Human corneal fibrosis: an in vitro model.
OBJECTIVE
Corneal injury may ultimately lead to a scar by way of corneal fibrosis, which is characterized by the presence of myofibroblasts and improper deposition of extracellular matrix (ECM) components. TGF-beta1 is known to stimulate overproduction and deposition of ECM components. Previously, an in vitro three-dimensional (3-D) model of a corneal stroma was developed by using primary human corneal fibroblasts (HCFs) stimulated with stable vitamin C (VitC). This model mimics corneal development. The authors postulate that with the addition of TGF-beta1, a 3-D corneal scar model can be generated.


METHODS
HCFs were grown in four media conditions for 4 or 8 weeks: VitC only; VitC+TGF-beta1 for the entire time; VitC+TGF-beta1 for 1 week, then VitC only for 3 or 7 weeks; and VitC for 4 weeks, then VitC+TGF-beta1 for 4 weeks. Cultures were analyzed with TEM and indirect immunofluorescence.


RESULTS
Compared with the control, addition of TGF-beta1 increased construct thickness significantly, with maximum increase in constructs with TGF-beta1 present for the entire time-2.1- to 3.2-fold at 4 and 8 weeks, respectively. In all TGF-beta-treated cultures, cells became long and flat, numerous filamentous cells were seen, collagen levels increased, and long collagen fibrils were visible. Smooth muscle actin, cellular fibronectin, and type III collagen expression all appeared to increase. Cultures between weeks 4 and 8 showed minimal differences.


CONCLUSIONS
Human corneal fibroblasts stimulated by VitC and TGF-beta1 appear to generate a model that resembles processes observed in human corneal fibrosis. This model should be useful in examining matrix deposition and assembly in a wound-healing situation.
Karamichos
Dimitris
D
Department of Ophthalmology, Harvard Medical School, Schepens Eye Research Institute, Boston, Massachusetts 02114, USA.
Guo
Xiaoqing Q
XQ
Hutcheon
Audrey E K
AE
Zieske
James D
JD
D000199:Actins / Q000378:metabolism; D001205:Ascorbic Acid / Q000494:pharmacology; D002452:Cell Count; D002478:Cells, Cultured; D024061:Collagen Type III / Q000378:metabolism; D003319:Corneal Stroma / Q000473:pathology*; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005353:Fibronectins / Q000378:metabolism; D005355:Fibrosis / Q000473:pathology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
D000199:Actins; D024061:Collagen Type III; D005353:Fibronectins; D053773:Transforming Growth Factor beta1; D001205:Ascorbic Acid