title: TGF-β3 stimulates stromal matrix assembly by human corneal keratocyte-like cells.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Schepens Eye Research Institute/Massachusetts Eye and Ear and the Department of Ophthalmology Harvard Medical School, Boston, Massachusetts.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Karamichos
authors.forename: Celeste B
authors.initials: CB
authors.lastname: Rich
authors.forename: Ramin
authors.initials: R
authors.lastname: Zareian
authors.forename: Audrey E K
authors.initials: AE
authors.lastname: Hutcheon
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Ruberti
authors.forename: Vickery
authors.initials: V
authors.lastname: Trinkaus-Randall
authors.forename: James D
authors.initials: JD
authors.lastname: Zieske
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D060527:Corneal Keratocytes / Q000187:drug effects* / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D046529:Microscopy, Electron, Transmission; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053782:Transforming Growth Factor beta3 / Q000494:pharmacology*
chemical_list: D015415:Biomarkers; D053782:Transforming Growth Factor beta3
keywords: TGF-β3; extracellular matrix; human corneal keratocytes
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