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Ex Vivo Corneal Organ Culture Model for Wound Healing Studies
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Proteolytic processing of connective tissue growth factor in normal ocular tissues and during corneal wound healing

Paulette M Robinson1, Tyler S Smith, Dilan Patel

  • 1Department of Obstetrics and Gynecology, Institute for Wound Research, University of Florida, Gainesville, Florida 32610-0294, USA

Investigative Ophthalmology & Visual Science
|November 10, 2012
PubMed
Abstract

Insights

Connective tissue growth factor (CTGF) is processed into smaller fragments during corneal wound healing. This proteolytic processing of CTGF is crucial for regulating scar formation in the cornea.

Area of Science:

  • Ophthalmology
  • Cell Biology
  • Biochemistry

Background:

  • Connective tissue growth factor (CTGF) is a fibrogenic cytokine.
  • CTGF is upregulated by TGF-β and mediates key fibrotic actions, including extracellular matrix synthesis and fibroblast differentiation into myofibroblasts.

Purpose of the Study:

  • To investigate the role of proteolytic processing of CTGF.
  • Examine CTGF processing in human corneal fibroblasts (HCF) stimulated with TGF-β, normal ocular tissues, and wounded corneas.

Main Methods:

  • Western blot analysis of CTGF processing in HCF cultures, animal eyes, and wounded rat corneas.
  • Tandem mass spectrometry to identify a 21-kDa CTGF fragment.
  • 5' Rapid Amplification of cDNA Ends (RACE) to assess CTGF splice variants.

Main Results:

  • TGF-β-stimulated HCF produced CTGF fragments (25, 21, 18, 13 kDa) alongside full-length 38-kDa CTGF.
  • A 21-kDa CTGF fragment containing the middle hinge region was identified in conditioned medium.
  • Normal ocular tissues showed abundant C-terminal 25- and 21-kDa CTGF fragments.
  • All CTGF forms (38, 25, 21 kDa) were present during rat corneal wound healing, peaking on day 11.

Conclusions:

  • Proteolytic processing of 38-kDa CTGF occurs during corneal wound healing.
  • This processing has significant implications for the regulation of corneal scar formation.

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