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1From the Laboratory for Oculo-Cerebrospinal Investigation, Division of Neurosurgery, Children’s Memorial Medical Center, and the 5Department of Ophthalmology, Northwestern University Medical School, Chicago, Illinois; the 2Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois; 3Alcon Research, Ltd., Fort Worth, Texas; and the 4University of North Texas Health Science Center, Fort Worth, Texas.
PURPOSE. Current glaucoma research targets neuroprotective therapies for retinal ganglion cells (RGCs) in primary open-angle glaucoma (POAG). The purpose of this study was to determine whether the 32-kDa ectodomain fragment of CD44—soluble CD44 (sCD44)—which is increased in the aqueous of patients with POAG, affects RGC and trabecular meshwork (TM) cell survival in vitro.
METHODS. sCD44 was isolated from human or fetal calf serum (FCS) by urea solubilization and immunoprecipitation. A transformed rat RGC-like cell line (RGC-5), human and bovine TM cells, and control cells were grown in Dulbecco’s modified Eagle’s medium containing 10% FCS until confluent and then were incubated in medium containing 0.1% FCS and treated with various doses of purified sCD44 and 17-
-methyl testosterone (17--MT). The cytotoxicity of sCD44 was verified by heat-inactivation, pretreatment with a pan-caspase inhibitor, and coadministration of anti-CD44 neutralizing antibody or hyaluronic acid (HA). Cell viability was assessed by trypan blue staining, cell counting, and phase-contrast microscopy.
RESULTS. There was a statistically significant dose- and time-dependent decrease in the number of cells and viability in the RGC-5 and TM cells treated with sCD44. Within 12 hours of sCD44 treatment, RGC-5 and TM cells displayed cell rounding, detachment, and swelling. sCD44-induced cell death was cell specific. Smooth muscle cells were resistant to sCD44, whereas human cortical neuronal-like cells were susceptible to sCD44 after 24 hours, but recovered. The cytotoxicity of sCD44 was blocked by heat-inactivation, pretreatment with a pan-caspase inhibitor, or coadministration of anti-CD44 antibody or HA. 17--MT prevented sCD44 cytotoxicity in both RGC-5 and TM cells.
CONCLUSIONS. The results indicate that exogenous sCD44 adversely affects RGC-5 and TM cell survival in vitro by activating proapoptotic pathways.
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