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Originally published In Press as doi:10.1167/iovs.08-2590 on December 5, 2008
 (Investigative Ophthalmology and Visual Science. 2009;50:1801-1807.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-2590
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Functional CNTF Receptor {alpha} Subunit Restored by Its Recombinant in Corneal Endothelial Cells in Stored Human Donor Corneas: Connexin-43 Upregulation

Shay-Whey M. Koh,1,2 Jordan Celeste,1 and C. Y. Paul Ku1

1From the Departments of Ophthalmology and Visual Sciences and 2Physiology, University of Maryland, Baltimore, Maryland.

PURPOSE. Ciliary neurotrophic factor (CNTF) is undergoing testing in human clinical trials to rescue degenerating retina, whereas studies show that the CNTF-binding {alpha}-subunit of the CNTF receptor (CNTFR{alpha}) is released from injured tissues. Here, the recombinant human (rh) CNTFR{alpha} was shown to restore functional CNTFR{alpha} in human corneal endothelial (CE) cells that lost endogenous CNTFR{alpha} during corneal storage

METHODS. In CE cells of stored human donor corneas, endogenous CNTFR{alpha} levels were quantified (by Western blot analysis), CNTF stimulation leading to the upregulation of connexin-43 was demonstrated, and the effectiveness of rhCNTFR{alpha} (8.3 nM) in augmenting the CNTF (0.83 nM) effect was tested. Paired human donor corneas were used as vehicle versus CNTF-treated or CNTF- versus (rhCNTFR{alpha}+CNTF)-treated (24 hours, 37°C), followed by analysis of CE cell connexin-43 mRNA and protein by semiquantitative RT-PCR and Western blot analysis, respectively. After 90-minute incubation with stored human corneas, rhCNTFR{alpha} incorporation into the CE membrane fraction was demonstrated by Western blot analysis

RESULTS. CE cell CNTFR{alpha} levels decreased as corneal storage time increased. CE cell connexin-43 mRNA levels in CNTF-treated and (rhCNTFR{alpha}+CNTF)-treated paired corneas averaged (mean ± SEM) 0.26 ± 0.08 and 0.58 ± 0.21, respectively (P = 0.029; eight pairs; storage time ≥25 days). rhCNTFR{alpha} augmentation was confirmed at the protein level. In corneas with short storage times (≤9 days) that retained abundant endogenous CNTFR{alpha}, rhCNTFR{alpha} decreased the effectiveness of CNTF. rhCNTFR{alpha} was incorporated into CE membranes

CONCLUSIONS. rhCNTFR{alpha} acted as a surrogate to the lost endogenous membrane-bound CNTFR{alpha} in CNTF signaling, suggesting the potential of an adjuvant rhCNTFR{alpha} therapy in CNTF-therapy.






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