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Dimitrios Karamichos, Celeste B. Rich, Ramin Zareian, Audrey E. K. Hutcheon, Jeffrey W. Ruberti, Vickery Trinkaus-Randall, James D. Zieske; TGF-β3 Stimulates Stromal Matrix Assembly by Human Corneal Keratocyte-Like Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(10):6612-6619. doi: https://doi.org/10.1167/iovs.13-12861.
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We have previously shown that TGF-β3 (T3) stimulates extracellular matrix (ECM) assembly while maintaining antifibrotic characteristics in a model using human corneal fibroblasts (HCFs). This model, however, requires non-physiological levels of serum. In the current study, we tested whether T3 could stimulate human corneal keratocytes (HCKs) in vitro to assemble a functional ECM, while maintaining their characteristics.
Human corneal keratocytes and HCFs were isolated and cultured using 1% or 10% serum, respectively ±T3. The constructs were processed for indirect immunofluorescence (IF), transmission electron microscopy (TEM), and qRT-PCR, analyzing for keratocyte marker, keratocan, and ECM components, collagen (col) types I, III, and V.
Quantitative reverse transcriptase PCR data showed that keratocan, col I, and V were all upregulated in HCKs compared with HCFs, whereas col III was expressed at low levels in HCKs. Transforming growth factor beta 3 stimulation further enhanced the level of change. Without T3, HCK constructs were very thin, approximately 5 μm; however, as with HCFs, upon stimulation with T3, HCK constructs increased in thickness by approximately 5-fold. Cell counts and ECM production revealed that HCKs assembled more ECM per unit area compared with HCFs, and IF revealed downregulation of fibrotic markers, col III, and thrombospondin-1, with T3 stimulation. Transmission electron microscopy data revealed aligned ECM with long fibrils for all conditions except HCK Controls. Human corneal keratocytes+T3 also showed denser collagen fibrils with more consistent fibril diameter.
Overall, the data suggests that it is possible to stimulate matrix secretion and assembly by HCKs in vitro by using a single growth factor, T3.
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