March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Rac Inhibition Stimulates Fibroblastic Transformation Of Corneal Keratocytes In 3-D Collagen Matrices
Author Affiliations & Notes
  • Matthew Petroll
    Ophthalmology, Univ Texas Southwestern Med Ctr, Dallas, Texas
  • Neema Lakshman
    Ophthalmology, Univ Texas Southwestern Med Ctr, Dallas, Texas
  • Footnotes
    Commercial Relationships  Matthew Petroll, None; Neema Lakshman, None
  • Footnotes
    Support  NIH Grants R01 EY013322 and P30 EY020799, and Research to Prevent Blindness, Inc.
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1082. doi:
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      Matthew Petroll, Neema Lakshman; Rac Inhibition Stimulates Fibroblastic Transformation Of Corneal Keratocytes In 3-D Collagen Matrices. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1082.

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

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The small GTPases Rho and Rac are central players in the regulation of cytoskelatal organization in a variety of cell types. The goal of this study was to investigate the role of Rac1 in regulating corneal keratocyte phenotypes in response to specific wound healing cytokines.


Rabbit corneal keratocytes were seeded within 3-D collagen matrices that were compacted using external compression. Buttons (6 mm diameter) were punched out and cultured in serum-free media, PDGF BB, IGF, FGF2 or TGFβ1, with or without the Rac1 inhibitor NSC23766 (50 μM) and/or the Rho kinase inhibitor Y-27632 (10 μM). After 1 to 4 days, cells were labeled for F-actin and α-SM-actin, and imaged using confocal microscopy.


Corneal keratocytes in basal media within compressed matrices exhibited a broad, convoluted cell body and thin dendritic processes. Exposure to PDGF and IGF produced keratocyte elongation, without inducing stress fiber formation. In contrast, cells cultured in FGF2 lost their dendritic extensions and developed intracellular stress fibers. TGFβ induced formation of stress fibers expressing α-smooth muscle actin, suggesting a myofibroblastic phenotype. Addition of NSC23766 to basal media (Fig. 1), PDGF (Fig. 2) or IGF resulted in a loss of dendritic processes and formation of stress fibers at both 1 and 4 days of culture. NSC23766 also enhanced stress fiber formation in FGF, and increased the proportion of cells expressing α-SM-actin in TGFβ. Treatment with Y-27632 blocked the induction of stress fibers under all conditions studied.


Taken together, the results suggest that Rac1 plays a critical role in maintaining the quiescent corneal keratocyte phenotype, whereas Rho kinase is required for fibroblastic and myofibroblastic transformation of these cells.  


Keywords: cornea: stroma and keratocytes • cytoskeleton • growth factors/growth factor receptors 

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