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Alison R Romisher, Alhanoof A Alnwibit, William J Vines, Yana Sharpadskaya, A Sue Menko, Janice L Walker; Hyaluronic Acid and its Receptor RHAMM in an Ex Vivo Mock Cataract Surgery Model Under Fibrotic Promoting Conditions. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3196.
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© ARVO (1962-2015); The Authors (2016-present)
Hyaluronic Acid (HA) is an extracellular matrix molecule well-known for its role in promoting fibrosis. Here, we examined expression and localization of HA and RHAMM, the receptor of hyaluronan mediated motility, in a model of mock cataract surgery cultured under conditions that promote fibrosis.
Following mock cataract surgery lens explants are flattened on a rigid tissue culture dish. Mesenchymal leader cells populate the outside wound edge and direct the injured lens epithelium onto the culture substrate, the Extra Capsular Zone (ECZ). The leader cells are induced to become myofibroblasts within 3 days and myofibroblasts persist and expand over time. Cells were fixed with formaldehyde, and the organization and distribution of HA, RHAMM, α-tubulin and vimentin determined by confocal microscopy imaging following immunofluorescence labeling; F-actin was labeled with fluorescent-conjugated phalloidin. To best resolve pericellular HA we used a formalin acetic alcohol fixative. An association between RHAMM and vimentin was investigated using the Proximal Ligation Assay (PLA), which reveals protein-protein interactions within 30-40 nm in situ.
Our results revealed that RHAMM and its ligand HA are widely distributed in both the mesenchymal leader cells and the epithelial cells that had migrated onto the ECZ. RHAMM often exhibited a punctate distribution. Interestingly, by culture day 6 when leader cells had acquired a myofibroblast phenotype, HA was highly organized in extracellular cables co-linear with actin stress fibers. Using more stringent fixative conditions we discovered that RHAMM also had a filamentous distribution that was co-localized with HA cables that appeared to follow along actin stress fibers. RHAMM did not co-distribute with microtubules in these cultures. However, we discovered that in the protrusions extended by the leader cells at the edge of the ECZ RHAMM was highly enriched and co-localized with vimentin, an intermediate filament protein linked to the progression of fibrosis. Further investigation using PLA revealed that this co-localization reflected an interaction between RHAMM and vimentin, both before and after these cells transition to myofibroblasts.
The distribution of HA and RHAMM suggests that these molecules play an important role in the regulation of leader cell function and the progression of fibrosis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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