Functional CNTF receptor alpha subunit restored by its recombinant in corneal endothelial cells in stored human donor corneas: connexin-43 upregulation.
OBJECTIVE
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 (CNTFRalpha) is released from injured tissues. Here, the recombinant human (rh) CNTFRalpha was shown to restore functional CNTFRalpha in human corneal endothelial (CE) cells that lost endogenous CNTFRalpha during corneal storage


METHODS
In CE cells of stored human donor corneas, endogenous CNTFRalpha levels were quantified (by Western blot analysis), CNTF stimulation leading to the upregulation of connexin-43 was demonstrated, and the effectiveness of rhCNTFRalpha (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 (rhCNTFRalpha+CNTF)-treated (24 hours, 37 degrees 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, rhCNTFRalpha incorporation into the CE membrane fraction was demonstrated by Western blot analysis


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


CONCLUSIONS
rhCNTFRalpha acted as a surrogate to the lost endogenous membrane-bound CNTFRalpha in CNTF signaling, suggesting the potential of an adjuvant rhCNTFRalpha therapy in CNTF-therapy.
Koh
Shay-Whey M
SW
Department of Ophthalmology and Visual Sciences, University of Maryland, Baltimore, Maryland, USA. skoh@umaryland.edu
Celeste
Jordan
J
Ku
C Y Paul
CY
D015153:Blotting, Western; D002457:Cell Extracts; D002462:Cell Membrane; D002809:Chondroitin Sulfates; D020934:Ciliary Neurotrophic Factor / Q000378:metabolism / Q000494:pharmacology; D053739:Ciliary Neurotrophic Factor Receptor alpha Subunit / Q000502:physiology*; D045424:Complex Mixtures; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism; D015925:Cryopreservation; D016895:Culture Media, Serum-Free; D003911:Dextrans; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D005839:Gentamicins; D006801:Humans; D009926:Organ Preservation; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014019:Tissue Donors; D015854:Up-Regulation
D002457:Cell Extracts; D020934:Ciliary Neurotrophic Factor; D053739:Ciliary Neurotrophic Factor Receptor alpha Subunit; D045424:Complex Mixtures; D018031:Connexin 43; D016895:Culture Media, Serum-Free; D003911:Dextrans; D005839:Gentamicins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002809:Chondroitin Sulfates; C075736:Optisol