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Macromolecular Differences in the Extracellular Matrix Between Normal and Keratoconus Human Corneas: An Ex Vivo Study
Murilo B Peres1, Joyce L Covre1, Larissa R da Rosa1
1Departamento de Oftalmologia e Ciências Visuais da Universidade Federal de São Paulo (Unifesp), São Paulo, Brazil.
Purpose:
To analyze proteoglycans and other extracellular matrix macromolecules from normal (NL) and keratoconus (KC) human corneas.
Methods:
Corneas from eye banks (NL) and penetrating keratoplasty (KC) were obtained. Halves of the corneas were fixed and prepared for immunofluorescence using confocal microscopy; the other halves were subjected to macromolecule extraction with GuHCl for protein quantification, macromolecule identification by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) and western blotting, and quantification by enzyme-linked immunosorbent assay (ELISA).
Results:
In the study population (N = 8 for NL and N = 12 for KC), there was no difference in sex, laterality, wet weight, or preservation time, but only in age. Western blotting detected keratan sulfate (KS), decorin, and lumican in both groups, with structural differences in decorin in the KC group. The mean protein concentration by corneal wet weight was significantly lower in the KC group. The mean concentrations of decorin, lumican, and KS did not show any difference between the groups when normalized by wet weight but showed a higher concentration in the KC group when normalized by μg of extracted protein. Immunofluorescence analysis revealed a similar macromolecular distribution among the groups.
Conclusions:
In corneas with KC, non-collagenous proteins decreased without affecting decorin, lumican, or KS levels, which were relatively increased. In addition, decorin was highly glycosylated.
