TY - JOUR
T1 - TGF-β3 stimulates stromal matrix assembly by human corneal keratocyte-like cells
AU - Karamichos, Dimitrios
AU - Rich, Celeste B.
AU - Zareian, Ramin
AU - Hutcheon, Audrey E.K.
AU - Ruberti, Jeffrey W.
AU - Trinkaus-Randall, Vickery
AU - Zieske, James D.
N1 - Funding Information:
The concepts and algorithms discussed above were developed under the sponsorship of USAF Rome Air Development Center, Contract F30602-79-C-0249.
PY - 2013
Y1 - 2013
N2 - PURPOSE. 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.
AB - PURPOSE. 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.
KW - Extracellular matrix
KW - Human corneal keratocytes
KW - TGF-β3
UR - http://www.scopus.com/inward/record.url?scp=84885199331&partnerID=8YFLogxK
U2 - 10.1167/iovs.13-12861
DO - 10.1167/iovs.13-12861
M3 - Article
C2 - 24022012
AN - SCOPUS:84885199331
SN - 0146-0404
VL - 54
SP - 6612
EP - 6619
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 10
ER -