Lactate treatment causes NF-kappaB activation and CD44 shedding in cultured trabecular meshwork cells.

PURPOSE: To challenge human trabecular meshwork (TM) cells using lactate to mimic cell stress and observe the effects on cell viability, NF-kappaB, and membrane type 1 matrix metalloproteinase (MT1-MMP) expression and the ectodomain shedding of soluble (s)CD44. METHODS: Human TM cells grown in 10% fetal calf serum (FCS) were incubated in 0.1% FCS with 1, 10, or 40 mM lactate or PBS for 5 and 30 minutes and 1, 3, and 6 hours. Cell viability was determined with trypan blue staining. NF-kappaB and MT1-MMP expression was evaluated through Western blot analysis of medium and the cytoplasmic and nuclear fractions. Media sCD44 concentration was determined by enzyme-linked immunosorbent assay and Western blot analysis. RESULTS: The TM cell viability was significantly decreased after incubation for 3 hours with 40 mM lactate (P < 0.01) and 6 hours with 10 and 40 mM lactate (P < 0.001). Western blot analysis showed an increased NF-kappaB p50 and MT1-MMP expression and activity by 5 minutes in lactate-treated TM cells compared with that of control cells. At 6 hours, NF-kappaB p65 was increased in nuclear fraction of lactate-treated compared with control cells. Treatment with 1 mM lactate caused an increase in the media concentration of both the 32 and 55 kDa sCD44 at 3 (P < 0.05) and 6 hours (P < 0.01). CONCLUSIONS: Lactate treatment resulted in dose- and time-dependent effects on human TM cell viability, translocation of NF-kappaB, and activation of MT1-MMP. Increased shedding of sCD44 occurred with the l mM dose of lactate. Lactate treatment of human TM cells in culture offers a useful cell model to examine the stress responses that occur in glaucoma.

Aqueous humor sCD44 concentration and visual field loss in primary open-angle glaucoma.

PURPOSE: To correlate aqueous humor soluble CD44 (sCD44) concentration, visual field loss, and glaucoma risk factors in primary open-angle glaucoma (POAG) patients. METHODS: Aqueous samples were obtained by paracentesis from normal and glaucoma patients who were undergoing elective surgery and analyzed for sCD44 concentration by enzyme-linked immunosorbent assay. RESULTS: In normal aqueous (n=124) the sCD44 concentration was 5.88+/-0.27 ng/mL, whereas in POAG aqueous (n=90) the sCD44 concentration was 12.76+/-0.66 ng/mL, a 2.2-fold increase (P<0.000001). In POAG patients with prior successful filtration surgery (n=13), the sCD44 concentration was decreased by 43% to 7.32+/-1.44 (P=0.001) in comparison with POAG patients without filtration surgery; however, the sCD44 concentration in the prior successful filtration subgroup with no medications and normal intraocular pressure was 12.62+/-3.81 (P=0.05) compared with normal. The sCD44 concentration of normal pressure glaucoma patients was 9.19+/-1.75 ng/mL, a 1.6-fold increase compared with normal (P=0.02). Race and intraocular pressure pulse amplitude were significant POAG risk factors in this cohort of patients. In both normal and POAG patients with mild and moderate visual field loss, sCD44 concentration was greater in African Americans than in whites (P=0.04). CONCLUSIONS: sCD44 concentration in the aqueous of POAG patients correlated with the severity of visual field loss in all stages in white patients and in mild to moderate stages in African American patients. sCD44 concentration in aqueous is a possible protein biomarker of visual field loss in POAG.

Soluble CD44 is cytotoxic to trabecular meshwork and retinal ganglion cells in vitro.

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-alpha-methyl testosterone (17-alpha-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-alpha-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.

Hypophosphorylation of aqueous humor sCD44 and primary open-angle glaucoma.

PURPOSE: The ectodomain of CD44, the principal receptor for hyaluronic acid (HA), is shed as a 32-kDa fragment-soluble CD44 (sCD44)-which is cytotoxic to trabecular meshwork (TM) cells and retinal ganglion cells (RGCs) in culture. The purpose of this study was to characterize sCD44 further by determining the phosphorylation of aqueous humor sCD44 in normal and primary open-angle glaucoma (POAG). METHODS: Aqueous humor samples of patients were subjected to CD44 enzyme-linked immunosorbent assay (ELISA) and two-dimensional (2-D) polyacrylamide gel electrophoresis, followed by Western blot analysis with anti-CD44, anti-serine/threonine, and anti-tyrosine phosphospecific antibodies, to determine sCD44 concentration, isoelectric point (pI), and phosphorylation, respectively. The bioactivity of hypophosphorylated sCD44 was tested in cell culture and HA affinity columns. RESULTS: Two-dimensional Western blot analysis revealed that the representative pI of the 32-kDa sCD44 was 6.96 +/- 0.07 in POAG versus 6.38 +/- 0.08 in normal (P < 0.0004). Enzymatic dephosphorylation of sCD44 resulted in a basic shift in the pI. The normal aqueous humor sCD44 was positive for serine-threonine phosphorylation; however, POAG sCD44 was hypophosphorylated. Hypophosphorylated sCD44 was more toxic to TM and RGC cells than standard sCD44, and hypophosphorylated sCD44 had decreased affinity to HA, particularly with increased pressure. CONCLUSIONS: POAG aqueous is characterized by posttranslational change in the pI of sCD44 and hypophosphorylation, which clearly distinguished POAG from normal aqueous humor. The high toxicity and low HA-binding affinity of hypophosphorylated sCD44 may represent specific pathophysiologic features of the POAG disease process.

Reconstitution of trabecular meshwork GAGs: influence of hyaluronic acid and chondroitin sulfate on flow rates.

PURPOSE: This study was undertaken to determine whether the concentration of hyaluronic acid (HA) and of chondroitin sulfate (CS) occurring in the normal and the primary open-angle glaucoma (POAG) trabecular meshwork (TM) influences flow rates in vitro as a function of pressure. METHODS: We tested 100, 500, and 4000 kDa molecular weight HA, CS, reconstituted normal and POAG TM HA-CS and juxtacanalicular connective tissue (JCT) HA-CS in a micro test chamber to determine initial and steady-state flow rates. The resistance and permeability (Ko) were calculated; Linear Newtonian mechanics were used to determine the possible contributions of the hydrophobic interactions of HA. RESULTS: Initial flow rates increased in the pressure range of 5 to 20 mm Hg for the three HA preparations and the flow rates declined in the pressure range of 20 to 40 mm Hg. Flow rates of reconstituted normal TM and JCT were optimum at 10 mm Hg and then declined with increasing pressure. Flow rates of reconstituted POAG TM and JCT were optimum only at 5 mm Hg and then declined. The steady-state rate of POAG JCT HA-CS at 10 mm Hg was slow: the transition time (ie, the time required to start an increase in flow rate) was 29 hours and the lag time (ie, the time required to obtain steady-state flow rate) was 17 hours. The maximum flow rate in POAG JCT HA-CS decreased by 37.2% from the normal JCT HA-CS. The calculated resistance of reconstituted POAG JCT HA-CS was approximately 18% of the total resistance of the human JCT compared with 10% in the normal JCT. CONCLUSIONS: Hyaluronic acid and CS contribute to flow resistance and influence flow rate in vitro. The influence of HA is particularly sensitive to an increase in the pressure gradient, which may be caused by unfolding of the hydrophobic interactions of HA polymers that further entangles the HA polymer. The POAG JCT HA-CS concentrations represent a significant factor in outflow resistance in POAG, particularly at higher pressures.

sCD44 internalization in human trabecular meshwork cells.

PURPOSE: To determine whether soluble CD44 (sCD44), a likely biomarker of primary open-angle glaucoma (POAG), is internalized in cultured human trabecular meshwork (TM) cells and trafficked to mitochondria. METHODS: In vitro, 32-kD sCD44 was isolated from human sera, biotinylated, and dephosphorylated. TM cells were incubated for 1 hour at 4°C with biotinylated albumin (b-albumin), biotin-labeled sCD44 (b-sCD44), or hypophosphorylated biotin-labeled sCD44 (-p b-sCD44) in the presence or absence of unlabeled sCD44, hyaluronic acid (HA), and a selected 10-mer HA binding peptide. The slides were warmed for 1 or 2 hours at 37°C, and 125 nM MitoTracker Red was added for the last 20 minutes of the incubation. The cells were washed, fixed, incubated with anti-biotin antibody and FITC-labeled goat anti-mouse antibody, and examined under a confocal microscope. RESULTS: TM cell membranes were positive for b-sCD44 after 4°C incubation. When the temperature was raised to 37°C, b-sCD44 or -p b-sCD44 appeared in the cytoplasm. The internalization of b-sCD44 was blocked by excess unlabeled sCD44, HA, and a 10-mer HA-binding peptide. Double label experiments with b-sCD44 or -p b-sCD44 and MitoTracker Red indicated partial overlap. The percent co-localization of MitoTracker Red at 2 hours and FITC -p b-sCD44 was 17.4% (P < 0.001) and for FITC b-sCD44 was 11.7% (P < 0.001) compared with b-albumin. The influence of putative CD44 phosphorylation sites on mitochondrial trafficking was determined by TargetP 1.1. CONCLUSIONS: sCD44 is internalized by TM cells and trafficked in part to mitochondria, which may be a factor in the toxicity of sCD44 in the POAG disease process.