title: Stromal edema in klf4 conditional null mouse cornea is associated with altered collagen fibril organization and reduced proteoglycans.
abstract: OBJECTIVE
  Klf4, one of the highly expressed transcription factors in the mouse cornea, plays an important role in maturation and maintenance of the ocular surface. In this study, the structure and proteoglycan composition of the Klf4 conditional null (Klf4CN) corneal stroma was investigated, to further characterize the previously reported Klf4CN stromal edema.
  
  
  METHODS
  Collagen fibril spacing and diameter were calculated from scattering intensity profiles from small angle synchrotron x-ray scattering patterns obtained across the cornea along a vertical meridian at 0.5-mm intervals. Collagen fibril organization and proteoglycans were visualized by electron microscopy (EM), with or without the cationic dye cuprolinic blue. Proteoglycans and glycosaminoglycans were further analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and immunoblot analysis. Q-RT-PCR was used to measure the transcript levels.
  
  
  RESULTS
  In the central cornea, the average collagen interfibrillar Bragg spacing increased from 44.5 nm (SD +/-1.8) in wild-type to 66.5 nm (SD +/-2.3) in Klf4CN, as measured by x-ray scattering and confirmed by EM. Mean collagen fibril diameter increased from 32 nm (SD +/-0.4) in wild-type to 42.3 nm (SD +/-4.8) in Klf4CN corneal stroma. Downregulation of proteoglycans detected by EM in the Klf4CN stroma was confirmed by FACE and immunoblot analysis. Q-RT-PCR showed that, whereas the Klf4CN corneal proteoglycan transcript levels remained unchanged, matrix metalloproteinase (MMP) transcript levels were significantly upregulated.
  
  
  CONCLUSIONS
  The Klf4CN corneal stromal edema is characterized by increased collagen interfibrillar spacing and increased diameter of individual fibrils. The stroma also exhibits reduced interfibrillar proteoglycans throughout, which is possibly caused by increased expression of MMPs.
authors.affiliation: Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff University, Cardiff, Wales, United Kingdom.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Young
authors.forename: Shivalingappa K
authors.initials: SK
authors.lastname: Swamynathan
authors.forename: Craig
authors.initials: C
authors.lastname: Boote
authors.forename: Mary
authors.initials: M
authors.lastname: Mann
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Quantock
authors.forename: Joram
authors.initials: J
authors.lastname: Piatigorsky
authors.forename: James L
authors.initials: JL
authors.lastname: Funderburgh
authors.forename: Keith M
authors.initials: KM
authors.lastname: Meek
mesh_terms: D000818:Animals; D003094:Collagen / Q000378:metabolism* / Q000648:ultrastructure; D015715:Corneal Edema / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D003319:Corneal Stroma / Q000378:metabolism* / Q000648:ultrastructure; D015536:Down-Regulation; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000502:physiology*; D015151:Immunoblotting; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics*; D051379:Mice; D008854:Microscopy, Electron; D011509:Proteoglycans / Q000378:metabolism* / Q000648:ultrastructure; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014961:X-Ray Diffraction; D016335:Zinc Fingers / Q000502:physiology
chemical_list: C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D011509:Proteoglycans; D003094:Collagen