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PubMed1996

Dexamethasone inhibits trabecular cell retraction.

Glucocorticosteroids such as dexamethasone (Dex) are known to cause an increased resistance to aqueous outflow in the intact and cultured eye. We investigated whether Dex treatment of cultured endothelial or trabecular meshwork (TM) cells might interfere with the cell separations and retraction induced by the facility-enhancing agents ethacrynic acid (ECA), cytochalasin B and the calcium chelator EGTA. Our hypothesis was that Dex-induced changes in the response of our model cells in vitro might serve as a paradigm for those produced in the cells of the outflow pathway, perhaps through influencing the changing dimensions of the pathway for aqueous humor through the juxtacanalicular tissue and/or inner wall of Schlemm's canal. We treated calf pulmonary artery endothelial (CPAE) and human and porcine TM cells with Dex (1-100 microns, 1-9 days), and then assessed monolayer and cytoskeletal integrity by immunofluorescence microscopy for tubulin and direct fluorescence staining for F-actin after exposure to the agents named above. We found that Dex-pretreated CPAE and TM cells gradually (over 5-7 days) became refractory to the effects of both ECA and EGTA, but not to cytochalasin B. Despite the preservation of general cell shape and attachment after ECA in Dex-treated cells, microtubule disruption still took place as in controls. Dex-treated cells also demonstrated a reorganization of filamentous actin staining after ECA and EGTA. Combination experiments of ECA and EGTA in Dex-treated cells suggested that the Dex effects were due to a greater strength of cell-to-cell and cell-to-substrate attachment, possibly due to interference with the normal cellular signaling required for coordinated cellular retraction and junctional disruption.

Actins↗
PubMed1999

Clinical features of isolated noncompaction of the ventricular myocardium: long-term clinical course, hemodynamic properties, and genetic background.

OBJECTIVES: A nationwide survey was conducted to clarify the clinical features of isolated noncompaction of the ventricular myocardium (INVM) in Japanese children in comparison with features previously described in patients with INVM. BACKGROUND: Isolated noncompaction of the ventricular myocardium is a rare disorder characterized by an excessively prominent trabecular meshwork. It is accompanied by depressed ventricular function, systemic embolism and ventricular arrhythmia. METHODS: A questionnaire specifically designed for this study was sent to 150 hospitals in Japan where a pediatric cardiology division exists. RESULTS: Twenty-seven patients were diagnosed by two-dimensional echocardiography, their ages ranging from one week to 15 years at presentation, with follow-up lasting as long as 17 years. The gross anatomical appearance and the extension of noncompacted myocardium predominantly at the apex observed on two-dimensional echocardiograms were similar to observations reported previously. Dissimilarities included a greater number of asymptomatic patients at initial presentation, a longer clinical course with gradually depressed left ventricular function, no systemic embolism, and rare ventricular tachycardia in the Japanese children. Cardiac catheterization disclosed normal left ventricular end-diastolic volume and increased left ventricular end-diastolic pressure in most cases, consistent with restrictive hemodynamics. A higher incidence of Wolff-Parkinson-White syndrome was found in the children, whereas left bundle branch block was rarer than reported in adults. Familial recurrence was high (44%) and included many women. CONCLUSIONS: In Japanese children, INVM can be found by screening examinations at asymptomatic stage, and it might have a longer dinical course with gradually depressed left ventricular function and restrictive hemodynamics. The pattern of familial recurrence we observed implies that INVM is a distinctive clinical entity with a heterogeneous genetic background.

Adolescent↗
PubMed2005

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.

Aqueous Humor↗
PubMed2003

Involvement of the Erk-MAP kinase pathway in TNFalpha regulation of trabecular matrix metalloproteinases and TIMPs.

PURPOSE: TNFalpha is a strong modulator expression of trabecular meshwork (TM) matrix metalloproteinase (MMP) and tissue inhibitor (TIMP). Laser trabeculoplasty appears to rely on this process to restore normal aqueous humor outflow facility. Thus, studies were conducted to determine whether the extracellular signal-regulated kinase (Erk)-mitogen-activated protein (MAP) kinase signal-transduction pathway is involved. METHODS: Porcine TM cells were treated with TNFalpha, and changes in MMPs and TIMPs were evaluated by zymography and Western immunoblot assay. Phosphospecific antibodies to proteins from the Erk pathway were used to evaluate responses to treatment with TNFalpha. Inhibitors of Mek, the kinase that activates Erk, and of protein kinase C (PKC) isoforms were used to define pathway involvement. RESULTS: Treatment with TNFalpha increased MMP-1, -3, and -9 and TIMP-1, whereas expression of MMP-2 was not affected and expression of TIMP-2 was decreased. Erk and Mek were rapidly phosphorylated after treatment with TNFalpha, and c-Raf-1 showed a significant bandshift. A specific inhibitor of Mek blocked the TNFalpha induction of the MMPs and TIMPs and the phosphorylation of Erk. An inhibitor of the PKC- micro isoform, which also blocks the effects of MMP-TIMP of TNFalpha, did not affect phosphorylation of Erk. CONCLUSIONS: The components of this MAP kinase pathway in the TM are dramatically affected by TNFalpha and inhibition of Erk's phosphorylation blocks the changes in MMP and TIMP expression. PKC micro, which is also required in this transduction process, does not appear to be upstream from Erk in the signaling cascade. Manipulation of this and related TM signal-transduction pathways may provide targets for developing improved glaucoma treatments.

Animals↗
PubMed2005

Configuration of the drainage angle, intraocular pressure, and optic disc cupping in subjects with chronic angle-closure glaucoma.

OBJECTIVE: To investigate the relationship between drainage angle configuration with untreated intraocular pressure (IOP) and optic disc cupping in subjects with chronic angle-closure glaucoma (CACG). DESIGN: Prospective, observational study. PARTICIPANTS: Two hundred seventy-five Asian subjects with CACG who participated in a randomized controlled trial that investigated the IOP-reducing effect of latanoprost and timolol. METHODS: Chronic angle-closure glaucoma was defined as the presence of glaucomatous optic neuropathy (with or without a visual field defect), an anterior chamber angle in which the pigmented trabecular meshwork was not visible for at least 180 degrees on gonioscopy, and evidence of peripheral anterior synechiae (PAS) in association with elevated IOP of 21 mmHg or more. Static and dynamic gonioscopy were performed, the angles were graded in each quadrant according to the Shaffer scheme, and the number of clock hours of PAS was recorded. The untreated IOP and vertical cup-to-disc ratio were correlated with mean angle width and extent of PAS. MAIN OUTCOME MEASURES: Mean angle width, clock hours of PAS, IOP, and vertical cup-to-disc ratio. RESULTS: Most subjects were female (75%), and the mean age was 62.9+/-9.4 years. The mean angle width was 0.77+/-0.53 and the mean number of clock hours of PAS was 4.77+/-3.2 hours. Untreated IOP correlated with angle width (r = -0.23; P<0.001) and clock hours of PAS (r = 0.22; P<0.001). Vertical cup-to-disc ratio also correlated with angle width (r = -0.17; P = 0.004) and PAS (r = 0.28; P<0.001). Performing a multiple linear regression using baseline IOP as the outcome variable with age, gender, clock hours of PAS, and angle width as predictors, there was a 0.39-mmHg (95% confidence interval, 0.15-0.63) increase in baseline untreated IOP for each unit increase in clock hours of PAS (P = 0.002). CONCLUSIONS: In subjects with CACG, the extent of PAS and a narrower width of the drainage angle were associated with higher untreated IOP and a larger vertical cup-to-disc ratio.

Adult↗
PubMed1998

Q-switched 532-nm Nd:YAG laser trabeculoplasty (selective laser trabeculoplasty): a multicenter, pilot, clinical study.

OBJECTIVE: To investigate the safety and efficacy of a new laser procedure using a q-switched 532-nm neodymium (Nd):YAG laser, also called "selective laser trabeculoplasty," to lower intraocular pressure (IOP) in patients with open-angle glaucoma (OAG). The laser parameters were set to selectively target pigmented trabecular meshwork (TM) cells without coagulative damage to the TM structure or nonpigmented cells. DESIGN: Nonrandomized, prospective, clinical trial. PARTICIPANTS: Thirty eyes of 30 patients with uncontrolled OAG (OAG group) and 23 eyes of 23 patients with uncontrolled OAG treated previously with argon laser trabeculoplasty (ALT group) were observed for 4 to 26 weeks. Forty-four of the 53 eyes were observed for 26 weeks. INTERVENTION: Patients were treated with the Coherent Selecta 7000 (Coherent, Inc, Palo Alto, CA) frequency-doubled q-switched Nd:YAG laser (532 nm). A total of approximately 50 nonoverlapping spots were placed over 180 degrees of the TM at energy levels ranging from 0.6 to 1.2 mJ per pulse. After surgery, patients were maintained with the identical drug regimen as that before treatment. RESULTS: Both the OAG and ALT groups showed similar IOP reductions over time. Seventy percent of patients in each group responded to treatment with an IOP reduction of least 3 mmHg. At 26 weeks of follow-up, mean IOP reduction was 5.8 mmHg (23.5%, P < 0.001) for the OAG group and 6.0 mmHg (24.2%, P < 0.001) for the ALT group. The untreated eye showed a 9.7% (P < 0.001) reduction of IOP at 26 weeks. However, the IOP difference between the treated and untreated eyes was statistically significant at P < 0.003. Transient IOP elevation of 5 mmHg or greater was seen in 24% of patients. CONCLUSION: The selective laser trabeculoplasty appears to be a safe and effective method to lower IOP in patients with OAG and patients treated previously with ALT. A reduction of IOP can be achieved without coagulation of the TM.

Adult↗
PubMed2001

Anterior chamber angle anomalies associated with signs of pigment dispersion in a group of black probands and their first-degree relatives.

PURPOSE: To evaluate the presence of iridocorneal angle anomalies associated with signs of pigment dispersion among a group of black probands and their first-degree relatives. METHODS: Eleven blacks who exhibited pigment dispersion syndrome (PDS) signs, including Krukenberg's spindles, moderate-to-heavy trabecular meshwork pigmentation, and peripheral lenticular/zonular pigmentation, received a complete eye examination that included gonioscopy. Overt iridocorneal angle abnormalities other than pigment deposition were photo-documented. Twenty-two first-degree relatives (6 siblings, 15 children, and 1 parent) of these probands underwent similar examination. RESULTS: Among the subjects, 5 of 11 probands (age range 13 to 59 years) and 4 of 22 relatives (age range 14 to 51 years) exhibited definite angle anomalies. The nine subjects with angle anomalies belonged to 7 of 11 separate families. The four relatives with angle anomalies did not have PDS according to our criteria, but two other relatives from two additional families did have signs consistent with clinical PDS. Types of angle anomalies included (1) peripheral anterior synechia-like lesions; (2) a prominent concavity of the peripheral iris at the 6-o'clock meridian; (3) a whitish, thread-like band of tissue near the iris base; (4) a prominent, externally-visible Schwalbe's line at the 6-o'clock meridian; and (5) a prominent, web-like confluence of exaggerated iris processes 360 degrees along the angle wall. Iris processes and prominent Schwalbe's lines were not considered abnormal or anomalous unless they were clearly atypical. CONCLUSIONS: Iridocorneal angle anomalies, other than pigment deposition, were common among our black group who exhibited signs of pigment dispersion as well as among their first-degree relatives. Further consideration should be given to the possibility that among some patient groups, overt goniodysgenesis could be part of a constellation of signs indicative of a pigment dispersion syndrome. Potentially, clinically detectable goniodysgenesis could prove to be a useful marker to identify certain subjects who carry PDS genetic traits but who have not expressed complete phenotypical disease.

Adolescent↗
PubMed1998

Effect of beta-adrenergic agonists on paracellular width and fluid flow across outflow pathway cells.

PURPOSE: To determine whether the adrenergic agonists epinephrine and isoproterenol regulate fluid flow across endothelial cells cultured from the human aqueous outflow pathway and to evaluate associated cellular mechanisms. METHODS: Confluent monolayers of human trabecular meshwork (TM) or Schlemm's canal endothelial (SCE) cells were grown on porous filter supports. The monolayers were perfused with media while fluid flow, expressed as hydraulic conductivity (HC = microl/min/mm Hg/cm2), was continuously measured in preparations treated with isoproterenol, epinephrine, or control medium. Morphometric ultrastructural methods were used to measure the area occupied by the intercellular space and by each cell. RESULTS: SCE cells and TM cells exposed to isoproterenol or epinephrine responded with an increase in transendothelial fluid flow. Dose-response curves for both adrenergic agonists showed that HC increased linearly as a function of the log of the isoproterenol and epinephrine concentration. At 10(-4) M isoproterenol, the HC increased threefold, and threshold conditions were reached at 10(-9) M. The increase in HC was apparent after isoproterenol had been applied for 1 hour, reached a peak in 3 to 4 hours, and declined gradually to return to baseline conditions in 10 to 12 hours. Morphometric analyses showed that the area occupied by the intercellular space increased fourfold when isoproterenol was used at 10(-4) M, whereas the cell area decreased as a function of the concentration of adrenergic agonist. Epinephrine's effects on HC and cell morphology were blocked by pretreatment with equimolar concentrations of the nonselective beta-blocker, timolol. CONCLUSIONS: Epinephrine and isoproterenol increase flow through the paracellular pathway of SCE and TM cells through a beta-receptor mediated response that widens the intercellular space and reduces cell area. These findings support the hypothesis that epinephrine decreases the intraocular pressure in glaucoma therapy by promoting fluid flow across the SCE and TM cells lining tissues of the major aqueous outflow pathway.

1-Methyl-3-isobutylxanthine↗
PubMed2004

Long-term, targeted genetic modification of the aqueous humor outflow tract coupled with noninvasive imaging of gene expression in vivo.

PURPOSE: To address a problem impeding research into glaucoma-associated genetic mutations and glaucoma gene therapy and achieve permanent, targeted transgene expression in the trabecular meshwork (TM). Lentiviral vectors are known to transduce human donor eye TM ex vivo, but efficacy in vivo has not been shown. More generally in the field of gene therapy, the authors hypothesized that distinctive properties of the intraocular aqueous circulation could facilitate solving problems of accessibility, targeting, and scale that have hindered realization of gene therapy in other settings. METHODS: A domestic cat model was developed in which long-term in vivo studies were performed. After dose-response studies in primary human TM cells, 19 cats received anterior chamber (AC) injections of stepped doses (10(6)-10(8) transduction units) of lentiviral vectors encoding different marker transgenes (beta-galactosidase, Aequorea victoria green fluorescent protein [GFP], or Renilla reniformis GFP). Animals were monitored serially for transgene expression and IOP. RESULTS: High-grade, stable transgene expression in the TM was achieved and monitored noninvasively over time in living animals. Extensive expression resulted after a single transcorneal injection, persisted for at least 10 months (time of death in the present studies), and was targeted to the TM. The initial IOP did not differ significantly from the IOP at the end of the study (P = 0.4). Aequorea GFP was superior to Renilla GFP. Vectors were effective enough to cause GFP-specific overexpression cytotoxicity at the highest dose, which was solved by dose reduction. CONCLUSIONS: High-grade transgene expression in this large-animal model persisted stably for at least 10 months after a single transcorneal lentiviral vector injection, was highly targeted, and could be monitored serially and noninvasively in living animals. These studies provide a basis for developing realistic disease models and administering glaucoma gene therapy.

Animals↗
PubMed2003

The role of protein kinase C in modulation of aqueous humor outflow facility.

The elevated intraocular pressure that is commonly associated with glaucoma is believed to arise due to impairment of trabecular meshwork (TM) function. Although the TM and Schlemm's canal (SC) comprise the major route for aqueous humor outflow, little is known about the potential signaling mechanisms involved in the regulation of aqueous outflow. Based on knowledge regarding the role of protein kinase C (PKC) in vascular biology, we sought to understand the contribution of the PKC pathway towards outflow function by studying the modulation of contractile and morphological characteristics of TM and SC cells. We investigated the involvement of PKC in regulation of myosin light chain (MLC) phosphorylation, formation of actin stress fibers and integrin-ECM adhesions (focal adhesions) in human TM and SC cells and correlated these changes with aqueous outflow facility measured in an enucleated porcine whole eye perfusion model. Expression and distribution of PKC isoforms (alpha and epsilon ) in TM and SC cells and tissues was confirmed by Western blot and immunohistochemical analysis, respectively. Both, pharmacological activators (phorbol-12-myristate 13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and inhibitors (staurosporine and GF109203X) of PKC were found to induce changes in cell shape (retraction and rounding up) and cytoskeletal organization in human TM and SC cells. While PMA and PDBu produced an increase in formation of actin stress fibers and focal adhesions and in MLC phosphorylation, PKC inhibitors were observed to induce contrasting effects in these cells. Intriguingly, both PDBU and GF109203X caused increases in aqueous outflow facility in the perfusion model. The PKC inhibitor (GF109203X) increased outflow by 46% while the PKC activator (PDBu) only increased outflow by 27%. These results suggest that PKC might play an important role in modulation of aqueous outflow facility by regulating MLC phosphorylation and thereby, the morphological and cytoskeletal characteristics of TM and SC cells.

Actins↗
PubMed2006

Functional analysis of the glaucoma-causing TIGR/myocilin protein: integrity of amino-terminal coiled-coil regions and olfactomedin homology domain is essential for extracellular adhesion and secretion.

TIGR/MYOC mutations account for 2-4% of the primary open-angle glaucoma (POAG) patients. More than 90% of the known mutations are located within its carboxy-terminus olfactomedin-homology (Olf) domain (amino acids (aa) 245-504). In vitro and in vivo studies showed that several Olf domain mutations prevented myocilin secretion. To investigate if intracellular sequestration was a characteristic feature shared by a majority of the mutations, we analyzed the secretion status of 36 myocilin variants. These encompassed 26 glaucoma-causing mutations and 10 non-disease associated or undefined polymorphisms. As several variants were found to be secreted, we tested for their adhesion to the extracellular matrix (ECM) and/or cell surface. Myocilin variants were generated by site-directed mutagenesis of a vector encoding the human MYOC cDNA. COS-7 or immortalized human trabecular meshwork cells were transfected with wild-type or mutated MYOC constructs. Myocilin levels were estimated by immunoprecipitation and/or immunoblotting. All variants showed identical behaviors in both cell lines; the truncated R46X polypeptide being the only variant which could not be detected in our assays. Of the 35 variants monitored, 20 remained sequestered intracellularly. All of them encoded disease-causing polypeptides carrying Olf domain mutations. Of the 15 variants secreted into the culture medium, six (6) were POAG mutants (of which three (3) located within the Olf domain) while the remaining nine (9) were non-disease causing or undefined polymorphisms. Three (3) of the six (6) secreted mutations caused familial POAG; these were the R126W, T377M and A427T mutants. Both, the T377M and A427T mutants located within the Olf domain. When cells were cultured at 30 degrees C, a process known to facilitate protein folding, 11 of the 20 sequestered mutants were released in the extracellular medium. Out of the 15 secreted variants tested for their adhesion to the ECM and/or cell surface, only the R82C and L95P polypeptides displayed loss of their adhesive properties. Deletion experiments revealed that the coiled-coil (aa 78-105) and leucine zipper (aa 114-183) motifs were essential for adhesion. These experiments demonstrate that intracellular sequestration might be the primary mechanism contributing to myocilin-related POAG as it was associated with more than 80% of the disease-causing mutants tested in our study. A second mechanism may involve abnormal interaction(s) between myocilin and ECM and/or cell surface proteins. Our data further revealed the importance of the olfactomedin-homology domain for myocilin secretion and the significant role of the N-terminal region for its extracellular interactions.

Amino Acids↗
PubMed1999

Effects of Na-K-2Cl cotransport regulators on outflow facility in calf and human eyes in vitro.

PURPOSE: Cultured human trabecular meshwork (TM) cells possess substantial Na-K-Cl activity, which is involved in the regulation of TM cell volume. The hypothesis in the present study was that drugs that affect the cotransporter might alter aqueous humor outflow facility (C) in the intact eye. The effects of agents and conditions known to modulate Na-K-CI cotransport activity and/or TM cell volume on C in perfused anterior segments were investigated. METHODS: Human and calf eyes were dissected and perfused, and C was determined according to standard published methods. Perfusates with modified osmolarity were used to cause alterations in TM cell volume. Cl-free perfusate and/or bumetanide (10(-5) M) was used to inhibit Na-K-Cl cotransport activity, and vasopressin (10(-7) M, 10(-8) M) was used to stimulate cotransport activity. RESULTS: In human eyes, hypo-osmotic perfusate decreased C 12%, whereas hyper-osmotic perfusate increased C 44%. These changes lasted approximately 30 minutes, after which C began to normalize. Inhibition of Na-K-Cl cotransport using Cl-free medium or bumetanide resulted in facility increases of 27% and 22%, respectively. There was an additive increase in C with bumetanide plus Cl-free media. Stimulating Na-K-Cl cotransport with 10(-8)M and 10(-7)M vasopressin resulted in 28% and 35% decreases in C, respectively. The results were similar in calf eyes: Cl-free medium or bumetanide resulted in 41% and 52% increases in C, whereas 10(-8) M and 10(-7) M vasopressin resulted in 14% and 19% decreases in C, respectively. CONCLUSIONS: Modulation of Na-K-Cl cotransport results in changes in C that may be mediated in part by cell volume changes.

Aged↗
PubMed1999

Ganglion cell losses underlying visual field defects from experimental glaucoma.

PURPOSE: To investigate the relationship between ganglion cell losses and visual field defects caused by glaucoma. METHODS: Behavioral perimetry and histology data were obtained from 10 rhesus monkeys with unilateral experimental glaucoma that was induced by argon laser treatments to their trabecular meshwork. After significant visual field defects had developed, the retinas were collected for histologic analysis. The ganglion cells were counted by light microscopy in cresyl violet-stained retina sections, and the percentage of ganglion cell loss (treated to control eye counts) was compared with the depth of visual field defect (treated to control eye thresholds) at corresponding retinal and perimetry test locations. Sensitivity losses as a function of ganglion cell losses were analyzed for Goldmann III, white and Goldmann V, and short- and long-wavelength perimetry test stimuli. RESULTS: The relationship between the proportional losses of ganglion cells and visual sensitivity, measured with either white or colored stimuli, was nonlinear. With white stimuli, the visual sensitivity losses were relatively constant (approximately 6 dB) for ganglion cell losses of less than 30% to 50%, and then with greater amounts of cell loss the visual defects were more systematically related to ganglion cell loss (approximately 0.42 dB/percent cell loss). The forms of the neural-sensitivity relationships for visual defects measured with short- or long-wavelength perimetry stimuli were similar when the visual thresholds were normalized to compensate for differences in expected normal thresholds for white and colored perimetry stimuli. CONCLUSIONS: Current perimetry regimens with either white or monochromatic stimuli do not provide a useful estimate of ganglion cell loss until a substantial proportion have died. The variance in ganglion cell loss is large for mild defects that would be diagnostic of early glaucoma and for visual field locations near the fovea where sensitivity losses occur relatively late in the disease process. The neural-sensitivity relationships were essentially identical for both white and monochromatic test stimuli, and it therefore seems unlikely that the higher sensitivity for detecting glaucoma with monochromatic stimuli is based on the size-dependent susceptibility of ganglion cells to injury from glaucoma.

Animals↗
PubMed1998

Isolation, culture, and characterization of endothelial cells from Schlemm's canal.

PURPOSE: An important goal in glaucoma research has been to understand the functional contribution of trabecular meshwork (TM) and Schlemm's canal (SC) endothelia to aqueous humor outflow resistance. To date, TM cells are routinely cultured and used as a model by several laboratories. However, there has been only limited success in isolating SC cells. The current objective was to develop a technique for selective isolation and culture of endothelial cells from human SC. METHODS: The anterior chamber of human cadaveric eyes was cut into eight equal and radially symmetric wedge-shaped pieces. Using a dissecting microscope, a gelatin-coated suture (6-0 sterile nylon monofilament) was gently inserted into the lumen of SC and advanced into the canal. The cannulated pieces of tissue were placed in culture medium and maintained for 3 weeks. Sutures were removed from SC and cells seeded onto 3-cm culture plates. Morphology, growth characteristics, and expression of endothelial surface antigens and other cellular markers were evaluated. RESULTS: Of the 20 pairs of eyes that were cannulated, primary cells were obtained from 13. All SC cell isolates had a fusiform morphology; formed nonoverlapping, linearly arranged monolayers; and were contact inhibited. Schlemm's canal cell isolates reacted with antibodies specific for CD44 (hyaluron receptor), CD54 (intercellular adhesion molecule-1, ICAM-1), tissue-type plasminogen activator, and TM-inducible glucocorticoid-responsive protein-myocilin (TIGR-MYOC). Unlike TM cells, however, TIGR-MYOC protein was not induced in SC cells after long-term dexamethasone treatment. Schlemm's canal cells endocytosed low-density lipoprotein and acetylated low-density lipoprotein, and in the presence of Matrigel organized into multicellular tubelike structures. CONCLUSIONS: Cannulation of SC with gelatin-coated suture material is an effective method for the isolation of human SC cells and provides a cellular model to study the potential role of SC cells in aqueous humor outflow function.

Adult↗
PubMed2001

Modulation of aqueous humor outflow facility by the Rho kinase-specific inhibitor Y-27632.

PURPOSE: The goal of this study was to investigate the role of Rho kinase in the modulation of aqueous humor outflow facility. Rho kinase, a critical downstream effector of Rho GTPase is recognized to control the formation of actin stress fibers, focal adhesions, and cellular contraction. METHODS: Expression of Rho GTPase, Rho kinase, and other downstream targets of Rho GTPase were determined in human trabecular meshwork (HTM) and Schlemm's canal (SC) primary cell cultures by Western blot analysis. The Rho kinase-specific inhibitor (Y-27632)-induced changes in actin stress fibers, focal adhesions, and protein phosphotyrosine status were evaluated by staining with rhodamine-phalloidin, anti-paxillin, and anti-phosphotyrosine antibodies, respectively. Myosin light-chain phosphorylation was determined by Western blot analysis. Y-27632-induced changes in SC cell monolayer permeability were quantitated using a colorimetric assay to evaluate horseradish peroxidase diffusion through SC cell monolayers grown in transwell chambers. Aqueous humor outflow facility was measured using enucleated porcine eyes and a constant-pressure perfusion system. RESULTS: Treatment of HTM and SC cells with Y-27632 (10 microM) led to significant but reversible changes in cell shape and decreases in actin stress fibers, focal adhesions, and protein phosphotyrosine staining. SC cell monolayer permeability increased (by 80%) in response to Y-27632 (10 microM) treatment, whereas myosin light-chain phosphorylation was decreased in both HTM and SC cells. Aqueous humor outflow facility increased (40%-80%) in enucleated porcine eyes perfused with Y-27632 (10-100 microM), and this effect was associated with widening of the extracellular spaces, particularly the optically empty area of the juxtacanalicular tissue (JCT). The integrity of inner wall of aqueous plexi, however, was observed to be intact. CONCLUSIONS: Based on the Rho kinase inhibitor-induced changes in myosin light-chain phosphorylation and actomyosin organization, it is reasonable to conclude that cellular relaxation and loss of cell-substratum adhesions in HTM and SC cells could result in either increased paracellular fluid flow across Schlemm's canal or altered flow pathway through the JCT, thereby lowering resistance to outflow. This study also suggests Rho kinase as a potential therapeutic target for the development of drugs to modulate intraocular pressure in glaucoma patients.

Actins↗
PubMed2001

Evaluation of the myocilin (MYOC) glaucoma gene in monkey and human steroid-induced ocular hypertension.

PURPOSE: Glucocorticoid-induced ocular hypertension (the steroid response) may result in optic nerve damage that very closely mimics the pathologic course of primary open angle glaucoma (POAG). In addition, patients with glaucoma and their relatives are much more likely to exhibit the steroid response than unaffected individuals, suggesting a potential link between the steroid response and POAG. Recently, the expression of a gene (MYOC) in the trabecular meshwork was shown to be steroid-induced. MYOC variations thought to be disease-causing also were found in 3% to 5% of POAG cases. The purpose of this study was to determine whether some variations in MYOC might be involved in steroid-induced ocular hypertension. METHODS: Seventy human steroid responders and 114 control subjects were screened for variations in the coding sequence and promoter of MYOC. Also, topical doses of dexamethasone (DEX) were administered to cynomolgus monkeys to determine their steroid responsiveness, and the MYOC orthologue was cloned from the cynomolgus monkey. RESULTS: Overall, 109 instances of 20 different sequence variations were identified in the human myocilin gene. However, only four of these (each observed in a single individual) met the study criteria for a possible phenotype-altering variation. Three of these were present in steroid responders and one in a control patient, a distribution that was not statistically significant (P: = 0.3). In addition, the allele frequency of a closely flanking marker was compared between the steroid responders and the control subjects, and no evidence for linkage disequilibrium was observed. Reproducible and reversible ocular hypertension was induced in approximately 40% of the monkeys treated with DEX, similar to that seen in man. Ten monkeys were screened for MYOC mutations with single-strand conformation polymorphism (SSCP) analysis. Overall, 37 instances of 13 different sequence variations were observed. Four of these changes met the study criteria for a possible phenotype-altering variation, and these were equally distributed between responder and nonresponder monkeys. CONCLUSIONS: This study identified no statistically significant evidence for a link between MYOC mutations and steroid-induced ocular hypertension.

Administration, Topical↗
PubMed2003

Tissue differential microarray analysis of dexamethasone induction reveals potential mechanisms of steroid glaucoma.

PURPOSE: To identify myocilin (TIGR/MYOC) properties that are specific to the human trabecular meshwork (HTM). To search for genes highly expressed in dexamethasone (DEX)-induced HTM cells that are barely expressed or absent in DEX-induced cells from other tissues. METHODS: TIGR/MYOC induction by DEX (10(-7) M for 8-10 days) was analyzed by Northern and Western blot analyses in HTM, human umbilical vein endothelial cells, HeLa cells, and human embryonic skeletal muscle cells and optic nerve head (ONH) astrocytes at confluence. Processing and secretion were analyzed after the cells were infected with adenoviruses overexpressing wild-type and mutant forms of TIGR/MYOC. Affymetrix U95Av2 GeneChips (n = 6) and software were used to compare paired expression profiles of HTM, HTM-DEX, ONH astrocytes, and ONH astrocytes-DEX. Identification of HTM-DEX-specific genes (compared with ONH astrocytes-DEX) was performed by selecting genes with the highest fold change values (>/=20). Genes with fold change values of four or more were matched with loci linked to glaucoma, by using gene databases. RESULTS: TIGR/MYOC induction by DEX occurred only in HTM cells. Secretory and glycosylation characteristics remained the same across cell types. Expression profile analysis revealed multiple genes differentially upregulated in HTM-DEX including, in addition to TIGR/MYOC, a serine protease inhibitor (alpha1-antichymotrypsin), a neuroprotective factor (pigment epithelium-derived factor), an antiangiogenesis factor (cornea-derived transcript 6), and a prostaglandin synthase (prostaglandin D(2) synthase). Fifteen of the 249 genes with fold change values of four or more mapped to glaucoma-linked loci. CONCLUSIONS: The induction of TIGR/MYOC by DEX is HTM-specific, whereas its secretory and glycosylation characteristics are ubiquitous. The known functions of HTM-DEX-specific genes reveal the presence of protective and damaging mechanisms for regulation of IOP during DEX treatment. Besides TIGR/MYOC, other HTM-DEX-specific genes may be good candidates for linkage to glaucoma.

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PubMed2005

Excimer laser trabeculotomy: a new, minimally invasive procedure for patients with glaucoma.

BACKGROUND: Excimer laser trabeculotomy (ELT) ab interno is a new surgical technique to reduce intraocular pressure (IOP) in patients with glaucoma or ocular hypertension. Our purpose was to examine IOP reduction and the use of antiglaucoma drugs (AGD) in patients treated with ELT and to evaluate the safety of this laser treatment. METHODS: To increase the outflow of aqueous humor, ten microperforations of the trabecular meshwork were performed by an endoscope-guided photoablative laser probe (Excimer laser, AIDA, TUI-Laser, Munich; pulse energy: 1.2 mJ at fiber tip, pulse duration: 60 ns, repetition rate: 20 Hz). Average operation time usually was about 2 min. In our pilot study, one group of patients without cataract underwent ELT, the other group with cataract underwent phacoemulsification (PHACO) plus ELT. IOP, visual acuity, and AGD were determined preoperatively (T0) and 2-4 months (T1), 5-7 months (T2), 11-13 months (T3), and 22-26 (T4) months after surgery. Treatment was defined to be successful if (1) postoperative IOP was < or =21 mmHg, and (2) IOP reduction was at least 20%. RESULTS: ELT reduced the IOP from 24.1+/-0.7 (n=69) mmHg preoperatively to 18.8+/-0.4 (T1, n=66), 20.0+/-0.5 (T2, n=51), 18.8+/-0.8 (T3, n= 37), and 16.8+/-1.0 (T4, n=15) mmHg, respectively. However, 28% of the eyes needed repeat surgery due to insufficient IOP reduction. According to Kaplan-Meier statistics, the success rate was 60% (T1), 49% (T2), and 46% (T3), respectively. The number of AGD was 1.9+/-0.1 (T0), 1.2+/-0.2 (T1), 1.3+/-0.2 (T2), 1.8+/-0.2 (T3), and 1.5+/-0.3 (T4). Combined phacoemulsification plus ELT reduced the IOP from 22.4 mmHg+/-0.6 (T0, n=57) to 16.5+/-0.4 (T1, n=52), 16.1+/-0.5 (T2, n= 40), 16.4+/-0.4 (T3, n= 35), and 12.8+/-1.5 (T4, n=4) mmHg, respectively; 7% of the eyes treated with the combined procedure needed repeat surgery due to insufficient IOP reduction. According to Kaplan-Meier statistics, the success rate was 85% (T1), 74% (T2), and 66% (T3), respectively. The number of AGD was 1.1+/-0.2 (T0), 0.9+/-0.2 (T1), 1.1+/-0.2 (T2), 1.2+/-0.2 (T3), and 1.8+/-0.9 (T4). CONCLUSIONS: ELT, especially in combination with phacoemulsification, is a new, promising, minimally invasive laser treatment to reduce IOP for at least 1-2 years. ELT alone is less effective in IOP reduction.

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