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(Investigative Ophthalmology and Visual Science. 2004;45:1109-1116.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0660
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Involvement of CTGF in TGF-ß1–Stimulation of Myofibroblast Differentiation and Collagen Matrix Contraction in the Presence of Mechanical Stress

Qian Garrett,1 Peng T. Khaw,1 Timothy D. Blalock,2 Gregory S. Schultz,2 Gary R. Grotendorst,3 and Julie T. Daniels1

1From the Wound Healing Research Unit, Institute of Ophthalmology, London, United Kingdom; the 2Institute for Wound Research, University of Florida, Gainesville, Florida; and the 3Department of Anatomy and Cell Biology, University of Miami, Miami, Florida.

PURPOSE. This study was undertaken to investigate the role of connective tissue growth factor (CTGF) in fibroblast-to-myofibroblast differentiation and fibroblast-mediated collagen matrix contraction in the presence of mechanical stress.

METHODS. An in vitro three-dimensional contraction model of human corneal-fibroblast–seeded collagen lattices (FSCLs) in the presence of mechanical stress generated by attaching the lattices to the culture well was used to measure FSCL contraction. FSCLs were treated with CTGF; TGF-ß1; serum-free (SF) control medium; or TGF-ß1 plus antisense oligodeoxynucleotides to CTGF; TGF-ß1 plus scrambled-sequence oligodeoxynucleotide to CTGF; or TGF-ß antibody. Expression of {alpha}-smooth muscle actin ({alpha}-SMA) by fibroblasts in FSCLs was detected by immunostaining and confocal microscopy, whereas ELISA was used for the fibroblasts cultured on plastic. The conditioned media were analyzed by ELISA for CTGF production.

RESULTS. Exogenous CTGF stimulated significantly less collagen matrix contraction and myofibroblast differentiation than TGF-ß1, but similar to that stimulated by SF. TGF-ß1 stimulated fibroblasts to express CTGF. CTGF antisense oligodeoxynucleotide inhibited TGF-ß1–stimulated myofibroblast differentiation and FSCL contraction. Exogenous CTGF circumvented the inhibitory effects of CTGF antisense on FSCL contraction. TGF-ß antibody significantly inhibited FSCL contraction and myofibroblast differentiation under mechanical stress and SF control conditions.

CONCLUSIONS. In the presence of mechanical stress, CTGF is necessary for TGF-ß1–stimulation of myofibroblast differentiation and subsequent collagen matrix contraction, but CTGF alone is not sufficient to induce myofibroblast differentiation and collagen matrix contraction. Thus, TGF-ß1 appears to regulate multiple genes that are essential for fibroblast-mediated contraction of stressed matrix, one of which is CTGF.




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