Abstract
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.
Ciliary neurotrophic factor (CNTF) was discovered in an extract of eye tissues consisting of ciliary body, iris, and choroid and was characterized as a survival factor for chick ciliary ganglion neurons.
1 2 It has since been shown to exert neurotrophic activities in various neuronal injury models, including axotomy-induced motor neuron degeneration and retinal ganglion cell apoptosis in vivo.
3 4 Recently, because the beneficial effects of CNTF treatment have been observed in a variety of animal models of photoreceptor cell degeneration,
5 a phase 1 (safety) human clinical trial was conducted in which encapsulated cells engineered to secrete CNTF were implanted into the vitreous cavity of the eyes of patients with retinitis pigmentosa.
6 Although the precise CNTF targets have not been identified, this therapy demonstrated a trend of beneficial effects on the visual outcome of the participants. Because CNTFRα, the CNTF-binding subunit of the CNTF receptor complex, has been localized to retinal pigment epithelial cells, rods and cones, inner nuclear cells, and retinal ganglion cells,
7 these cells are potential targets of CNTF therapy. However, to the best of our knowledge, the status of CNTFRα in the CNTF therapy-targeted tissues has never been addressed. It is a distinct possibility that CNTFRα is lost during the progression of degenerative eye diseases, which CNTF therapy is designed to treat, and the inclusion of CNTFRα in CNTF therapy may further improve the outcome. This possibility must be addressed given that phase 2 and 3 clinical trials are under way or have been conducted to test the efficacy of CNTF in the treatment of macular degeneration, retinitis pigmentosa, Usher type 2 and 3, and choroideremia.
8 9 10 11
Although the mechanism has not been established, studies describe the circumstances in which CNTFRα is lost from the tissues under stress. CNTFRα is lost from the fibers of the optic nerve after optic nerve injury, which may explain why CNTF is not effective in promoting regeneration of the injured optic nerve.
12 Although CNTF does not have a signal peptide sequence for secretion and has been postulated as an injury factor released only after injury
13 and CNTFRα lacks a transmembrane domain and is anchored to the cell membrane through a glycosylphosphatidylinositol anchor,
14 we have previously demonstrated that CNTF is released in a complex with CNTFRα by sublethal oxidative stress-injured corneal endothelial (CE) cells in corneal organ cultures.
15 In addition, CNTFRα is released by the skeletal muscle in response to peripheral nerve injury.
16 Furthermore, CNTFRα has been detected in cerebrospinal fluid samples of patients with degenerative diseases of the central nervous system,
16 and the level detected in the urine of patients with amyotrophic lateral sclerosis is 4.4-fold that of healthy persons.
17
Recombinant human (rh) CNTFRα has been shown to bind with CNTF at a 1:1 stoichiometry and transduces signal as the membrane-anchored CNTFRα.
18 CNTF in combination with CNTFRα (but not alone) transduces signals upregulating connexin-43 expression in glioma and astrocytes, cells that do not express CNTFRα.
19 20 The present study was designed to demonstrate that rhCNTFRα can restore in cells with diminished endogenous CNTFRα functional levels of CNTFRα, augmenting the effect of CNTF on these cells.
The corneal endothelium, a neural crest-derived tissue,
21 22 consists of a single cell layer of CE cells. CE cells in fresh human donor corneas express CNTFRα.
23 As a common clinical practice, human donor corneas for transplantation are stored in the preservation medium in the eye bank (for only a few days) before they are transplanted into recipient eyes. We have found in CE cells in stored human donor corneas a functional CNTF/CNTFRα signaling pathway that induces the expression of vasoactive intestinal peptide,
23 which is a trophic
24 and differentiation-maintaining factor of CE cells.
25 We have also found that the level of CE cell CNTFRα gradually diminishes during human donor cornea storage. The diminishing level of CNTFRα in stored human donor corneas in which the CE cells remain viable provides a unique opportunity to examine whether functional CNTFRα can be restored by rhCNTFRα.
Whereas CNTF/CNTFRα signaling leads to the upregulation of connexin-43 expression in astrocytes and in C6 glioma cells,
18 19 connexin-43 is expressed in the corneal endothelium of rat,
26 27 rabbit,
28 and human.
29 Recently, connexin-43 has been shown to play an important role in wound repair of the corneal endothelium.
30 In the present study we demonstrated that stimulation of the CNTF/CNTFRα signaling pathway in the corneal endothelium also upregulated connexin-43 expression and that, in the presence of rhCNTFRα, CE cells that lost endogenous CNTFRα during human donor cornea storage were more responsive to stimulation by CNTF.
Media used were as follows: medium A—Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, and 20 mM HEPES; medium B—DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, and 0.292 mg/mL l-glutamine; complete medium B—medium B plus 5% fetal calf serum and fungizone (250 ng/mL amphotericin; Invitrogen, Grand Island, NY).
Viable human corneoscleral explants (human donor corneas) stored in medium (Optisol-GS; Bausch & Lomb Surgical, Irvine, CA) at 4°C were obtained from the Lions Eye Institute for Transplant and Research (Tampa, FL). These corneas were deemed not suitable for transplantation because of their less than optimal CE cell densities and the advanced ages of the donors. In addition, using the same procedure as that used by the eye bank, fresh human donor corneoscleral explants were retrieved from cadavers (within 30 hours of death) in the Anatomy Board of the State of Maryland (Baltimore, MD). Cadavers were deidentified and were not considered human subjects by the Human Research Protection Office of the University of Maryland School of Medicine.
Corneal endothelium was scraped from the corneas using a razor blade and homogenized in a glass homogenizer in the solubilization buffer containing 50 mM HEPES, pH 7.2, 10% glycerol, 1.25% triton X-100, 300 mM NaCl, 0.2 mM EGTA, 0.2 mM MgSO4, 100 μM sodium orthovanadate, and protease inhibitor cocktail (one tablet of Complete Mini [Roche Diagnostics, Mannheim, Germany] per 10 mL buffer).
Samples of CE cell extracts and membrane preparation were electrophoresed under reducing conditions using preformed Tris/glycine polyacrylamide gradient gels (NuPage; Novex, San Diego, CA) and electrophoretically transferred to nitrocellulose membranes. For the detection of CNTFRα, nitrocellulose membranes were immunostained with an affinity-purified goat anti-human CNTFRα primary antibody (catalog no. AF-303-NA; R&D Systems) and an anti-goat IgG-alkaline phosphatase conjugate secondary antibody (catalog no. 401512; Calbiochem, La Jolla, CA). CNTFRα on nitrocellulose membranes was detected by a chromogenic method using an alkaline phosphatase substrate solution made from tablets (Fast Red TR/Naphthol AS-MX; Sigma, St Louis, MO). For the detection of connexin-43 and actin (internal standard), chemiluminescence was used with affinity-purified rabbit anti-connexin-43 (catalog no. AB19012; Chemicon, Temecula CA) and mouse monoclonal anti-actin (catalog no. CP01; Ab-1; Calbiochem, La Jolla, CA) primary antibodies, horseradish peroxidase-linked anti-mouse and anti-rabbit IgG secondary antibody, and horseradish peroxidase substrate (ECL kit; Amersham Pharmacia, Piscataway, NJ).
rhCNTFRα Augmentation of CNTF-Upregulated Connexin-43 mRNA Expression in CE Cells of Human Donor Corneas Stored in Storage Medium
Increased CE Cell Membrane-Bound CNTFRα Found in Long-term Stored Human Donor Corneas after Short-term Incubation with rhCNTFRα