June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
ECM stiffness regulates the myofibroblastic differentiation of cultured primary keratocytes via subcellular changes in contractility and focal adhesion assembly.
Author Affiliations & Notes
  • Daniel Maruri
    The Department of Bioengineering, The University of Texas at Dallas, Richardson, Texas, United States
  • Miguel Miron-Mendoza
    The Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • David Schmidtke
    The Department of Bioengineering, The University of Texas at Dallas, Richardson, Texas, United States
  • Matthew Petroll
    The Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Victor Varner
    The Department of Bioengineering, The University of Texas at Dallas, Richardson, Texas, United States
  • Footnotes
    Commercial Relationships   Daniel Maruri, None; Miguel Miron-Mendoza, None; David Schmidtke, None; Matthew Petroll, None; Victor Varner, None
  • Footnotes
    Support  NIH Grant R01EY030190, P30 EY030413, and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 935. doi:
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      Daniel Maruri, Miguel Miron-Mendoza, David Schmidtke, Matthew Petroll, Victor Varner; ECM stiffness regulates the myofibroblastic differentiation of cultured primary keratocytes via subcellular changes in contractility and focal adhesion assembly.. Invest. Ophthalmol. Vis. Sci. 2021;62(8):935.

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

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Abstract

Purpose : Previous studies have suggested that focal adhesion size is correlated with the stiffness of ECM, but it remains unclear how signaling downstream of focal adhesion formation modulates changes in corneal keratocyte morphology and mechanical activation.

Methods : Polyacrylamide (PA) hydrogels of varying stiffness were fabricated on glass coverslips, functionalized with type I collagen, and used as substrata for two-dimensional (2D) cell culture. The gels were plated with primary corneal keratocytes (NRKs) in either serum-free media or media containing exogenous TGF-β1. In some experiments focal adhesion kinase (FAK) inhibitor (PF-573228) was also added to the culture media. Afterward, NRKs were fixed and stained for F-actin, as well as markers for myofibroblastic activation (α-SMA), contractility (pMLC), or focal adhesions (vinculin). Focal adhesion size and traction stresses exerted by NRKs were also measured.

Results : Treatment with TGF-β1 elicited distinct cellular phenotypes when NRKs were cultured on gels of varying stiffness. Cells cultured on either stiff (10 kPa) PA gels or collagen-coated glass coverslips formed abundant stress fibers, exhibited elevated levels of α-SMA immunofluorescence, and exerted large traction forces. These cells also formed abundant focal adhesions distributed across the entire cell body. NRKs cultured on soft (1 kPa) substrata, however, exhibited behaviors more indicative of a quiescent phenotype, even in the presence of TGF-β1. They formed fewer stress fibers, retained a more elongated morphology, and exerted significantly lower traction forces, with small focal adhesions, localized primarily at the tips of cellular projections. pMLC immunofluorescence further revealed stiffness-dependent differences in subcellular contractility, with contractions localized in the tips of cellular projections in cells cultured on soft substrata. Traction force maps correlated strongly with these patterns of pMLC immunofluorescence. FAK inhibition blocked myofibroblast transformation and the associated phenotypic changes.

Conclusions : Taken together, these data suggest that mechanotransductive signaling downstream of FAK activation is required for TGF-β1 induced myofibroblast transformation of corneal keratocytes.

This is a 2021 ARVO Annual Meeting abstract.

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