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Originally published In Press as doi:10.1167/iovs.08-2525 on November 14, 2008
 (Investigative Ophthalmology and Visual Science. 2009;50:1129-1136.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-2525
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Colocalization of Increased Transforming Growth Factor-β-Induced Protein (TGFBIp) and Clusterin in Fuchs Endothelial Corneal Dystrophy

Ula V. Jurkunas,1,2,3 Maya Bitar,2,3 and Ian Rawe2

1From the Massachusetts Eye and Ear Infirmary, Boston, Massachusetts; 2Schepens Eye Research Institute, Boston, Massachusetts; and the 3Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.

PURPOSE. To investigate the differential expression of TGFBIp in normal human and Fuchs endothelial corneal dystrophy (FECD) endothelial cell-Descemet’s membrane (HCEC-DM) complex, and to asses the structural role of TGFBIp and clusterin (CLU) in guttae formation.

METHODS. HCEC-DM complex was dissected from stroma in normal and FECD samples. Proteins were separated by 2-D gel electrophoresis and subjected to proteomic analysis. N-terminal processing of TGFBIp was detected by Western blot analysis with two separate antibodies against the N- and C-terminal regions of TGFBIp. Expression of TGFBI mRNA was compared by using real-time PCR. Subcellular localization of TGFBIp and CLU in corneal guttae was assessed by fluorescence confocal microscopy.

RESULTS. A major 68-kDa fragment and a minor 39-kDa fragment of TGFBIp were identified on 2-D gels. Western blot analysis revealed an age-dependent proteolytic processing of the TGFBIp N terminus resulting in the increased formation of 57-kDa (P = 0.04) and 39-kDa (P = 0.03) fragments in older donors. FECD HCEC-DM showed a significant increase in the 68-kDa (P = 0.04), 57-kDa (P = 0.01), and 39- kDa (P = 0.03) fragments of TGFBIp. Real-time PCR analysis revealed that TGFBI mRNA was significantly increased (P = 0.04) in FECD samples. TGFBIp formed aggregates at the lower portions of guttae, next to Descemet’s membrane, whereas CLU localized mostly on top of the TGFBIp-stained areas at the level of the endothelial cell nuclear plane.

CONCLUSIONS. The overexpression of proaggregative protein CLU, and proadhesive protein TGFBIp, have been colocalized in the guttae. Such findings provide us with a better understanding of the major contributors involved in the aberrant cell-extracellular matrix interactions seen in the guttae of patients with FECD.






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