M. J. Nolan

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

PurposeTo correlate aqueous humor soluble CD44 (sCD44) concentration, visual field loss, and glaucoma risk factors in primary open-angle glaucoma (POAG) patients. MethodsAqueous 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. ResultsIn 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) ConclusionssCD44 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.

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.

CD44 and Primary Open Angle Glaucoma

In our view, primary open-angle glaucoma (POAG) is a common neurodegenerative disease caused by a variety of molecular defects and/or cellular insults that result in cell stress and death of the trabecular meshwork (TM) and retinal ganglion cells (RGC). One potential biological marker of POAG is CD44, which is one of the adhesion/homing molecules. Direct evidence for CD44’s very central role in POAG includes: (1) aqueous humor of patients with POAG contains an increased amount of the soluble extracellular 32-kDa fragment of CD44 (sCD44) in comparison with the aqueous humor of age-matched normal individuals; (2) increased levels of sCD44 in the aqueous correlates with the extent of visual field loss in POAG patients; (3) sCD44, particularly hypo-phosphorylated sCD44, is a potent and specific toxic protein to TM and RGC in vitro; and (4) overexpression of both full-length CD44 and truncated sCD44 in transgenic mouse eyes is sufficient to cause ocular hypertension. The increase in intraocular pressure (IOP) lasted more than 90 days accompanied by optic nerve damage. The overexpression of CD44 may thus be the first documented animal model that closely mimics the human disease POAG. Other models have been cytodestructive and nonphysiologic.