Abstract
Purpose :
The intermediate filament (IF) vimentin has been associated with myofibroblast transformation of corneal keratocytes and the development of fibrosis in vivo. Studies in other systems suggest it can play a central role in regulating key aspects of cell mechanical behavior, including mechanosensing, polarization, and migration. In this study, we investigate the role of vimentin in mediating spreading and migration of corneal fibroblasts in 3D collagen matrices.
Methods :
To assess 3D cell spreading, human corneal fibroblasts were embedded in fibrillar collagen matrices. To assess 3D cell migration, compacted cell-populated collagen matrices were embedded in cell-free collagen matrices. Once polymerized, samples were cultured for 24h in serum-free media (S-), S- plus PDGF (50 ng/ml), or S- plus TGFβ (5 ng/ml). Withaferin A (WFA), a vimentin polymerization inhibitor, was added at a concentration of 2 μM based on initial dose response experiments. 3D and 4D imaging were used to assess cell-matrix mechanical interactions, global matrix contraction, and live cell spreading and migration. F-actin labeling was used to assess cell morphological changes.
Results :
During initial cell spreading in PDGF, corneal fibroblasts randomly extended and retracted dendritic processes; these protrusions disappeared over time as cells elongated. When WFA was present, cells maintained their protrusive activity but did not elongate. As shown in Figure 1, cells treated with WFA had a significant decrease in both length and area, and a less elongated morphology (as indicated by a higher shape factor). In contrast, during culture in S- or TGFβ (which do not induce cell elongation or migrational polarity), WFA induced an increase in cell length and area. Global matrix contraction in response to PDGF was reduced by 39% when WFA was present (P < 0.035), and cell migration assays showed a reduction of 40%.
Conclusions :
Our results suggest that cells cultured with WFA are unable to polarize and elongate in response to PDGF, which results in a reduction in migration through 3D collagen matrices. In addition, inhibition of cell-induced matrix reorganization by WFA suggests that polymerization of IFs is important for development of cell tractional forces. Overall, vimentin may mediate key aspects of cell mechanical activity during corneal wound healing.
This is a 2021 ARVO Annual Meeting abstract.