we have developed the first‐in‐class imaging probe for vimentin that binds and labels vimentin in cells and in vivo. The use of WFA‐Verde as an imaging reagent for fibrosis biomarker detection in live animals is being currently pursued as this will afford us a non‐invasive means to image disease and injury ultimately in humans.Support or Funding InformationThis work was funded by NIH grant R01EY16782 and by the John A. and Florence Mattern Solomon Endowed Chair in Vision Biology and Eye Diseases.