June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
The effects of vimentin knockout on corneal fibroblast mechanical behavior in 3D collagen matrices
Author Affiliations & Notes
  • Miguel Miron Mendoza
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Emi Nakahara
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Meet Paresh Bhatt
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • John Hulleman
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Matthew Petroll
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Footnotes
    Commercial Relationships   Miguel Miron Mendoza None; Emi Nakahara None; Meet Bhatt None; John Hulleman None; Matthew Petroll None
  • Footnotes
    Support  NIH R01 EY013322 and P30 EY030413
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 105 – A0203. doi:
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    • Get Citation

      Miguel Miron Mendoza, Emi Nakahara, Meet Paresh Bhatt, John Hulleman, Matthew Petroll; The effects of vimentin knockout on corneal fibroblast mechanical behavior in 3D collagen matrices. Invest. Ophthalmol. Vis. Sci. 2022;63(7):105 – A0203.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The intermediate filament vimentin has been linked to myofibroblast transformation of corneal keratocytes and the development of fibrosis during wound healing. Studies in other systems suggest it can also regulate key aspects of cell mechanical activity such as polarization, mechanosensing and focal adhesion dynamics. In this study, we investigate the role of vimentin on corneal fibroblast spreading in 3D culture.

Methods : A CRISPR/Cas9 RNP targeting vimentin was electroporated into human corneal fibroblasts, followed by screening to identify vimentin KO cell lines. Vimentin KO was verified by Western blotting and immunostaining. To assess cell spreading and mechanical activity, cells were embedded in 3D fibrillar collagen matrices. Samples were cultured for 4h or 24h in defined serum-free media (S-) or S- supplemented with PDGF BB (to stimulate cell spreading). 3D and 4D DIC imaging were used to assess cell mechanical behavior, global matrix contraction and live cell spreading. F-actin labeling was used to assess cytoskeletal organization and morphological changes.

Results : Both control (clone F11) and vimentin KO (clones F5 and G12) cells were able to spread and elongate within 3D collagen matrices in response to PDGF. Vimentin KO cells produced a similar percentage of matrix contraction as control cells (PDGF control = 33.3 ± 9.8; and PDGF KO = 37.2 ± 6.4; P = 0.89), and this was significantly higher than in S- media (S- control = 15.2 ± 3.0, P = 0.049 compared to PDGF; and S- KO = 11.4 ± 12.3, P < 0.01 compared to PDGF). Live cell imaging showed that during initial cell spreading in PDGF, all cell lines formed dendritic cell extensions that generated localized tractional forces by displacing collagen fibers. While the number of dendritic cell processes was similar (control = 15.3 ± 1.0; and KO = 13.2 ± 3.0, P = 0.08), motility and turnover of dendritic cell processes was more rapid in KO cells.

Conclusions : Corneal fibroblasts are able to spread, elongate and generate tractional forces in response to PDGF in the absence of vimentin within 3D collagen matrices. However, the increased activity and turnover of dendritic processes suggest underlying differences in the mechanics of cell spreading. The KO cell lines developed here should allow the role of vimentin in modulating corneal fibroblast behavior to be further assessed using a range of culture conditions and mechanical assays.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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