March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Distinctive Effects Of Tgfβ1, Fgf2, Igf1 And Igf2 On Growth And Extracellular Matrix Gene Expression By Primary Human Corneal Keratocytes
Author Affiliations & Notes
  • Sherri-Gae Scott
    Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
  • Shukti Chakravarti
    Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
  • Footnotes
    Commercial Relationships  Sherri-Gae Scott, None; Shukti Chakravarti, None
  • Footnotes
    Support  EY11654
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1100. doi:
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      Sherri-Gae Scott, Shukti Chakravarti; Distinctive Effects Of Tgfβ1, Fgf2, Igf1 And Igf2 On Growth And Extracellular Matrix Gene Expression By Primary Human Corneal Keratocytes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1100.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : The keratocytes are specialized mesenchymal cells, central to corneal homeostasis. Their growth and abilities to express extracellular matrix (ECM) genes during corneal development and repair are regulated by growth factor signals. Here we tested the effects of TGFβ1, FGF2, IGF-I and IGF-II on primary human keratocytes with respect to growth and expression of collagens and proteoglycans.

Methods: : Keratocytes, isolated by serial collagenase L (Sigma) digestion from transplant-unsuitable human corneas (Tissue Banks International) were cultured in DMEM/F12 with insulin, selenium, transferrin (ITS) and phosphoascorbic acid (2011, Exp. Eye Res.). After ITS depletion the keratocytes were treated with 1) FGF2 10ng/ml, 2) TGFβ1 2ng/ml, 3) FGF2 and TGFβ1, 4) IGF-I 15ng/ml and 5) IGF-II 15ng/ml. Cell growth was determined using AqueousOne (Promega) and expression of COL3A1, COL5A1, lumican (LUM), biglycan (BGN), decorin (DCN), vimentin (VIM), keratin 12 (KRT12) determined by semi-quantitative RT-PCR and qRT-PCR.

Results: : The keratocytes assumed a typical dendritic morphology in DMEM/F12+ITS and appeared fibroblastic in TGFβ1. The mesenchymal origin of keratocytes was confirmed by VIM expression and lack of epithelial KRT12. In ITS-DMEM/F12 cell growth increased by 3-fold after 24h of plating; growth was uninhibited by TGFβ1, but inhibited by FGF2. Cell growth was promoted by IGF-I and inhibited by IGF-II. Profibrogenic TGFβ1 promoted a 5-6 fold increase in COL5A1 and BGN transcripts relative to 18s RNA, while FGF2 counteracted these increases. FGF2 and TGFβ1, alone or together blocked COL3A1 expression. TGFβ1 inhibited DCN expression by 3-fold while FGF2 had no detectable effect. Expression of LUM was decreased slightly by TGFβ1, unaffected by FGF2 and promoted by IGF-I.

Conclusions: : The growth and profibrogenic effects of TGFβ1 were counteracted by FGF2. Insulin and IGF are known to promote somatic cell growth; the unexpected decrease in growth by IGF-II may be related to its higher affinity for IGF- IIR/M6P and its pro-apoptotic functions.

Keywords: cornea: stroma and keratocytes 
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