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PubMed2005

CB2 cannabinoid receptors in trabecular meshwork cells mediate JWH015-induced enhancement of aqueous humor outflow facility.

PURPOSE: To study the effects of JWH015, a CB2-selective agonist, on aqueous humor outflow facility, to investigate whether functional CB2 cannabinoid receptors are expressed in trabecular meshwork cells, and to study whether these receptors are involved in the enhancement of outflow facility induced by JWH015. METHODS: A porcine anterior segment perfused organ culture model was used to measure the effects of JWH015 on aqueous humor outflow facility. Immunofluorescence microscopy, Western blot analysis, and mitogen-activated protein (MAP) kinase activity assays were used to study the expression of CB2 cannabinoid receptors on cultured porcine trabecular meshwork cells and the coupling of these receptors to p42/44 MAP kinase. RESULTS: The outflow facility was increased dose dependently within 1 hour after adding 10, 30, and 100 nM of JWH015, a CB2 agonist. In addition, the effect of 100 nM of JWH015 was completely blocked by SR144528, a selective CB2 antagonist. Furthermore, the outflow-enhancing effect of JWH015 was blocked by pretreatment with PD98059, an inhibitor of the p42/44 MAP kinase pathway. In immunofluorescence microscopy and Western blot studies, positive signals were detected on cultured porcine trabecular meshwork cells with an anti-CB2 antibody. In MAP kinase assays, treatment of porcine trabecular meshwork cells with 100 nM of JWH015 activated p42/44 MAP kinase activity. Pretreatment with SR144528 blocked the effect of JWH015 on p42/44 MAP kinase activity. CONCLUSIONS: The data from this study demonstrate that the CB2-selective cannabinoid agonist JWH015 increases aqueous humor outflow facility. The results also indicate that functional CB2 cannabinoid receptors are expressed in trabecular meshwork cells, and these receptors are involved in the enhancement of outflow facility induced by JWH015.

Animals↗

PubMed2002

[Effect of pressure on proliferation and apoptosis of bovine trabecular meshwork].

OBJECTIVE: To investigate the effect of pressure on the expression of cell cycle and apoptosis related gene bcl-2 and bax mRNA by bovine trabecular meshwork cells. METHOD: Twenty mm Hg, 40 mm Hg, 60 mm Hg and 80 mm Hg(1 mm Hg = 0.133 kPa) pressure were given to the cultured trabecular meshwork cells respectively in the treatment groups. No pressure was given in the control groups. Each pressure sustained for 24 and 48 hours. Proliferation index, apoptosis index, bcl-2 and bax mRNA expression of trabecular meshwork cells were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling, flow cytometry and in situ hybridization. RESULTS: In 24 hour groups, there were no significant differences between the apoptosis index of control group and pressure </= 40 mm Hg groups. But in 40 mm Hg groups, the proliferative index decreased and bax mRNA expression increased significantly. The apoptosis index increased remarkably when the pressure was >/= 60 mm Hg. In 48 hour groups, apoptosis index, proliferative index and bax mRNA expression had significant differences from that of the control groups when pressure was >/= 40 mm Hg. CONCLUSION: Pressure >/= 40 mm Hg sustaining for a period of time can suppress trabecular meshwork cell proliferation and induce apoptosis through influencing bcl-2 family members.

Animals↗

PubMed1990

Ascorbic acid modulates the production of fibronectin and laminin by cells from an eye tissue-trabecular meshwork.

Ascorbic acid is a significant component in the aqueous humor of the eye, in which trabecular meshwork cells are immersed. Using immunostaining and Western blot analysis, the effects of ascorbic acid on the extracellular matrix production by cultured bovine trabecular meshwork cells were examined. It was found that, when the cultures were treated with 100, 250, and 500 micrograms/ml of ascorbic acid, the amount of fibronectin and laminin produced was enhanced. An enzyme-linked immunosorbent assay for quantitation further showed that the fibronectin and laminin levels in ascorbate-treated cultures were higher than those found in control cultures. These results indicate that ascorbic acid promotes fibronectin and laminin production and suggest that this substance may be one of the factors modulating basal lamina assembly in the trabecular meshwork.

Animals↗

PubMed1998

[Electrophysiological specification of trabecular meshwork by dielectric dispersion method].

In an attempt to observe the dielectric behavior of the trabecular meshwork and to estimate its pumping of aqueous humor quantitatively, we measured tissue impedance of the anterior portion of 22 enucleated eyeballs from 11 rabbits non-invasively with a minute coaxial surface probe. Serial measurements of five points near the limbus revealed a significant decrease of the peak value (p < 0.05, paired t-test) on loss tangent function at a point 0.5 mm from the limbus towards the sclera. We considered that the decrease in the peak value at the point was due to broadening of the dielectric dispersion reflecting the microstructure of trabecular meshwork beneath the point. After removing the corneal epithelium, the limbal epithelium, and the conjunctiva, we also observed a decrease in the peak value at the same point, which reinforced our assumption that we can identify the trabecular meshwork electrophysiologically by means of dielectric dispersion.

Animals↗

PubMed1995

Cultured human trabecular meshwork cells express aquaporin-1 water channels.

The identification and characterization of aquaporin-1 water channels and other related proteins has provided a molecular explanation for the enhanced permeability of a variety of epithelial tissues. Previously, we documented the distribution of aquaporin-1 in the human eye, which included the trabecular meshwork; the primary outflow channel for aqueous humor. The goal of this study was to determine if aquaporin-1 could be detected in cultures of human trabecular meshwork cells. Using primers specific for aquaporin-1, reverse transcription combined with polymerase chain reaction yielded a product of the appropriate size with total RNA prepared from the human trabecular meshwork cells. The presence of this product and its size (298 base pairs), is consistent with the presence of an aquaporin-1 message in these cells. Indirect immunofluorescence microscopy with affinity purified antibodies against a fusion protein containing the carboxy tail of aquaporin-1 showed specific labeling of the plasma membrane and immunoblotting identified a band of Mr 28,000 which agrees with the molecular size of aquaporin-1. The presence of aquaporin-1 in human trabecular meshwork cells, the predominant cell-type of the primary outflow region of the human eye, suggests that water channels may be involved with the movement of aqueous fluid out of the eye. In addition, the existence of aquaporin-1 on cultures of human trabecular meshwork cells provides an in vitro model to study the endogenous expression of aquaporin-1 and its possible role in the regulation of aqueous outflow.

Adult↗

PubMed1995

Early changes in matrix metalloproteinases and inhibitors after in vitro laser treatment to the trabecular meshwork.

Extracellular matrix turnover in the trabecular meshwork may play a role in regulating aqueous humor outflow. Laser trabeculoplasty is a common treatment for open-angle glaucoma. The mechanism of this treatment is not understood. We investigated changes in the levels and expression of the matrix metalloproteinases and their tissue inhibitors (TIMPs) in this tissue using cultured human anterior segment explants and standard clinical-parameter laser treatment. Medium gelatinase A activity levels are relatively high for sham-treated controls and are not changed dramatically following laser treatment. Medium gelatinase B and stromelysin activity levels are low in sham-treated explants and increase significantly by 24 h after treatment. TIMP1 levels, as assessed by immunoblots of Western transfers, are initially low. However, by 24 h TIMP1 levels have increased significantly. Using semi-quantitative reverse transcription and the polymerase chain reaction, mRNA levels of stromelysin, gelatinase B and TIMP1 are shown to increase after laser treatment, while gelatinase A and TIMP2 remain relatively constant. The increases in trabecular stromelysin and gelatinase B in response to laser trabeculoplasty may have important implications for the mechanism of action of this treatment for open-angle glaucoma.

Adolescent↗

PubMed1998

Ultrastructural identification of substance P containing nerves in the guinea pig trabecular meshwork.

We aimed to provide a detailed analysis of substance P (SP)-containing nerves in the trabecular meshwork by ultrastructural immunohistochemistry and capsaicin treatment for chemical ablation of the sensory nerves. Numerous myelinated and unmyelinated nerves were observed inside the sheets and intertrabecular spaces of the trabecular meshwork, and the inner side of Schlemm's canal. SP-like immunoreactive products were identified in some of these nerves and associated with numerous vesicles of different sizes, a few mitochondria and numerous neurotubili. After the capsaicin treatment, SP-like immunoreactive nerves persisted and no degenerated SP-like immunoreactive nerves were noted. On the basis of the ultrastructural features and the results of capsaicin treatment, it seems that these nerves are most probably autonomic in origin. Autonomic efferent SP-containing nerves are confirmed directly located in the trabecular meshwork; these have not been previously described at electron microscopic level. SP in autonomic efferent nerves might act as a neurotransmitter or neuromodulator for intraocular pressure regulation.

Animals↗

PubMed1999

Distribution of proteoglycans in the human trabecular meshwork: histochemical electron microscopic findings with cuprolinic blue.

The distribution of sulfated proteoglycans (PGs) in the normal human trabecular meshwork was studied by histochemical electron microscopy using the cationic dye, cuprolinic blue (CB).. The trabecular meshwork was obtained from human enucleated eyes and incubated for three days. After incubation, they were stained with 0.2% CB at a critical electrolyte concentration and prepared for histochemical electron microscopy. Ultrastructurally, PG-CB complexes were found as small punctate or filamentous structures, and were associated with collagen fibrils in the cores of the trabecular beams and the basal laminae of trabecular endothelial cells. In addition, large filamentous PG-CB complexes were mainly associated with areas of amorphous extracellular matrix between the collagen fiber bundles and in the fine fibrillar material near the basal laminae of endothelial cells of Schlemm's canal. This investigation resulted in an illustration of the ultrastructural distribution of PGs in the human trabecular meshwork. Further studies will be needed to specify the nature of PGs and their role in the aqueous outflow system.

Cells, Cultured↗

PubMed1990

Human trabecular meshwork phagocytosis. Observations in an organ culture system.

Perfusion organ culture of the trabecular meshwork was used to study the phagocytic ability of human trabecular cells. Cultured eyes were challenged with blood, latex microspheres, or zymosan particles for periods of 1 hour to 7 days. Trabecular cells were capable of ingesting all three types of particles. The presence of a foreign particle did not necessarily induce a phagocytic response, however, as free particles were seen in the intertrabecular spaces and Schlemm's canal. In contrast to studies in animals which indicate trabecular cell migration from the eye may play a role in the removal of foreign debris, limited human trabecular cell migration was observed. The effect of the culture process on trabecular cell phagocytosis was also studied, using adult cats. One eye received a phagocytic challenge in vivo with the fellow eye later receiving the phagocytic challenge in vitro. Phagocytosis was demonstrated in each eye, although more cells were involved with phagocytosis in vivo. The additional cells involved in vivo may have been recruited by an accompanying inflammation. Organ culture of human trabecular meshwork may be useful in the study of trabecular cell phagocytosis, and it allows separation of the effects of inflammation from the potential effects of phagocytosis itself.

Aged↗

PubMed2000

H-7 effects on the structure and fluid conductance of monkey trabecular meshwork.

OBJECTIVE: To determine the effects of H-7 (1-[5-isoquinoline sulfonyl]-2-methyl piperazine) on the structure and fluid conductance of the trabecular meshwork of live cynomolgus monkeys. METHODS: Fluid outflow was measured by constant pressure perfusion of the anterior chamber with cationized and noncationized gold solution with or without H-7 in opposite eyes. The eyes were fixed by infusing Ito solution and enucleated. Anterior segments were cut into 4 sections, fixed in immersion solution, and embedded in epoxy resin-812. Trabecular meshwork morphologic features were studied by light and electron microscopy. RESULTS: H-7 affected trabecular meshwork organization and increased fluid outflow. H-7 expanded the intercellular spaces in the juxtacanalicular meshwork, accompanied by removal of extracellular material. The inner wall cells of the Schlemm canal became highly extended, yet cell-cell junctions were maintained. Colloidal gold particles were detected only in limited areas along the subcanalicular region in control eyes; after H-7 treatment, gold was widely seen along the entire inner canal wall. Most inner wall cells in H-7-treated eyes, but only few cells in control eyes, contained gold-loaded vesicles. CONCLUSION: H-7 inhibits cell contractility, leading to "relaxation" of the trabecular outflow pathway, expanding the draining surface, and permitting more extensive flow through the meshwork. CLINICAL RELEVANCE: By inhibiting cellular contractility and relaxing the trabecular meshwork, the protein kinase inhibitor H-7 increases outflow facility and reduces intraocular pressure and thus has potential as an ocular hypotensive antiglaucoma medication. Arch Ophthalmol. 2000;118:955-962

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

[Selective excimer laser ablation of the trabecular meshwork. Clinical results].

PURPOSE: Examination of the efficacy of pore formation in the trabecular meshwork by excimer laser to reduce intraocular pressure in glaucoma eyes. PATIENTS AND METHODS: In 27 consecutive eyes with chronic simple glaucoma and 8 eyes with low-tension glaucoma, 3 to 5 pores were ablated into the trabecular meshwork with an excimer laser (308 nm, 35-55 mJ/mm2), creating an open communication between the anterior chamber and Schlemm's canal. This was accomplished by the use of a 400-micron quartz fiber and a modified Trokel goniolens. All patients were candidates for trabeculectomy because visual fields continued to deteriorate in spite of maximum medication. RESULTS: Intraocular pressure was median reduced by 7 mmHg (range 10.5 to 1.5 mmHg) in 22 of 27 eyes with chronic simple glaucoma over a median follow-up of 7 months. In 12 eyes, further medication has to be continued, yet at a lower dose and lower level of intraocular pressure. In five eyes therapy failed. In three of these eyes, a trabeculectomy had to be performed. In eight eyes with low-tension glaucoma, a median reduction of intraocular pressure of 5 mmHg (range 10 to 0.5 mmHg) was accomplished over a median follow-up of 7 months. In five of these eyes, further medication on a lower level was continued. No further surgery was necessary. CONCLUSIONS: With the microsurgical method of pinpoint ablation of the trabecular meshwork by excimer laser, intraocular pressure was reduced in 30 of 35 eyes over a median follow-up period of 7 months. These results encourage us to continue the development of this procedure, perhaps with a microendoscope. The minimal trauma to the eye of this procedure leaves all other options of surgery open.

Follow-Up Studies↗

PubMed1989

Dexamethasone induces specific proteins in human trabecular meshwork cells.

Previous studies have demonstrated the presence of high-affinity, glucocorticoid-specific receptors in explants of human outflow tissue and in cultured trabecular meshwork. Glucocorticoid-induced responses of scleral fibroblasts and trabecular meshwork cells were evaluated in this study. Incubation of human trabecular meshwork (HTM) and scleral fibroblast (HS) cells with 10(-7) M dexamethasone (DEX) results in a 60% inhibition of prostaglandin production. The effects of glucocorticoid treatment on cellular and secreted proteins using [35S] methionine incorporation were evaluated. Treatment of HTM cells cultured from two normal individuals with DEX induced the expression of [35S] methionine-labelled cellular proteins of 35, 65 and 70 kD, and secreted proteins of 40, 90 and 100 kD. Under the same experimental conditions, a 70 kD molecular weight cellular protein was induced in the HS cells. There were no apparent DEX-induced alterations in HS-secreted proteins. Since a functional common response to glucocorticoid treatment in both HS and HTM cells was inhibition of prostaglandin production, the dexamethasone-induced expression of the 70 kD protein in these cells may be related to this effect. Further studies are required to elucidate specific roles of the steroid-induced proteins in the effects of glucocorticoids on HTM and HS cells.

Cells, Cultured↗

PubMed1987

Human trabecular meshwork organ culture. A new method.

A new method has been developed for organ culture of human trabecular meshwork. Human eyebank eyes are sectioned at the equator and lens and vitreous are removed. The anterior segments are placed in a modified culture dish, cornea side up, and then sealed in place. Culture media (Dulbecco's modified Eagle media) are perfused through a cannula built into the bottom of the dish. The sealed system forces media to flow through the trabecular meshwork, Schlemm's canal, and exit through the normal limbal pathways. Intraocular pressure can be maintained at normal (or elevated) levels throughout the culture period. Corneal clarity is maintained. Trabecular cells remain in normal position on trabecular beams and maintain many of their usual morphologic characteristics. Cultures appear to be viable for up to 4 weeks. This technique should allow study of intact human trabecular meshwork in a controlled experimental fashion.

Aged↗

PubMed2002

Adenosine A1 receptor modulation of MMP-2 secretion by trabecular meshwork cells.

PURPOSE: Studies have shown that adenosine A(1) agonists can lower IOP in rabbits, mice, and monkeys, and this response is mediated in part by increases in outflow facility. The purpose of this project was to evaluate the response of trabecular meshwork cells to the addition of the adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA). METHODS: The human trabecular meshwork (HTM-3) cell line and primary cultures of bovine trabecular meshwork (BTM) cells were used in these studies. Cells were treated with CHA, and the secretion of matrix metalloproteinase (MMP)-2 or the activation of extracellular signal-regulated kinase (ERK1/2) was determined. RESULTS: Treatment of HTM-3 and BTM cells with CHA (0.1 micro M) resulted in a time-dependent secretion of MMP-2 that was measurable as early as 30 minutes after treatment and reached a maximum by 2 hours. This CHA-induced secretion of MMP-2 was inhibited by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) and by the ERK1/2 pathway inhibitor U0126. Treatment of HTM-3 cells with CHA produced a rapid dose-dependent activation of ERK1/2 with an EC(50) of 5.7 nM. The CHA-induced activation of ERK1/2 was inhibited by pretreatment with the adenosine A(1) antagonist CPT and by the ERK pathway inhibitor U0126. CONCLUSIONS: The addition of the adenosine A(1) agonist CHA stimulates the secretion of MMP-2 from trabecular meshwork cells. This secretory response involves the activation of adenosine A(1)-linked stimulation of ERK1/2. These results provide evidence for the existence of functional adenosine A(1) receptors in the trabecular cells and that the activation of these receptors stimulates secretion of MMP-2.

Adenosine↗

PubMed1989

Monoclonal antibodies and lectins as probes for investigation of the cell biology of human trabecular meshwork: a preliminary report.

Two classes of ligand-binding molecules, (1) monoclonal antibodies (MABs) and (2) lectins, were used as probes to investigate the cell biology of human trabecular cells. We raised MABs by using human trabecular meshwork as the immunogen. One MAB (KAa25D7) consistently recognized two polypeptides with molecular weights of approximately 42 and 49 kilodaltons. The former comigrated with actin and the latter was shown by fractionation and extraction studies to be a macromolecular complex. Four lectins (wheat germ agglutinin, concanavalin A, Phaseolus vulgaris leukoagglutinin I, and Ulex europaeus agglutinin I) were used to identify the glycosylated polypeptides in human trabecular meshwork on Western blots. Compared to normal age-matched controls, there was a significant decrease in binding of the lectins to most polypeptides of the trabecular meshwork samples that were obtained from 4 eyes of 2 patients with primary open-angle glaucoma. Our preliminary studies show that lectins applied to Western blots and MABs are useful methods to probe the cell biology of human trabecular meshwork in health and disease.

Adult↗

PubMed1980

Potential artifacts in scanning electron microscopy of the trabecular meshwork in glaucoma.

We examined the trabecular meshwork surface by scanning electron microscopy to investigate possible artifacts appearing during collection and preparation of trabeculectomy specimens. As controls, trabecular tissue samples from normal and glaucomatous patients were dissected from whole eyes obtained at enucleation or at autopsy. Our results suggest that the major contaminants are released during decompression of the globe at trabeculectomy; these consist of an amorphous substance, a fibrillar material, and, occasionally, red blood cells. Small tissue samples may also abe contaminated by the conducting adhesive used to fasten them to metal stubs. This silver paint may coat the surface of the meshwork and appear to be a smooth substance blocking the outflow of aqueous humor. We were unable to confirm the finding reported by others that an amorphous blocking material is present in the trabecular interstices in patients with open-angle glaucoma.

Adhesives↗

PubMed1984

Histoautoradiographic and biochemical studies on human and monkey trabecular meshwork and ciliary body in short-term explant culture.

Explants of two types of chamber angle tissue derived from five human autopsy eyes and five freshly enucleated monkey eyes were kept under tissue culture conditions for 1-3 days and then incubated with labelled 14C-glucosamine for 40-48 h. In all specimens an intense labelling was seen histoautoradiographically within the area of the trabecular meshwork, especially in the cribriform layer and the outer wall of Schlemm's canal. Large quantities of silver grains were also found at the tips of the ciliary processes where the nonpigmented epithelium showed the most intense labelling. Measuring the total radioactivity of the two types of specimens in the tissue-localized and the medium-released glycosaminoglycans (GAGs), we found that the type 1 specimens containing only trabecular meshwork work and corneosclera "excrete" relatively more GAGs into the medium than the type 2 specimens consisting of corneosclera, trabecular meshwork and ciliary body. Biochemical analysis of these GAGs revealed that 57%-70% of the tissue GAGs were hyaluronic acid, 16%-33% heparan sulphate and 12%-22% various types of chondroitin sulphate. The findings show that both the ciliary epithelium and the trabecular meshwork possesses the ability to synthesize large amounts of GAGs, probably in the form of proteoglycans. The differences between the in vitro behaviour of type 1 and type 2 specimens indicate a functional interrelationship between the ciliary body and the trabecular meshwork, at least with regard to the GAG metabolism.

Animals↗

PubMed1981

Freeze-fractured replica of Schlemm's canal and the trabecular meshwork in the primate.

Using a freeze-fracture technique, the fine structure of the Schlemm's canal and trabecular meshwork in the monkey was studied with special reference to intercellular junctions. Gap junctions were observed between the endothelial cells of Schlemm's canal and between the trabecular meshwork cells. Maculae occludentes were rarely observed between the endothelial cells of the inner wall of Schlemm's canal. The trabecular meshwork cells appeared to act in an electrotonic and metabolic syncytium.

Animals↗