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PubMed2000

Responses of different cell lines from ocular tissues to elevated hydrostatic pressure.

BACKGROUND/AIMS: Mechanical forces are thought to induce cellular responses through activation of signalling pathways. Cells within the intraocular environment are exposed to constant changes in the levels of intraocular pressure. In this study, an attempt was made to determine the acute effects of elevated hydrostatic pressure on different intraocular cells grown in culture. METHODS: Different cell lines derived from ocular tissues including non-pigmented and pigmented ciliary epithelium, trabecular meshwork, retina, and lamina cribrosa were incubated in a pressurised chamber at 50 mm Hg in a culture incubator at 37 degrees C for up to 6 hours. Control cells were incubated at atmospheric pressure. The viability of the cells was examined using their intracellular esterase activity. The morphology and cytoskeleton of the cells were investigated using microscopy and phalloidin staining. Adenylyl cyclase activity was assessed by measuring the conversion of [(3)H]-cAMP from [(3)H]-ATP in response to elevated hydrostatic pressure for 1-6 hours. In addition, at the end of incubation period under elevated hydrostatic pressure the recovery of adenylyl cyclase activity to control levels was examined. RESULTS: Cell viability did not change following exposure to elevated hydrostatic pressure for 6 hours. Cells subjected to elevated hydrostatic pressure demonstrated morphological differences characterised by a more rounded shape and a redistribution of actin stress fibres that was most prominent in lamina cribrosa astrocytes. A time dependent increase in basal adenylyl cyclase activity, and a decrease in maximum forskolin stimulated activity were observed in all cell lines following exposure to elevated hydrostatic pressure. CONCLUSION: These observations demonstrate that cell lines from different ocular tissues are sensitive to changes in external pressure in vitro. They exhibit morphological and cytoskeletal changes as well as significant alterations of intracellular adenylyl cyclase activity following exposure to acute and sustained levels of elevated hydrostatic pressure of up to 6 hours' duration.

Actins↗

PubMed2005

Effects of travoprost on aqueous humor dynamics in monkeys.

PURPOSE: To determine the mechanism by which travoprost, a prodrug of a prostaglandin F2alpha analog, reduces intraocular pressure (IOP) in cynomolgus monkey eyes. METHODS: One eye each of 12 monkeys was treated with laser burns to the trabecular meshwork to elevate IOP. At least 4 months later (Baseline Day), IOP was measured by pneumatonometry (9:00 AM and 11:45 AM), and aqueous flow and outflow facility were determined by a fluorophotometric method. Uveoscleral outflow was calculated. Both eyes were treated with travoprost 0.004% at 9:00 AM and 5:00 PM for two days and at 9:30 AM on the third day (Treatment Day), when measurements were repeated as on Baseline Day. Statistical analyses were performed using two-tailed, paired t tests. RESULTS: On Treatment Day compared with Baseline Day, IOP in hypertensive eyes was reduced at 2.25 hours (25.8 +/- 11.2 vs 33.7 +/- 13.2 mm Hg; mean +/- standard error of the mean [SEM]; P = 0.02) and 16 hours (26.3 +/- 10.2 vs 35.1 +/- 13.6 mm Hg; P = 0.02) after treatment. The increase in uveoscleral outflow was not significant. In normotensive eyes, IOP was reduced at 2.25 hours (19.0 +/- 3.7 vs 23.0 +/- 4.0 mm Hg; P = 0.03) and 16 hours (20.7 +/- 5.4 vs 23.4 +/- 5.3 mm Hg; P = 0.01) after treatment, and uveoscleral outflow was significantly (P = 0.02) increased (1.02 +/- 0.43 vs 0.35 +/- 0.72 microL/min). CONCLUSION: Travoprost reduces IOP in normotensive monkey eyes by increasing uveoscleral outflow. The IOP reduction in hypertensive eyes is probably via the same mechanism, although the increased uveoscleral drainage did not reach statistical significance. Travoprost had no effect on aqueous flow or outflow facility.

Animals↗

PubMed2003

Ocular hypotensive FP prostaglandin (PG) analogs: PG receptor subtype binding affinities and selectivities, and agonist potencies at FP and other PG receptors in cultured cells.

Natural prostaglandins (PGs) such as PGD2, PGE2, PGF2(2alpha), and PGI2 exhibited the highest affinity for their respective cognate receptors, but were the least selective agents when tested in receptor binding assays. Travoprost acid ([+]-fluprostenol) was the most FP-receptor-selective compound, exhibiting a high affinity (Ki = 35 +/- 5 nM) for the FP receptor, and minimal affinity for DP (Ki = 52,000 nM), EP1 (Ki = 9540 nM), EP3 (Ki = 3501 nM), EP4 (Ki = 41,000 nM), IP (Ki > 90,000 nM), and TP (Ki = 121,000 nM) receptors. Travoprost acid was the most potent PG analog tested in FP receptor functional phosphoinositide turnover assays in the following cell types: human ciliary muscle (EC50 = 1.4 nM), human trabecular meshwork (EC50 = 3.6 nM), and mouse fibroblasts and rat aortic smooth muscle cells (EC50 = 2.6 nM). Although latanoprost acid exhibited a relatively high affinity for the FP receptor (Ki = 98 nM), it had significant functional activity at FP (EC50 = 32-124 nM) and EP1 (EC50 = 119 nM) receptors. Bimatoprost acid was less selective, exhibiting a relatively high affinity for the FP (Ki = 83 nM), EP1 (Ki = 95 nM), and EP3 (Ki = 387 nM) receptors. Bimatoprost acid exhibited functional activity at the EP1 (EC50 = 2.7 nM) and FP (EC50 = 2.8-3.8 nM in most cells) receptors. Bimatoprost (nonhydrolyzed amide) also behaved as an FP agonist at the cloned human FP receptor (EC50 = 681 nM), in h-TM (EC50 = 3245 nM) and other cell types. Unoprostone and S-1033 bound with low affinity (Ki = 5.9 microM to > 22 microM) to the FP receptor, were not selective, but activated the FP receptor. In conclusion, travoprost acid has the highest affinity, the highest FP-receptor-selectivity, and the highest potency at the FP receptor as compared to the other ocular hypotensive PG analogs known so far, including free acids of latanoprost, bimatoprost, and unoprostone isopropyl ester.

Amides↗

PubMed1998

Mutations in CYP1B1, the gene for cytochrome P4501B1, are the predominant cause of primary congenital glaucoma in Saudi Arabia.

The autosomal recessive disorder primary congenital glaucoma (PCG) is caused by unknown developmental defect(s) of the trabecular meshwork and anterior chamber angle of the eye. Homozygosity mapping with a DNA pooling strategy in three large consanguineous Saudi PCG families identified the GLC3A locus on chromosome 2p21 in a region tightly linked to PCG in another population. Formal linkage analysis in 25 Saudi PCG families confirmed both significant linkage to polymorphic markers in this region and incomplete penetrance, but it showed no evidence of genetic heterogeneity. For these 25 families, the maximum combined two-point LOD score was 15.76 at a recombination fraction of .021, with the polymorphic marker D2S177. Both haplotype analysis and homozygosity mapping in these families localized GLC3A to a 5-cM critical interval delineated by markers D2S2186 and D2S1356. Sequence analysis of the coding exons for cytochrome P4501B1 (CYP1B1) in these 25 families revealed three distinctive mutations that segregate with the phenotype in 24 families. Additional clinical and molecular data on some mildly affected relatives showed variable expressivity of PCG in this population. These results should stimulate a study of the genetic and environmental events that modify the effects of CYP1B1 mutations in ocular development. Furthermore, the small number of PCG mutations identified in this Saudi population makes both neonatal and population screening attractive public health measures.

Adult↗

PubMedDate not supplied

Non-invasive observation of repeated adenoviral GFP gene delivery to the anterior segment of the monkey eye in vivo.

BACKGROUND: Glaucoma is a group of chronic eye diseases often associated with an elevated intraocular pressure (IOP). If not controlled, the condition leads to blindness. The eye tissue responsible for maintaining aqueous humor resistance and thus normal IOP is the trabecular meshwork (TM). Adenoviral vectors are capable of transducing the TM in several rodent species. Because of the relevance of the non-human primate model in the study of glaucoma, gene transfer to the eyes of cynomolgus monkeys was investigated. METHODS: Four cynomolgus monkeys were injected with AdenoGFP into the anterior chamber: two monkeys received 10(9) pfu and the other two 10(7) pfu. One monkey received four consecutive injections into the same eye (10(7) pfu in each injection) over a 7-month period. In vivo gene transfer (fluorescence) and IOP were evaluated by standard clinical ophthalmic instruments (slit lamp biomicroscopy, gonioscopy and tonometry). Histopathology and cellular distribution were assessed postmortem. RESULTS: The first injection of the lower viral dose resulted in marked TM-preferred gene transfer visible non-invasively by in vivo gonioscopy. The expression of the transgene lasted for 3-4 weeks with little or no signs of clinical inflammation. Gene transfer was achieved on three sequential occasions (3-4 weeks each) but failed and induced substantial, albeit reversible, corneal abnormalities on the fourth occasion. CONCLUSIONS: Gene transfer to the TM and cornea can be monitored non-invasively in non-human primates, allowing correlation of gene transfer with physiological parameters. Because of ocular immune privilege, repeated anterior chamber administrations of adenoviral vectors expressing appropriate genes may have therapeutic potential for glaucoma.

Adenoviridae↗

PubMed1996

Aceclidine effects on outflow facility after ciliary muscle disinsertion.

Aceclidine increases outflow facility with little accommodative effect. To determine whether this dissociation resides in the ciliary muscle (CM) or trabecular meshwork (TM), we measured aceclidine effects on perfusion outflow facility in both eyes of 8 rhesus monkeys after unilateral disinsertion of the CM from the TM. Facility in the control eyes increased by approximately 250% following intravenous pilocarpine and by an additional approximately 250% following intracameral pilocarpine, relative to baseline and uncorrected for washout. In CM-disinserted eyes, the facility response to intravenous and intracameral pilocarpine averaged approximately 25% of that in contralateral controls. Cytochalasin B, which acts directly on the TM to increase facility but is not additive to maximal pilocarpine doses in normal eyes, had no additional effect beyond that of pilocarpine in control eyes but induced an additional 100% facility increase relative to baseline in CM-disinserted eyes. The accommodative response to carbachol in CM-disinserted eyes was approximately 80% of that in contralateral controls, consistent with retention of CM contractility and the gonioscopic appearance of shallow CM disinsertion. Intracameral aceclidine HCl doses of 5 and 50 micrograms increased outflow facility by approximately 80 and 250%, respectively, in control eyes, and by approximately 0 and 80% in CM-disinserted eyes. Either the low aceclidine dose affected facility via the CM, while the high dose exerted an additional effect on the TM, or aceclidine acted only via the CM, with the low dose being ineffective and the high dose modestly effective in CM-disinserted eyes because only a few CM-TM attachments remained.

Accommodation, Ocular↗

PubMed2001

Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility.

PURPOSE: To elucidate the roles of Rho-associated protein kinase (ROCK) in regulating intraocular pressure (IOP) and outflow facility in the rabbit eye. METHODS: A specific ROCK inhibitor Y-27632 was used. The IOP, the outflow facility, and the pupil diameter were determined before and after the topical, intracameral, or intravitreal administration of Y-27632 in rabbits. Western blot analysis was used to identify specific ROCK isoform in human trabecular meshwork (TM) cells and bovine ciliary muscle (CM) tissues. The cell morphology and distribution of actin filaments and vinculin in TM cells were studied by cell biology techniques. Carbachol (Cch)-induced contraction of isolated bovine CM strips after administration of Y-27632 was measured in a perfusion chamber. RESULTS: In rabbit eyes, administration of Y-27632 resulted in a significant decrease in IOP in a dose-dependent manner. An increase of the outflow facility and pupil size dilation was also observed in Y-27632-treated eyes. Western blot analysis revealed the presence of p160ROCK in human TM cells and bovine CM tissues. In cultured human TM cells, exposure to Y-27632 caused retraction and rounding of cell bodies as well as disruption of actin bundles and impairment of focal adhesion formation. Y-27632 in addition inhibited Cch-induced contraction of isolated bovine CM strips. CONCLUSIONS: Administration of Y-27632 caused a reduction in IOP and an increase in the outflow facility. The in vitro experiments suggest that the IOP-lowering effects of Y-27632 may be related to the altered cellular behavior of TM cells and relaxation of CM contraction. These studies suggest that ROCK inhibitors may have great potential to be developed for treatment of glaucoma and other ocular diseases.

Actins↗

PubMed2005

Degree of angle closure and the intraocular pressure-lowering effect of latanoprost in subjects with chronic angle-closure glaucoma.

OBJECTIVE: To examine the relationship between the configuration of the drainage angle and intraocular pressure (IOP)-lowering efficacy of latanoprost in subjects with chronic angle-closure glaucoma (CACG). DESIGN: Prospective observational case series. PARTICIPANTS: One hundred thirty-seven Asian subjects with CACG. METHODS: Study subjects had participated in a 12-week, randomized, double-masked study that assessed the IOP-reducing effect of latanoprost. Chronic angle-closure glaucoma was defined as optic neuropathy with or without a visual field defect, an anterior chamber angle in which the trabecular meshwork was not visible for at least 180 degrees on gonioscopy, and evidence of peripheral anterior synechiae (PAS) in association with a chronically elevated IOP. Static and dynamic gonioscopy was performed at baseline. The angles were graded in each quadrant according to the Shaffer scheme, and the number of clock hours of PAS was recorded. The change in daily IOP (defined as the mean of the 9:00 am and 5:00 pm IOP time point values) from baseline to week 12 was analyzed and correlated with mean angle width and extent of PAS. RESULTS: One hundred thirty-seven Asian subjects with CACG completed the study in the latanoprost-treated group. Most subjects were female (75%), and the mean age was 62.6+/-9.4 years. At baseline, the mean angle width was 0.84+/-0.55, and the mean number of clock hours of PAS was 4.67+/-2.95. After 12 weeks of treatment, latanoprost reduced IOP from 25.0+/-5.5 mmHg to 17.5+/-5.0 mmHg (P<0.001). The percent change in IOP produced by latanoprost was not associated with mean angle width (Spearman's r = 0.04, P = 0.64) or the number of clock hours of PAS (Spearman's r = -0.15, P = 0.08). CONCLUSIONS: In subjects with CACG, the IOP-reducing efficacy of latanoprost was not affected by the degree of angle narrowing or extent of synechial angle closure.

Adult↗

PubMed1996

Presence of functional type B natriuretic peptide receptor in human ocular cells.

PURPOSE: To study the effects of natriuretic peptides on cyclic guanosine monophosphate (cGMP) production and calcium mobilization in cultured human ocular cells. METHODS: Cultured simian virus 40-transformed (HTM-3) and nontransformed (HTM-16) human trabecular meshwork (TM) cells and nontransformed human ciliary muscle (CM) cells were used. Accumulation of cGMP in cells lysate was measured by radioimmunoassay. Intracellular calcium concentration was measured by microscope-based ratiofluorometry. RESULTS: Both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) increased the accumulation of cGMP in HTM-3, HTM-16, and CM cells. In the nontransformed TM cells, CNP was five times more efficacious (maximal effect of CNP was 497% +/- 44% that of ANP) and 10 times more potent than ANP (ANP, log [EC50] = -6.99 +/- 0.08; CNP, log [EC50] = -7.96 +/- 0.20). Similar results were seen in HTM-3 and CM cells. Under the assay conditions used, the peptides increased only the production of cGMP without changing its degradation rate. The peptide-induced increase of cGMP in the TM and CM cells correlated with suppression of carbachol-induced calcium mobilization in the cell. CONCLUSIONS: It is known that CNP, but not ANP, selectively activates the guanylyl cyclase associated with the type B natriuretic peptide receptor (NPR-B). Thus, the data suggest that NPR-B is the primary functional NPR in the TM and CM cells. The effects on cGMP and calcium produced by the activation of this receptor are expected to alter TM and CM contractility and may affect aqueous humor hydrodynamics and intraocular pressure.

1-Methyl-3-isobutylxanthine↗

PubMed2005

Temperature sensitive secretion of mutant myocilins.

Recent studies have demonstrated that glaucoma-causing mutant myocilin proteins are misfolded and retained in the endoplasmic reticulum of cells. We showed previously that P370L mutant myocilin is poorly secreted at 37 degrees C and prolonged expression of the protein in differentiated human trabecular meshwork cells results in abnormal morphology and cell killing. Culturing cells at a lower temperature, a condition known to facilitate protein folding, enhances secretion and reverses the cytotoxic effects. We wanted to determine if temperature sensitive secretion is a general property of myocilin missense mutants. Wild-type or mutant forms of myocilin were transiently expressed in HEK 293 cells cultured at either 37 or 30 degrees C and protein secretion was assessed by immunoblotting. Of 15 myocilin missense mutants tested, representing a range in severity of associated glaucoma phenotypes, 14 displayed increased secretion at 30 degrees C. The sole exception was K423E, which is associated with an unusual mode of glaucoma inheritance. Generally, there is an inverse relationship between the degree of mutant myocilin secretion at 30 degrees C and the severity of the associated glaucoma phenotype. Mutants that show abundant secretion at 30 degrees C such as T377M, G364V, I499F and D380A are associated with less virulent glaucoma phenotypes, while mutants such as P370L, I477N, and Y437H display little secretion at 30 degrees C and are associated with more virulent glaucoma phenotypes. We conclude that temperature sensitive secretion is a property of most olfactomedin-domain myocilin mutants. The correlation between temperature sensitive secretion and glaucoma phenotype likely reflects the intrinsic susceptibility to misfolding of individual mutant proteins. These results support the hypothesis that myocilin-induced glaucoma is a protein conformational disease. Facilitating mutant protein folding could be a new approach to development of therapies for this disease.

Cell Line↗

PubMed1998

Acute and chronic structural effects of pilocarpine on monkey outflow tissues.

PURPOSE: Determine the effects of supraclinical topical doses of pilocarpine on the structure of the trabecular meshwork (TM), ciliary muscle (CM), and ciliary processes in monkeys. METHODS: Cynomolgus monkeys received topical pilocarpine hydrochloride doses of 1 to 2 mg unilaterally once or twice daily for 1 to 211 days. Both eyes were examined by light and electron microscopy. RESULTS: After 1 dose, the TM was expanded and the flow pathways open. Some elastic anterior CM tendons were disconnected from the muscle tips and/or the tips themselves were disrupted; macrophage-like cells accumulated in these regions. In eyes that had long-term treatment, scar tissue at the muscle tips and disruption of myofibrils within muscle cells indicated regions of previous disconnection. Most collagen tendons inserting into the peripheral cornea were unaffected. With increasing treatment duration in younger monkeys, there was anteroposterior shortening of Schlemm's canal, TM densification due to collapse, and increased cellularity in the subendothelial region of Schlemm's canal. TM densification and Schlemm's canal shortening were not found in older monkeys, implicating posterior movement of the elastic young limbal region induced by strong CM contraction, rather than pilocarpine toxicity. In the ciliary body we found vasodilation, Greeff's vesicles, and blood-aqueous barrier breakdown in the anterior ciliary processes and vasodilation, edema, and round-cell infiltration in the posterior pars plana. CONCLUSIONS: Large pilocarpine doses induce morphologic changes in the monkey anterior segment, likely consequent to intense CM contraction. Changes in the TM and Schlemm's canal are seen only in young monkeys, apparently owing to mechanical deformation of the elastic limbal region.

Animals↗

PubMed2003

Ultrasound activates the TM ELAM-1/IL-1/NF-kappaB response: a potential mechanism for intraocular pressure reduction after phacoemulsification.

PURPOSE: Elevated intraocular pressure (IOP), the major causal risk factor for glaucoma, often decreases after cataract removal by phacoemulsification ultrasound. In this study, the hypothesis that ultrasound energy propagated through a fluid medium induces a stress response with the potential to lower IOP was investigated. METHODS: Normal and glaucomatous trabecular meshwork (TM) cell culture lines were initiated from tissue isolated from human cadaveric eyes or trabeculectomy specimens. Cultured cells were treated for 60 seconds with a phacoemulsification ultrasound probe set to a power of 70%. Activation of the TM cell-specific stress response was assayed by enzyme-linked immunosorbent assay (ELISA) and immunolocalization. RESULTS: Normal TM cell cultures did not release detectable levels of the stress response protein, IL-1alpha, into their culture medium. In contrast, IL-1alpha was easily detected after treatment with ultrasound energy. Consistent with earlier findings, glaucomatous TM cells produced IL-1alpha constitutively, and the level of expression was increased after treatment with phacoemulsification ultrasound. As was previously demonstrated, the stress-regulated transcription factor NF-kappaB was present in the cytoplasm of normal cells, but in the nucleus of glaucomatous cells. After treatment with ultrasound energy, NF-kappaB translocated to the nucleus in the normal cells. Endothelial leukocyte-adhesion molecule (ELAM)-1 was not detected in normal TM cells, but was constitutively present on glaucomatous TM cells, consistent with findings in previous work. ELAM-1 expression was induced in normal cells by ultrasound treatment. CONCLUSIONS: A potentially IOP-lowering stress response is induced in TM cells by ultrasound. The findings suggest that this response may be induced clinically during cataract removal by phacoemulsification, and may be one mechanism responsible for the reduction in IOP that often follows this procedure.

Cells, Cultured↗

PubMed2002

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.

Aged↗

PubMed2006

Regulation of connective tissue growth factor expression in the aqueous humor outflow pathway.

PURPOSE: Connective Tissue Growth Factor (CTGF) is an inducible secretory protein known to regulate proliferation and extracellular matrix production in various cell types. We hypothesize that CTGF plays a critical role in the physiological regulation of aqueous humor outflow through the trabecular meshwork (TM) by influencing extracellular matrix synthesis and organization. METHODS: To determine the expression of CTGF in tissues of the aqueous outflow pathway, cells obtained from human TM and Schlemm's Canal (SC) were analyzed by PCR and western blot analysis. To understand the regulation of CTGF expression in TM cells, TM cells were either treated with various physiologic factors or subjected to cyclical stretch prior to analysis of CTGF expression by RT-PCR and western blot analysis. To study the effect of increased intraocular pressure on CTGF production, we perfused porcine eyes at high pressure (50 mm Hg) for 5 h, followed by analysis of CTGF expression by RT-PCR and western blotting. RESULTS: Treatment of human TM cells treated with either serum or transforming growth factor-beta 1 led to a robust stimulation, compared to thrombin, lysophosphatidic acid (LPA), and dexamethasone, which elicited a relatively moderate induction of CTGF expression. Both high pressure perfusion and mechanical stretch were associated with increases in the levels of CTGF at the protein and transcript levels. CONCLUSIONS: This study demonstrates that CTGF expression in TM cells is modulated by several physiological agonists and by increased ocular pressure and mechanical stretch. These results suggest that the regulation of CTGF expression within tissues of the outflow pathway may play a role in the homeostasis of intraocular pressure, possibly by modulation of ECM production in these tissues.

Animals↗

PubMed2006

Optimized bacterial expression of myocilin proteins and functional comparison of bacterial and eukaryotic myocilins.

PURPOSE: To maximize the expression level of myocilin and its truncated proteins in Escherichia coli (E. coli) and to examine the biological effects of bacterially expressed myocilin as compared to eukaryotic myocilin on cultured human trabecular meshwork (TM) cells. METHODS: Myocilin full length (1-504 amino acids) and two truncated proteins, myocilin 1-270 and 271-504, were expressed and purified from an E. coli strain, Rosetta2(DE3)pLysS. The eukaryotic myocilin was purified from cultured medium of a transformed TM cell line (TM5) transduced with feline immunodeficiency virus that contains an internal cassette expressing full length myocilin. The morphology and adhesion of human TM cells plated either on fibronectin alone or on fibronectin/purified myocilin mixtures were assessed by phase contrast microscopy. Actin cytoskeleton was examined using Oregon Green phalloidin. Immunofluorescence staining for paxillin was also performed. RESULTS: The expression of full length and truncated myocilin proteins in Rosetta2(DE3)pLysS was markedly increased especially when the bacteria were grown in media supplemented with 1.0% glucose. Cell adhesion was impaired and microspikes were formed when TM cells were plated onto fibronectin/bacterial full length myocilin mixtures. Loss of actin stress fibers and focal adhesions was also observed. This myocilin phenotype was also seen with myocilin 1-270, but not with myocilin 271-504. The eukaryotic full length myocilin produced nearly identical de-adhesive effects as those of the bacterially expressed myocilin. CONCLUSIONS: The condition for a high level expression of full length and truncated myocilins in E. coli was optimized. The bacterial and eukaryotic recombinant full length myocilin produced similar biological consequence on TM cells. The myocilin phenotype appears to be largely due to the NH(2)-terminal half of the protein.

Adult↗

PubMed1996

Outflow characteristics of isolated anterior segments of human eyes.

PURPOSE: To evaluate the relationship between intraocular pressure (IOP) and flow through the trabecular meshwork in isolated anterior segments of human eyes. Outflow facility (C) is thought to decrease proportionally with increasing IOP in human eyes in vivo and in vitro. METHODS: Twenty-nine eviscerated human anterior segments were perfused with ascending and descending pressure sequences in a stepwise fashion (range, 4 to 40 mm Hg); 11 of the 29 eyes were treated similarly after 20 hours of perfusion at 12 mm Hg. Pressure-flow sequences of individual eyes were evaluated with a linear (constant C) and a nonlinear regression method (C decreasing with increasing IOP). In addition, in eight intact postmortem eyes, pressure-flow characteristics were determined, followed by perfusion of their isolated anterior segments. RESULTS: Pressure-flow sequences as determined by linear regression had an average correlation coefficient of 0.99. Average C (slope of the plot) was 0.26 +/- 0.03 microliter minute-1 mm Hg-1. There was no influence of direction of pressure sequence or time on C. To test for linearity, the hypothetical outflow obstruction coefficient (Q) was determined for each plot. Median Q of 29 eyes was 0.003 mm Hg-1, and in 45% (13 eyes) Q was negative, suggesting facilitation instead of obstruction. This indicates that the outflow obstruction coefficient is not a physiological parameter in isolated anterior segments. CONCLUSIONS: The relationship between IOP and flow through perfused isolated anterior segments of human eyes is linear between 4 and 40 mm Hg, indicating that within this range outflow facility is constant and does not decrease with increasing IOP.

Adult↗

PubMed1999

Pilocarpine induces an increase in the anterior chamber angular width in eyes with narrow angles.

AIM: To determine the mechanical effects of pilocarpine on the trabecular-iris angle opening in eyes with narrow angles, compared with its effects on healthy control subjects with wide angles. METHODS: A narrow angle was defined as 25 degrees or less of trabecular-iris angle on ultrasound biomicroscopic examination. The change in anterior chamber depth (ACD), trabecular-iris angle (TIA), angle opening distance (AOD, distance between trabecular meshwork and iris) measured at 250 microm and 500 microm from the scleral spur (AOD250 and AOD500), and iris thickness was determined in 30 eyes of 30 patients (13 men and 17 women, between 63 and 82 years (mean 70.4 years)) with narrow angles and in 30 sex and age matched control subjects with wide angles before and 1 hour after the instillation of 2% pilocarpine hydrochloride by ultrasound biomicroscopy. RESULTS: In all eyes with narrow angles, pilocarpine increased the TIA, AOD250, and AOD500; these changes increased significantly and linearly as the corresponding pretreatment values decreased (r = 0.807, p = 0.0001; r = 0.787, p = 0.0001; r = 0.852, p = 0.0001). Of 30 eyes with wide angles, 23 eyes whose ACD was 2670 microm and more showed a decrease in the TIA, AOD250, and AOD500; the changes in TIA, AOD250, and AOD500 also significantly correlated with the corresponding pretreatment values (r = 0.913, p = 0.0001; r = 0.882, p = 0.0001; r = 0.895, p = 0.0001). Pilocarpine induced a smaller decrease in ACD in eyes with narrow angles than in those with wide angles (p = 0.0001). There was a linear correlation between the increase in ACD change and the decrease in pretreatment ACD in eyes with narrow angles and those with wide angles (r = 0.781, p = 0.0003; r = 0.798, p = 0.0001). CONCLUSIONS: The finding that pilocarpine increases angular width in patients with narrow angles indicates that this agent is useful for treating patients with narrow angles and angle closure glaucoma. The prediction of the pilocarpine induced change in the angle may assist ophthalmologists in treating such patients.

Aged↗

PubMed2006

[Open angle glaucoma: epidemiology, pathogenesis and prevention].

There is growing evidence that reactive oxygen species (ROS) play a key role in the pathogenesis of primary open angle glaucoma (POAG). The occurrence of oxidative DNA damage in trabecular meshwork (TM) has been demonstrated by measuring the increase of 8-hydroxy-2'-deoxyguanosine, the most abundant DNA oxidative alteration, which is significantly increased in glaucoma-bearing subjects as compared with unaffected controls. Several lines of evidence support the hypothesis that ROS play a fundamental pathogenic role, including the following: (a) outflow resistance in the anterior chamber increases in the presence of high levels of hydrogen peroxide; (b) TM possesses abundant antioxidant activities; (c) significant increases in superoxide dismutase and glutathione peroxidase activities were detected in the aqueous humour of glaucoma patients; (d) hydrogen peroxide compromises TM integrity. The existence of a significant correlation between oxidative DNA damage and intraocular pressure in glaucoma patients has been reported. POAG patients appear to have a genetic predisposition rendering them susceptible to ROS-induced damage because of a more frequent deletion, as compared to controls, of the gene encoding for glutathione-S-transferase M1, a pivotal anti-oxidant activity. Furthermore, oxidative stress, occurring not only in TM but also in retinal cells, appears to be involved in the neuronal cell death that characterizes POAG. These considerations could bear relevance for POAG prevention and suggest that genetic analyses and the use of drugs or dietary measures attenuating the effects of ROS, if validated in future studies, could be useful tools contributing to the control of this disease.

Adrenergic beta-Antagonists↗