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Royce Mohan, Santosh Keshipeddy, Dennis Wright, Paola Bargagna-Mohan; A Novel Vimentin-Targeting Probe for Imaging Ocular Fibrosis. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1284.
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© ARVO (1962-2015); The Authors (2016-present)
Vimentin is a binding target for the small molecule withaferin A (WFA). Here we have synthesized a novel WFA fluorescent analog to investigate the dynamic motility of vimentin filaments in cell cultures and fibrotic expression of vimentin in vivo.
WFA was synthetically appended with a fluorescent tag to develop the imaging probe WFA-BODIPY-FL. In cell labeling assays, WFA-BODIPY-FL was added for 1 h, washed and cultured for 18 h. Cells were fixed and counter stained with antibody against serine 38 phosphorylated vimentin (pSer38vim). In cell spreading assays, rabbit corneal fibroblasts were trypsinized and plated on cover slips. After 1 h, WFA-BODIPY-FL (100 nM to 1 μM) was added to the cells for 5 min in serum-free medium, cells washed and allowed to spread. Serial images were collected every 5 seconds for 10 mins. For in vivo studies, 129Svev mice were subjected to the alkali injury model (JBC 2012, 287:989-1006) to promote corneal fibrosis. At 14 days post injury mice were injected with a single intraperitoneal injection of WFA-BODIPY-FL and sacrificed after 1 h. Cryosections from mouse eyes were fixed and stained with antibody against vimentin.
We demonstrate that WFA-BODIPY-FL labels vimentin intracellularly efficiently at 250 nM. Cells labeled for 18 h showed WFA-BODIPY-FL fluorescence overlapped with antibody staining for pSer38vim. In cell spreading assays, depolymerized soluble vimentin shows an abundance of fluorescence around the perinuclear region. Short squiggle- and dot-like vimentin structures showed bidirectional movement and increasingly extended towards the cell periphery. Some dot-like structures moved at rapid speeds of 0.5 micrometers/sec corroborating data from published transfection studies that used hybrid vimentin-green fluorescent protein expression. Finally, in injured mice injected with WFA-BODIPY-FL we found intense fluorescence in corneal myofibroblasts that co-stained for vimentin. Normal uninjured mice injected with WFA-BODIPY-FL did not show this intense green fluorescence in the corneal keratocytes.
We have developed a first-in-class in vivo imaging probe for studying the dynamic organization of vimentin filaments. WFA-BODIPY-FL is also bioavailable in mice and shows remarkable localization of myofibroblasts in fibrotic corneas. This biomarker-imaging reagent will be very useful as a diagnostic tool in ophthalmic applications for disease and injury-related fibrosis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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