Involvement of CTGF in TGF-beta1-stimulation of myofibroblast differentiation and collagen matrix contraction in the presence of mechanical stress.
OBJECTIVE
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-beta1; serum-free (SF) control medium; or TGF-beta1 plus antisense oligodeoxynucleotides to CTGF; TGF-beta1 plus scrambled-sequence oligodeoxynucleotide to CTGF; or TGF-beta 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-beta1, but similar to that stimulated by SF. TGF-beta1 stimulated fibroblasts to express CTGF. CTGF antisense oligodeoxynucleotide inhibited TGF-beta1-stimulated myofibroblast differentiation and FSCL contraction. Exogenous CTGF circumvented the inhibitory effects of CTGF antisense on FSCL contraction. TGF-beta 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-beta1-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-beta1 appears to regulate multiple genes that are essential for fibroblast-mediated contraction of stressed matrix, one of which is CTGF.
Garrett
Qian
Q
Wound Healing Research Unit, Institute of Ophthalmology, London, United Kingdom.
Khaw
Peng T
PT
Blalock
Timothy D
TD
Schultz
Gregory S
GS
Grotendorst
Gary R
GR
Daniels
Julie T
JT
D000199:Actins / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects*; D003094:Collagen / Q000378:metabolism*; D055513:Connective Tissue Growth Factor; D003315:Cornea / Q000166:cytology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D007124:Immunoenzyme Techniques; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D018613:Microscopy, Confocal; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D013314:Stress, Mechanical; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
D000199:Actins; C526317:CCN2 protein, human; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D016376:Oligonucleotides, Antisense; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D003094:Collagen