Quantitative analysis of collagen content and amino acids in trabecular meshwork.

The purpose of this study was to compare collagen content in the TM of normal and glaucomatous eyes, and to establish whether collagen levels change with age. Collagen content was measured in 30 normal and 27 age matched glaucoma trabeculectomy specimens by the sirius red dye binding technique, and in 14 normal and 15 age matched glaucoma specimens by amino acid analysis. Both dye binding data and amino acid analysis showed no statistical difference between normal and glaucoma samples. Age had no significant effect on mean optical densities or on the collagen-specific amino acids proline, hydroxyproline, and hydroxylysine. Amino acid variability, however, was statistically different between the two groups. These results indicate that mean collagen levels in the trabecular meshwork of glaucomatous eyes do not differ from those in normal eyes.

Localization of smooth muscle myosin-containing cells in the aqueous outflow pathway.

Cells containing smooth muscle myosin were localized in the human aqueous outflow pathway by immunohistochemical techniques. In the majority of eyes, immunoreactive cells were observed adjacent to the collector channels and slightly distal to the outer wall of Schlemm's canal. In a few eyes, smooth muscle myosin was localized to cells in the juxtacanalicular tissue and the trabecular meshwork. The immunoreactive cells from these regions may be true smooth muscle cells or pericytes, which can contain smooth muscle myosin. No obvious differences were observed in the pattern of distribution of smooth muscle myosin-containing cells in a comparison of age groups. In the majority of eyes, we observed an apparent direct insertion of the longitudinal portion of the ciliary muscle in the corneoscleral meshwork far internal to the scleral spur.

Beta adrenergic binding sites in the human eye: an autoradiographic study.

Beta adrenergic binding sites were localized and characterized in the human eye by means of "in vitro" autoradiography, using [125I] (-) iodocyanopindolol (125ICYP) as radioligand. Binding sites were visualized by apposition of isotope sensitive film to slide mounted eye sections. Receptor sites were present in the extraocular muscles, in the conjunctiva, in the epithelium and endothelium of the cornea, in the trabeculum and in the ciliary muscle. They were also present in the lens epithelium and in the retina. The pigmented ocular structures were heavily labelled but the binding was nonspecific. Characterization of these binding sites was achieved by testing the ability of selective adrenergic compounds to displace 125ICYP binding. These studies suggested that the majority of adrenergic binding sites in nonpigmented structures of human eye were of a beta2 type.

[Age-related changes in fibronectin of the eye outflow drainage system]. / Vozrastnaia dinamika fibronektina drenazhnogo apparata glaza.

The content of the fibronectin, an extracellular glycoprotein in the drainage outflow system of human eyes was determined by the indirect immunoperoxidase staining technique. The degree of fibronectin accumulation in ocular tissues was evaluated by quantitative morphometric analysis. It was shown that the fibronectin level was elevated in ageing. Increased deposit of fibronectin in trabecular tissues, mainly, in the inner wall of Schlemm's canal and juxta-canalicular zone was demonstrated along with ageing. Comparison of fibronectin accumulation in glaucoma and ageing support the idea that ageing is a risk factor of glaucoma.

Ultrastructural immunocytochemical localization of elastin in normal human trabecular meshwork.

Previous studies have suggested that hydrophobic moieties within the aqueous outflow channels might interact with certain aqueous components to retard outflow. While elastin is among the most hydrophobic proteins in the trabecular meshwork, it reacts poorly with conventional ultrastructural staining methods, so its potential role in regulating outflow could not be assessed. It was our goal to specifically localize elastin ultrastructurally using polyclonal antibodies against alpha elastin and its soluble precursor, tropoelastin. Human aorta served as a positive control. Preadsorption of the primary antibodies or their substitution with either normal rabbit serum or Tris buffer resulted in negligible labelling. With either antibody, only the electron-lucent elements in the center of elastic fibers of the trabecular meshwork were labelled, indicating that only these elements truly represent elastin. The pattern of elastin distribution within these fibers is most consistent with that found in tendons elsewhere in the body.

[Cytochemical localization of actin filaments in endothelial cells of rabbit trabecular meshwork].

Localization of actin filaments in the endothelial cells of rabbit trabecular meshwork was studied by the nitrobenzoxadiazole-phallacidin (NBD-ph) staining method for fluorescence microscopy and modified heavy meromyosin (HMM) decoration method for electron microscopy. Endothelial cells stained with NBD-ph exhibited intense fluorescence which was apparently associated with the basal plasma membrane area. By the modified HMM decoration method, labeled actin filaments were readily detected in the prefixed endothelial cells, because of the distinctive arrowhead-like appearance, observed beneath the basal plasma membrane facing the trabecular collagen sheet. The actin filaments were arranged with dual directionality within the bundle. In contrast, intermediate (10nm) filaments in the deeper region of endothelial cells were always unlabeled with HMM. The function of the actin filament bundles in endothelial cells may be to maintain the cell shape and provide contractility of the trabecular meshwork resulting in an alteration of the outflow resistance of aqueous humor drainage.

Analysis of the proteins of calf and cow trabecular meshwork: development of a model system to study aging effects and glaucoma.

The proteins from the trabecular meshwork (TM) of calf and cow eyes were analysed to determine if differences in composition were present and to examine whether this tissue could be used as a framework for the study of glaucoma. Differences in polypeptide composition or amount of protein could be detected with extractions using either acetic acid, neutral buffers and urea, or guanidine hydrochloride. In general, the results suggest that an aggregation of proteins may be occurring with aging. The acid-soluble fraction of both calf and cow TM resembled older human TM specimens with the most prominent protein around 68 kD. To test the utility of the bovine TM system, a mixed function oxidation system was used to determine how the proteins of the TM would react to oxidative stress. Aggregation of the proteins in calf TM as well as actin could be demonstrated, consistent with the idea that the aggregation seen in the cow TM might be the result of oxidation of this tissue. The present study lays a foundation for future work on bovine TM and is consistent with the hypothesis that aging changes in this tissue might be a result of oxidative processes.

Fibronectin detection in drainage outflow system of human eyes in ageing and progression of open-angle glaucoma.

The content of the fibronectin, an extracellular glycoprotein in the drainage outflow system of human eyes was determined by the indirect immunoperoxidase staining technique. The degree of fibronectin accumulation in ocular tissues was evaluated by quantitative morphometric analysis. It was shown that the fibronectin level was elevated in the ocular drainage outflow system of humans in ageing and was rapidly increased at different stages of primary open-angle glaucoma development. Increased deposit of fibronectin in trabecular tissues, mainly, in the inner wall of Schlemm's canal and juxtacanalicular, or cribriform part of trabecular meshwork, was demonstrated along with ageing and glaucoma disease progression. A hypothesis underlying the glaucomatous process as an adhesive impairment was proposed.

Immunomicroscopical study of type VI collagen in the trabecular meshwork of normal and glaucomatous eyes.

Cross-strained fiber bundles called long-spacing collagen or curly collagen occur in normal eyes in the trabecular meshwork. It can be seen in the basement membrane of the trabecular lamellae, in the sheath of the elastic-like fibers and underneath the inner wall of Schlemm's canal, where it forms part of the so called plaque material. The amount of this long-spacing collagen increases with age and is significantly more pronounced in glaucomatous eyes. Using immunohistochemical and immuno-electronmicroscopic methods, we have been able to show that type VI collagen is present in the aggregates called long-spacing collagen.

Tissue plasminogen activator in cultured human trabecular meshwork cells. Predominance of enzyme over plasminogen activator inhibitor.

Maintenance of patency of the trabecular meshwork, the major outflow channel of the anterior chamber of the eye, is necessary to prevent an excessive rise in intraocular pressure. Obstruction of flow due to clot formation results in severe glaucoma and damage to the optic nerve. We have found that human trabecular meshwork cells which have been passaged in tissue culture synthesize large amounts of tissue plasminogen activator (t-PA), based on functional, immunologic and molecular weight analysis. Trabecular cells express substantially more t-PA activity than vascular endothelium which produces t-PA for clot dissolution in the systemic circulation. Vascular cells produce excess t-PA inhibitor while trabecular cells make comparatively little. Trabecular meshwork cells are the first normal cell type reported in which the balance between t-PA and inhibitor is weighted towards the activator, indicating that fibrinolysis may be more important than clotting in the anterior chamber of the eye.

Human trabecular meshwork cells in culture: morphology and extracellular matrix components.

This study demonstrates the presence of laminin and collagen type IV in the extracellular space of human trabecular meshwork (HTM) cells in culture and its absence in cultures of fibroblasts from sclera adjacent to the outflow pathway. These basement membrane components can be detected by standard immunohistochemical techniques. Positive staining for these macromolecules is found only after the HTM cells have reached confluence. The presence of laminin and human collagen type IV in the early passages can serve as additional criteria for identification of HTM cells in culture. These cells provide a useful experimental system for studying the effects of drugs and other factors on the synthesis of components of the extracellular matrix.

Age-related changes in protein profiles of the normal human trabecular meshwork.

The protein profile of the trabecular meshwork from 44 normal human eyes of various ages, ranging from 5 months to 87 years, was investigated by using sodium dodecyl sulfate-polyacrylamide-gel (SDS-PAGE) electrophoresis and a highly sensitive silver-stain technique. Samples from each eye were analysed individually to avoid anomalies that may occur with tissue pooling. More than 20 protein bands were consistently visible at all ages and varied in molecular weight from 20,000 to 290,000 MW. The shifts which occurred in band prominence with increasing age included a consistent decrease in the intensity of bands at MWs 42,000 (tentatively identified as G-actin) and 58,000; a noticeable increase in the intensity of the bands at MWs 140,000 and 160,000 that correspond to alpha I and II components of type IV collagen; and a gradual disappearance of a faint band at MW 68,000 in eyes older than 31 years. Our study shows that molecular aging does occur in the trabecular meshwork. The data presented also provides a basis for comparison of the polypeptide alterations that may occur in primary open-angle glaucoma and in other disease processes.

Tissue plasminogen activator in the trabecular endothelium.

By quantitative analysis of tissue plasminogen activator (TPA) in the trabecular endothelium, corneal endothelium, and iris in the eyes of monkeys and dogs, we found significant levels of TPA activity. In a [125I]fibrin-coated well assay, the levels for the dog and monkey were, respectively: trabecular endothelium, 0.2 and 0.5; corneal endothelium, 0.8 and 0.5 IU per mg protein. The iris tissue showed high TPA activity, but its protein content could not be measured with the techniques employed. Activity in the aqueous humor was not detectable. By the ELISA technique, the values (in ng TPA/mg tissue protein) for the dog and monkey were, respectively: trabecular endothelium, 0.16 and 0.44; corneal endothelium, 0.48 and 0.92. Again, iris tissue showed high TPA activity, whereas the aqueous humor showed low activity (0.86 ng/ml). The data obtained with the two methods showed a reasonable consistency, although a direct comparison was not possible because two separate standards were used. The presence of TPA in the trabecular endothelium, corneal endothelium, and iris may be important in modulating the resistance to aqueous outflow under normal conditions as well as those of hyphema.

Beta-adrenergic receptors in human trabecular meshwork. Identification and autoradiographic localization.

Beta-adrenergic receptors were identified in slide-mounted sections of human trabecular meshwork by in-vitro labeling, light microscopic autoradiography. Autoradiograms were generated after incubation of slide-mounted tissue sections with 125I-cyanopindolol, a selective high-affinity probe for beta-adrenergic receptors. Experiments in which an excess of either unlabeled Practolol (beta 1 selective ligand) or Zinterol (beta 2 selective ligand) were included suggested that most of the receptors were of the beta 2 subtype. Data from displacement studies using increasing concentrations of the highly specific beta 1- and beta 2-adrenergic receptor antagonists, ICI-89,406 and ICI-118,551, respectively, confirmed the predominance of the beta 2-adrenergic receptors. Similar results obtained with cultured trabecular endothelial cells suggest that the beta-adrenergic receptors may be associated with the endothelial cells of the trabecular meshwork in vivo. These results provide anatomic evidence for the hypothesis that beta-adrenergic agents improve outflow by direct action on the trabecular meshwork and provide a rationale for the development of more selective beta 2-adrenergic agents to increase outflow facility.

Investigations of cytoskeletal elements in cultured bovine meshwork cells.

An ultrastructural, immunohistochemical, and functional study was conducted on cultured bovine meshwork cells. Particular emphasis was placed on the organization of the cytoskeleton, and the cells were viewed either as whole cells or following detergent extraction. For ultrastructural examination, several modes of viewing were adopted, including a detector situated above the specimens collecting secondary electrons (SE), a detector situated beneath the specimen collecting transmitted electrons (STEM), and conventional transmission electron microscopy at 100 KV (TEM). In whole cell mounts, information was obtained about the organization of the cytoskeleton and its relationship to other cytoplasmic organelles. Extraction procedures removed much of the plasma membrane and most organelles. The nucleus and cytoskeleton remained and stress fibers were prominent. Immunohistochemistry showed that the actin content of the cytoskeleton could be preserved after detergent extraction. Detergent-extracted cells decreased their surface area when exposed to MgATP in a dose-dependent manner. The decrease in surface area was associated with disassembly of cytoskeletal stress fibers and was optimal with 1 mM MgATP. Whether or not the change in surface area could be considered a "contractile event" was discussed.

Trabecular meshwork glycosaminoglycans in human and cynomolgus monkey eye.

The glycosaminoglycans (GAGs) extractable from the trabecular meshworks (TM) of human and non-human primate eye have been analyzed by sequential enzymatic degradation and cellulose acetate electrophoresis. For comparison, similar extracts of the cornea, sclera, iris, and ciliary body have also been analyzed. The distribution of glycosaminoglycans in human and in cynomolgus monkey TMs are similar, although not identical. The human TM contains hyaluronic acid (HA), chondroitin-4-sulfate and/or 6-sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), and an unidentified band of Alcian Blue staining material, which is resistant to the enzymes that we used. Based upon quantitation of the Alcian Blue staining intensities of extracted GAGs, which have been corrected by a relative dye-binding factor, the GAGs of the human TM include: 29.0% HA, 14.1% CS, 21.5% DS, 20.3% KS, and 15.0% HS. The cynomolgus monkey trabecular GAGs include: 12.8% HA, 14.3% CS, 15.2% DS, 42.1% KS, and 15.6% HS.

Fibronectin in human trabecular drainage channels.

Fibronectin, an extracellular glycoprotein, has been shown to be produced by human trabecular cells in culture by our group as well as Polansky and co-investigators. Studies of Rodrigues et al suggested that fibronectin may be one of several glycoproteins found in increased amounts in the corneoscleral trabecular meshwork of glaucomatous eyes. The authors have developed a sensitive immunoassay utilizing avidin-biotinylated enzyme complex (ABC) to detect low levels of fibronectin in frozen sections of human eyes. The authors have used this immunoassay together with a perfusion technique to demonstrate distribution patterns of fibronectin present in human aqueous drainage channels. The authors found that fibronectin is present in larger quantities in the aqueous drainage channels than in the surrounding tissues in 18 eyes from older patients.