The effect of various media and probe angles on the power output of the Cyclo G6 Glaucoma Laser System.

To evaluate the effect of various media and Iridex MicroPulse P3 (MP3) probe angles on the power output from the Cyclo G6 Glaucoma Laser (G6) System. A laser power meter was used to measure the power output (milliwatts, mW) of the Cyclo G6 System. Each of the ten trials consisted of measurements in six different media: no substrate, balanced salt solution (BSS), artificial tears (AT), tetracaine eye drop, lubricating ointment, and lidocaine gel. The output of the MP3 probe was measured at an angle of 90° and 45°, submerged in the respective media. The output was also measured with the probe held at 90° but above the medium. The mean power outputs with the probe being held at 90° to the sensor with no substrate, BSS, AT, tetracaine eye drop, lubricating ointment, and lidocaine gel were 358 ± 16.8 mW, 612 ± 14.2 mW, 613 ± 13.3 mW, 612 ± 14.0 mW, 620 ± 9.9 mW, and 610 ± 12.2 mW, respectively. These values were statistically higher than noncontact and 45° probe angles for each medium. The values between any two media (excluding no substrate) at a 90° probe angle with full contact were not statistically significant. The highest output of the G6 System was obtained with a 90° probe angle, with full contact and any of the coupling media.

Nailfold Capillary Hemorrhages: Microvascular Risk Factors for Primary Open-Angle Glaucoma.

BACKGROUND: Primary open-angle glaucoma (POAG) is associated with systemic microvascular dysfunction including hemorrhages and other abnormalities of the nailfold capillary bed. This study aimed to verify the specificity of nailfold capillary hemorrhages and other abnormalities as risk factors for POAG. METHODS: Nailfold video capillaroscopy was performed using a JH-1004 capillaroscope on the fourth and fifth digits of the nondominant hand in control (n = 277), POAG (n = 206), OHT (n = 57), and SG (n = 29) subjects. The number of hemorrhages, dilated capillaries >50 µm, and avascular zones ≥200 µm were counted and adjusted to counts per 100 capillaries. Descriptive analyses as well as univariate- and multivariable-adjusted logistic regression were performed comparing all groups with controls and POAG with OHT and SG. Subanalyses were conducted in POAG patients examining the association between nailfold capillary outcomes and previous glaucoma surgery, successful IOP control, or disease severity. RESULTS: All nailfold capillary outcomes were significantly increased in POAG, no outcomes were increased in SG, and only hemorrhages were mildly increased in OHT. Hemorrhages were significantly more frequent in POAG compared with both OHT (P < 0.0001) and SG (P=0.001). There were significant trends between higher numbers of hemorrhages and POAG compared with controls, OHT, and SG, with odds ratios of 18.3 (8.5-39.4), 9.1 (1.9-13.4), and 11.8 (1.7-7.3), respectively, for the presence of two or more hemorrhages per 100 capillaries. Hemorrhages were not significantly associated with previous glaucoma surgery, successful postoperative IOP control, or disease severity in POAG. CONCLUSIONS: These findings suggest that systemic microvascular dysfunction is frequent in POAG and occurs early in the disease process. The high specificity of nailfold hemorrhages makes them viable clinical risk factors for POAG.

Resting nailfold capillary blood flow in primary open-angle glaucoma.

BACKGROUND/AIMS: An altered haemodynamic profile for various ocular posterior segment capillary beds has been documented in primary open-angle glaucoma (POAG). POAG may also involve abnormal non-ocular blood flow, and the nailfold capillaries, which are not affected by elevated intraocular pressure (IOP), are readily assessable. METHODS: We measured resting nailfold capillary blood flow in 67 POAG and 63 control subjects using video capillaroscopy. Masked readers tracked blood column voids between consecutive, registered image sequence frames, measured vessel diameter and calculated blood flow. We used multiple logistic regression to investigate the relation between nailfold capillary blood flow and POAG. In secondary analyses, we stratified cases by maximum IOP and concurrent topical beta-blocker use. RESULTS: Mean (±SD) blood flow in picolitres per second was 26.8±17.6 for POAG cases and 50.1±24.2 for controls (p<0.0001). After adjustment for demographic and clinical factors including blood pressure and pulse, every picolitre per second increase in resting nailfold blood flow was associated with a 6% (95% CI 0.92 to 0.96) reduced odds of POAG (p<0.0001). Similar relations between nailfold capillary blood flow and POAG were found for cases stratified by maximum known IOP and for cases stratified by concurrent topical beta-blocker use. CONCLUSION: Reduced resting nailfold capillary blood flow is present in POAG independent of covariates such as blood pressure, pulse and IOP.

Nailfold Capillary Morphology in Alzheimer's Disease Dementia.

BACKGROUND: Cerebrovascular disease (CVD) is highly comorbid with Alzheimer's disease (AD), yet its role is not entirely understood. Nailfold video capillaroscopy (NVC) is a noninvasive method of live imaging the capillaries near the fingernail's cuticle and may help to describe further vascular contributions to AD. OBJECTIVE: To examine finger nailfold capillary morphology using NVC in subjects with AD dementia, mild cognitive impairment (MCI), and normal cognition (NC). METHODS: We evaluated nailfold capillary hemorrhages, avascular zones ≥100 microns, and degree of tortuosity in 28 NC, 15 MCI, and 18 AD dementia subjects using NVC. Tortuosity was measured with a semi-quantitative rating scale. To assess the relation between nailfold capillary morphological features and diagnostic grouping, univariate and multivariable logistic regression models were fit to the data. RESULTS: 56% of subjects with AD dementia compared to 14% with NC and 13% with MCI displayed moderate to severe tortuosity. Greater severity of tortuosity was associated with 10.6-fold (95% confidence interval [CI]: 2.4, 46.2; p = 0.0018) and 7.4-fold (95% CI: 1.3, 41.3; p = 0.023) increased odds of AD dementia relative to NC and MCI, respectively, after adjusting for multiple covariates. CONCLUSION: Greater nailfold capillary tortuosity was found in participants with AD dementia compared to those with MCI or NC. These data provide preliminary evidence of a systemic microvasculopathy in AD that may be noninvasively and inexpensively evaluated through NVC.

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Nailfold Capillary Abnormalities in Primary Open-Angle Glaucoma: A Multisite Study.

PURPOSE: There is considerable evidence for systemic vascular dysfunction in primary open-angle glaucoma (POAG). We performed nailfold capillary video microscopy to observe directly the nature of nonocular microvasculature abnormalities in POAG. METHODS: We enrolled 199 POAG patients and 124 control subjects from four sites. We used JH-1004 capillaroscopes to perform nailfold capillary video microscopy on the fourth and fifth digits of each subject's nondominant hand. Videos were evaluated for hemorrhages, dilated capillary loops > 50 µm, and avascular zones > 100 µm by graders masked to case status. Multivariable odds ratios (ORs) and 95% confidence intervals (CIs) for POAG were obtained by means of logistic regression analyses that were applied to data from all cases and controls. Corresponding estimates of moderate or severe POAG versus mild POAG (based on the Hodapp-Anderson-Parrish scale) were obtained among cases only. RESULTS: After controlling for demographic factors, family history of glaucoma, systemic diseases, and use of anticoagulation and antiplatelet therapy, for each 100 nailfold capillaries assessed, all types of microvascular abnormalities were significantly associated with POAG. Specifically, the presence of any dilated capillaries (OR = 2.9; 95% CI, 1.6-5.6), avascular zones (OR = 4.4; 95% CI, 1.7-11.3) and hemorrhages (OR = 12.2; 95% CI, 5.9-25.1) were associated with POAG. Among cases, the frequency of microvascular abnormalities was not associated with glaucoma severity (P ≥ 0.43). CONCLUSIONS: These data provided support for nonocular capillary bed abnormalities in POAG. Comparable vascular abnormalities in the optic nerve may render it susceptible to glaucomatous damage.

ABCB1 transporter and Toll-like receptor 4 in trabecular meshwork cells.

PURPOSE: The aqueous humor nourishes the avascular tissues of the anterior segment, and the trabecular meshwork (TM) plays a role in the efflux of endogenous substances and xenobiotics from the aqueous humor. ATP (ATP)-binding cassette (ABC) transporter superfamily members respond to stressors such as hypoxia, cytokine signaling, and aging. The innate immune system within the TM, particularly Toll-like receptor 4 (TLR4) and its ligands, e.g., low-molecular-weight hyaluronic acid (LMW-HA) and lipopolysaccharide (LPS), plays a significant role in maintaining a normal environment in the anterior chamber. We hypothesize that the innate immune system may interact with ATP-binding cassette sub-family members ABCB1 (p-glycoprotein and multidrug resistance protein 1) to detoxify xenobiotics from the aqueous humor and in the TM. METHODS: Cell lysates of human TM cells, RAW 264.7 macrophages, and PC12 cells were subjected to western blot analysis. The TM cells were positive for TLR4, ABCB1, and CYP3A5 and were negative for the ABCC1 transporter. Human TM cells and RAW 264.7 macrophages were plated on eight-well chamber slides at 5,000 cells/well overnight in 10% fetal bovine serum (FBS) cell growth medium. The medium was changed to 0.1% FBS 2 h before treatment. Cells were challenged with 1 and 10 mM lactate, 100 ng LMW-HA (20 kDa), 100 ng high-molecular-weight HA (HMW-HA, 1,000 kDa), 100 ng LPS, and/or 100 µM naloxone for 0.5, 1, 2, and 4 h. Calcein acetyoxymethyl ester (calcein AM; 0.25 µM) was added for 30 min as the reporting molecule. After calcein AM was administered, it was cleaved by an esterase into a fluorescent product that is normally transported out of the cell by ABCB1. Positive controls were 100 µM verapamil and 50 µM digoxin. After the challenge, the TM cells were fixed at 4 °C in 3% paraformaldehyde for 15 min, mounted with Vectashield and 4',6-diamidino-2-phenylindole (DAPI) mounting medium, and analyzed by a masked observer using a Leica confocal microscope and software. RESULTS: Verapamil, an ABCB1 inhibitor, significantly (p<0.001) increased fluorescent calcein retention in the cytoplasm of the TM and RAW 264.7 cells compared to the PBS control. Digoxin, an ABCB1 activator, increased calcein efflux (p<0.001). Lactate reduced ABCB1 activity. HMW-HA significantly (p<0.001) reduced ABCB1 activity, whereas LMW-HA decreased ABCB1 activity, and the HA effects were blocked by naloxone (p<0.001), a TLR4 inhibitor. LPS alone did not change ABCB1 activity whereas dephosphorylated LPS significantly (p<0.001) enhanced ABCB1 activity in the TM cells. ß-amyloid significantly reduced ABCB1 activity, and the ß-amyloid effects were blocked by naloxone. CONCLUSIONS: TM cells are responsive to ABCB1 inhibitors and activators. ABCB1 functional activity is affected by TLR4 agonists suggesting that modulation of TLR4 is important in ABCB1 function. The innate immune inflammatory response in the TM may play a role in the ABCB1 detoxification of potentially harmful constituents in the aqueous humor.

sCD44 overexpression increases intraocular pressure and aqueous outflow resistance.

PURPOSE: CD44 plays major roles in multiple physiologic processes. The ectodomain concentration of the CD44 receptor, soluble CD44 (sCD44), is significantly increased in the aqueous humor of primary open-angle glaucoma (POAG). The purpose of this study was to determine if adenoviral constructs of CD44 and isolated 32-kDa sCD44 change intraocular pressure (IOP) in vivo and aqueous outflow resistance in vitro. METHODS: Adenoviral constructs of human standard CD44 (Ad-CD44S), soluble CD44 (Ad-sCD44), and empty viral cDNA were injected into the vitreous of BALB/cJ mice, followed by serial IOP measurements. Overexpression of CD44S and sCD44 was verified in vitro by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Anterior segments of porcine eyes were perfused with the isolated sCD44. sCD44-treated human trabecular meshwork (TM) cells and microdissected porcine TM were examined by confocal microscopy and Optiprep density gradient with western blot analysis to determine changes in lipid raft components. RESULTS: Intravitreous injection of adenoviral constructs with either Ad-CD44S or Ad-sCD44 vectors caused prolonged ocular hypertension in mice. Eight days after vector injection, Ad-CD44S significantly elevated IOP to 28.3±1.2 mmHg (mean±SEM, n=8; p<0.001); Ad-sCD44 increased IOP to 18.5±2.6 mmHg (n=8; p<0.01), whereas the IOP of uninjected eyes was 12.7±0.2 mmHg (n=16). The IOP elevation lasted more than 50 days. Topical administration of a γ-secretase inhibitor normalized Ad-sCD44-induced elevated IOP. sCD44 levels were significantly elevated in the aqueous humor of Ad-CD44S and Ad-sCD44 eyes versus contralateral uninjected eyes (p<0.01). Anterior segment perfusion of isolated 32-kDa sCD44 significantly decreased aqueous outflow rates. Co-administration of isolated sCD44 and CD44 neutralizing antibody or of γ-secretase inhibitor significantly enhanced flow rates. sCD44-treated human TM cells displayed cross-linked actin network formation. Optiprep density gradient and western blot analysis of human TM cells treated with sCD44 showed decreased annexin 2 expression and increased phosphorylated annexin 2 and caveolin 1 expression. CONCLUSIONS: Our data suggest that sCD44 increases outflow resistance in vivo and in vitro. Viral overexpression of both CD44S and sCD44 is sufficient to cause ocular hypertension. Infusion of sCD44 in porcine anterior segment eyes significantly decreased flow rates. Notably, sCD44 enhanced cross-linked actin network formation. The elevated sCD44 levels seen in POAG aqueous humor may play an important causative role in POAG pathogenesis.

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.

Effect of chondroitin sulfate on intraocular pressure in rats.

PURPOSE: To study the effect of intracameral injections of chondroitin sulfate (CS) on intraocular pressure (IOP), retinal function, and histology in rats. METHODS: Acute or chronic injections of CS were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with vehicle. IOP was daily or weekly assessed by a tonometer. Retinal function was assessed by scotopic electroretinography (ERG) and the visual pathway by flash visual evoked potentials (VEPs), whereas the retinal and optic nerve head structure were examined by histologic analysis. RESULTS: A single injection of 8 mg (but not 2 or 4 mg) CS induced a significant increase of IOP. The increase of IOP induced by a single injection of 8 mg CS lasted for 7 days, whereas chronic (weekly) administration during 10 weeks induced a significant and sustained increase in IOP compared with eyes injected with vehicle. A significant decrease of scotopic ERG a- and b- wave amplitude was observed after 6 and 10 weeks of CS administration. Moreover, a significant decrease in scotopic flash VEP N2-P2 component amplitude was observed in eyes treated with CS for 6 and 10 weeks. A significant loss of ganglion cell layer cells and optic nerve axons was observed in eyes receiving CS for 10 weeks. CONCLUSIONS: These results suggest that exogenous CS simulates the accumulation of CS in primary open-angle glaucoma and that increased amounts of CS could play a key role in the IOP dysregulation characteristic of glaucoma.

Biomarkers of primary open-angle glaucoma.

Primary open-angle glaucoma (POAG) is a primary neuronal disease of the optic nerve without a definable cause, and is often associated with increased intraocular pressure. Worldwide, POAG is the second leading cause of blindness; there are 45 million people today with POAG and bilateral blindness is present in 4.5 million of these. In order to elucidate the possible etiologic factors in POAG, we have cataloged all known biomarkers in the aqueous humor, trabecular meshwork, optic nerve and blood into four categories, namely extracellular matrix (ECM), cell signaling molecules, aging/stress and immunity-related changes. We present a theoretical model to show possible signaling pathways of the ECM, cell signaling and innate immune response through activation of Toll-like receptor 4. Our article suggests that ECM and innate immune biomarkers are the lead candidates for developing the 'POAG biomarker signature'. We suggest that current research is critical to pinpoint the causes of the disease so that new treatment modalities can become available for better regulation of the intraocular pressure and neuroprotection of the optic nerve.

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.

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.

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.

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.

Aqueous humor in primary open-angle glaucoma contains an increased level of CD44S.

PURPOSE: To determine whether the cell adhesion molecule CD44, the principal receptor of hyaluronan, is altered in the aqueous humor and the anterior segment of patients with primary open-angle glaucoma (POAG). METHODS: The trabecular meshwork (TM), iris, ciliary body, and sclera of POAG and age-matched control eyes preserved in ethanol were microdissected and subjected to 1% Triton X-100 solubilization at 4 degrees C. Western blot analysis was performed using monoclonal antibodies that recognize either CD44H (hematopoietic; extracellular domain) or CD44S (soluble ectodomain). The concentration of soluble CD44S in aqueous and microdissected tissues was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: ELISA of soluble CD44S of aqueous from eyes of patients with POAG indicated that the concentration of soluble CD44S is increased in comparison with that of aqueous from normal eyes (P < 0.0003). Western blot analysis and densitometry of POAG iris and ciliary body revealed a statistically significant increase in the Triton X-100 extraction of CD44H. The predominant increases were in the 180-kDa (P < 0.001) and the 85-kDa (P < 0.001) forms. ELISA of soluble CD44S indicated that the concentration is statistically decreased in iris (P < 0.05), ciliary body (P < 0.001), and TM (P < 0.005) of POAG eyes. CONCLUSIONS: Increased amounts of soluble CD44S in POAG aqueous and Triton X-100-solubilized CD44H characterized POAG in the iris and ciliary body. These soluble CD44 isoforms may influence the activity of the transmembrane CD44H by acting as inhibitors of CD44H and, thereby, adversely influence the cell survival of TM and retinal ganglion cells in POAG.