W Matthew Petroll1, Lisha Ma, Linda Ly, Mridula Vishwanath. 1. Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, TX 75390-9057, USA. matthew.petroll@utsouthwestern.edu
Abstract
PURPOSE: To determine the structural and subcellular mechanical effects of Rho activation on corneal fibroblasts in three-dimensional collagen matrices. METHODS: Human corneal fibroblasts were plated at low density in 100-microm thick fibrillar collagen matrices and cultured for 1 or 2 days in serum-free media. Time-lapse imaging was then performed at 1- to 2-minute intervals with Nomarski differential interference contrast. After 1 hour, perfusion was switched to serum-free media containing 1 micromol/L lysophosphatidic acid (LPA). After an additional 30 to 60 minutes, the Rho kinase (ROCK) inhibitor Y-27632 was added to the perfusion media. Changes in cell structure and extracellular matrix deformation were measured with MetaMorph. RESULTS: Addition of LPA activated Rho and induced retraction of cell processes and cellular contraction, as indicated by decreases in cell length (-12.1%+/-7.0%; P<0.05) and cell area (-13.1%+/-13.5%; P=0.06). Force generation was greatest along the cell body in all cases, as indicated by the location of maximum extracellular matrix compression. Subsequent addition of Y-27632 resulted in relaxation of extracellular matrix stress, and reextension of cellular processes. CONCLUSIONS: The data show that Rho induces rapid contraction of corneal fibroblasts in three-dimensional collagen matrices. Forces are generated primarily along the cell body through a ROCK-dependent mechanism.
PURPOSE: To determine the structural and subcellular mechanical effects of Rho activation on corneal fibroblasts in three-dimensional collagen matrices. METHODS:Human corneal fibroblasts were plated at low density in 100-microm thick fibrillar collagen matrices and cultured for 1 or 2 days in serum-free media. Time-lapse imaging was then performed at 1- to 2-minute intervals with Nomarski differential interference contrast. After 1 hour, perfusion was switched to serum-free media containing 1 micromol/L lysophosphatidic acid (LPA). After an additional 30 to 60 minutes, the Rho kinase (ROCK) inhibitor Y-27632 was added to the perfusion media. Changes in cell structure and extracellular matrix deformation were measured with MetaMorph. RESULTS: Addition of LPA activated Rho and induced retraction of cell processes and cellular contraction, as indicated by decreases in cell length (-12.1%+/-7.0%; P<0.05) and cell area (-13.1%+/-13.5%; P=0.06). Force generation was greatest along the cell body in all cases, as indicated by the location of maximum extracellular matrix compression. Subsequent addition of Y-27632 resulted in relaxation of extracellular matrix stress, and reextension of cellular processes. CONCLUSIONS: The data show that Rho induces rapid contraction of corneal fibroblasts in three-dimensional collagen matrices. Forces are generated primarily along the cell body through a ROCK-dependent mechanism.