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Shay-Whey M. Koh, Jordan Celeste, C. Y. Paul Ku; Functional CNTF Receptor α Subunit Restored by Its Recombinant in Corneal Endothelial Cells in Stored Human Donor Corneas: Connexin-43 Upregulation. Invest. Ophthalmol. Vis. Sci. 2009;50(4):1801-1807. doi: 10.1167/iovs.08-2590.
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purpose. Ciliary neurotrophic factor (CNTF) is undergoing testing in human clinical trials to rescue degenerating retina, whereas studies show that the CNTF-binding α-subunit of the CNTF receptor (CNTFRα) is released from injured tissues. Here, the recombinant human (rh) CNTFRα was shown to restore functional CNTFRα in human corneal endothelial (CE) cells that lost endogenous CNTFRα during corneal storage
methods. In CE cells of stored human donor corneas, endogenous CNTFRα levels were quantified (by Western blot analysis), CNTF stimulation leading to the upregulation of connexin-43 was demonstrated, and the effectiveness of rhCNTFRα (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 (rhCNTFRα+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, rhCNTFRα incorporation into the CE membrane fraction was demonstrated by Western blot analysis
results. CE cell CNTFRα levels decreased as corneal storage time increased. CE cell connexin-43 mRNA levels in CNTF-treated and (rhCNTFRα+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). rhCNTFRα augmentation was confirmed at the protein level. In corneas with short storage times (≤9 days) that retained abundant endogenous CNTFRα, rhCNTFRα decreased the effectiveness of CNTF. rhCNTFRα was incorporated into CE membranes
conclusions. rhCNTFRα acted as a surrogate to the lost endogenous membrane-bound CNTFRα in CNTF signaling, suggesting the potential of an adjuvant rhCNTFRα therapy in CNTF-therapy.
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