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PubMed1998

Cultured human trabecular meshwork cells express functional growth factor receptors.

PURPOSE: To compare the mRNA expression of growth factor receptors in cultured human trabecular meshwork (HTM) cells with ex vivo HTM tissues and to determine whether HTM cells generate a physiologic response after exposure to exogenous growth factors. METHODS: The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of various growth factor receptor mRNAs using early passaged, cultured HTM cells from donors of several ages. RT-PCR on ex vivo HTM tissues from healthy donors and donors with glaucoma were also used to compare and contrast mRNA expression with cell culture results. After the exogenous administration of growth factors, cell proliferation and extracellular acidification rate studies were used to measure the functional responses of HTM cells to growth factors. RESULTS: Amplification products of the expected size for 15 growth factor receptors were detected in cultured HTM cells and in ex vivo HTM tissues. The administration of exogenous growth factors showed that (a) hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulinlike growth factor (IGF)-1, tumor necrosis factor (TNF) alpha, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and basic fibroblast growth factor (FGF-2) stimulated cell proliferation, whereas FGF-1 (acidic), transforming growth factor (TGF) alpha, interleukin (IL)-1alpha, nerve growth factor (NGF), and FGF-7 (keratinocyte growth factor [KGF]) had no significant influence on cell proliferation; (b) TGF-beta isoforms significantly inhibited EGF-stimulated trabecular meshwork cell proliferation; and (c) FGF-1 (acidic), TGF-alpha, EGF, IL-1alpha, IL-1beta, HGF, TNF-alpha, PDGF-AA, and IGF-1 significantly stimulated extracellular acidification, whereas FGF-2 (basic), FGF-7 (KGF), TGF-beta1-beta3 and NGF had no significant influence on extracellular acidification. CONCLUSIONS: These studies show that mRNA for numerous growth factor receptors can be detected in cultured HTM cells and in ex vivo HTM tissues. They also show that many of the receptors are functional, because exogenous growth factor administration elicits a physiologic response. In vivo, these receptors may be activated by growth factors present within the aqueous humor (aquecrine/paracrine) or by growth factors synthesized and released locally by trabecular meshwork cells themselves (autocrine). Specific growth factors acting through high-affinity receptors may be involved in maintaining the normal microenvironment of the HTM and also may be involved in the pathogenesis of primary open-angle glaucoma.

Adolescent↗
PubMed2005

Myocilin gene expression in the trabecular meshwork of rats in a steroid-induced ocular hypertension model.

PURPOSE: To investigate the expression pattern of myocilin in the trabecular meshwork of normal and dexamethasone-induced ocular hypertensive rat eyes. MATERIALS AND METHODS: An ocular hypertension model was generated by application of topical dexamethasone to rat eyes 4 times daily for 1, 2, and 4 weeks. Age-matched untreated eyes served as controls. The intraocular pressure (IOP) was monitored by electronic Tono-pen under anesthesia. The protein and mRNA levels of myocilin in the trabecular meshwork and endothelial lining of Schlemm's canal were investigated by immunohistochemistry and in situ hybridization, respectively. For semiquantitative evaluation, areas with positive staining were analyzed by a computer-assisted image-processing system with NIH software. RESULTS: The IOP in rat eyes was elevated after 2 weeks of topical dexamethasone treatment. Despite the IOP elevation, the protein and mRNA levels of myocilin in the trabecular meshwork and around Schlemm's canal in the steroid-treated eyes were not different from those of controls. CONCLUSION: No discernible changes in myocilin expression in the chamber angle indicate that myocilin may not be directly linked to ocular hypertension, at least not in rat eyes after relatively short-term steroid application.

Animals↗
PubMed1989

Effects of dopamine receptor activation on the level of cyclic AMP in the trabecular meshwork.

We examined the effects of dopamine and of a selective DA1 agonist, fenoldopam, on the levels of cyclic AMP in the trabecular meshwork, freshly excised from porcine and canine eyes. As measured by radioimmunoassay, fenoldopam at a concentration of 10(-5) M caused a 4-fold increase in the cyclic AMP content, from a basal level of 24.4 +/- 1.9 to 101.8 +/- 6.3 pmol/mg protein, of the trabecular meshwork samples from porcine eyes. In tissue samples from canine eyes, fenoldopam at a concentration of 10(-4) M increased the endogenous cyclic AMP level from a basal value of 26.0 +/- 4.6 to 64.2 +/- 5.7 pmol/mg protein. Dopamine, although less potent, produced a similar response. Preincubation with a DA1 receptor antagonist, SCH 23390, inhibited the increase in cyclic AMP levels by 90%. Such inhibition did not occur with the alpha- and beta-receptor antagonists phenoxybenzamine and propranolol, respectively. This investigation demonstrates that adenylate cyclase-coupled DA1 receptors are present in porcine and canine trabecular meshwork tissue.

Animals↗
PubMed2003

Carbohydrate binding proteins galectin-1 and galectin-3 in human trabecular meshwork.

PURPOSE: Galectins are a family of carbohydrate binding proteins involved in a variety of biological processes including cell-cell and cell-matrix interactions. We examined galectin-1 and galectin-3 to determine if galectins are expressed in the human trabecular meshwork and Schlemm's canal. METHODS: Human trabecular meshworks were dissected from donor eyes within 12 hr of death. Galectin-1 and galectin-3 expression was examined by RT-PCR and Western blot analysis. Immunohistochemistry of galectin-1 and galectin-3 was analyzed in normal and glaucomatous tissue. RESULTS: Expression of mRNA and protein of Galectin-1 (14 kDa) and galectin-3 (31 kDa) was found in the outflow pathway. Immunostaining revealed galectin-1 and galectin-3 throughout the meshwork, cells lining Schlemm's canal, and extracellular spaces in the inner and outer walls of the canal. Comparison of normal, POAG and PEX samples revealed no difference in location or intensity for either galectin-1 and galectin-3. CONCLUSION: Galectin-1 and galectin-3 are present in human trabecular meshwork of normal and glaucomatous eyes.

Aged↗
PubMed1996

[Establishment of in vitro culture of bovine trabecular meshwork cells and their phagocytosis].

OBJECTIVE: To study the role of abnormality of phagocytosis of trabecular meshwork cells in the pathogenesis of glaucoma. METHOD: In vitro trabecular meshwork cell culture was successfully established from excised bovine aqueous outflow pathway. Using latex microspheres 0.6 micron in diameter as markers, we investigated the phagocytosis of cultured bovine trabecular cells and its influential factors qualitatively and quantitatively. RESULTS: Immunohistochemical staining studies have shown that trabecular cells secrete fibronectin and laminin. It is shown that bovine trabecular cells in vitro had strong phagocytic ability to ingest the particles, the number of latex beads in cells was increased with the prolongation of time in culture, and by 24 hours of incubation, the average number of beads in each cell was 40.5 +/- 6.7, in the mean time marked morphological and damaging changes, even death, of trabecular cells occurred. Epinephrine and cortisone inhibited significantly the phagocytosis of cells (P < 0.05), while epidermal growth factor had no such effect. CONCLUSION: The phagocytosis of trabecular cells is important to keep aqueous humour outflow patent, and abnormal phagocytosis of trabecular cells might be related to the pathogenesis of primary glaucoma.

Animals↗
PubMed1980

[Electron microscopy studies of the trabecular meshwork in pigmentary glaucoma].

Trabeculectomy specimens from three patients with pigmentary glaucoma were investigated electron-microscopically. The pigment accumulations were highest in the uveal and inner corneoscleral trabecular meshwork. The outer lamellae and the cribriform region were hypochromic, while the endothelium of Schlemm's canal was unpigmented. The aqueous pathways of the inner trabecular meshwork were obstructed by hyperpigmented cells, cellular fragments and released pigment granules, those of the outer trabecular meshwork by depositions of extracellular material and collagen, by degenerative cells and by collapse of the trabecular lamellae.

Adult↗
PubMed1989

[Ultracytochemical study of Ca++-ATPase activity in rabbit trabecular meshwork].

Cytochemical localization of Ca++-ATPase activity in endothelial cells of rabbit trabecular meshwork was studied by the method of Ando et al. Ca++-ATPase activity was localized on the plasma membrane, mitochondria, endoplasmic reticulum, and the basal cytoplasmic matrix, which includes the actin filament bundles beneath the basal plasma membrane. Ca++-ATPase activity associated with membranous organelles was considerably reduced by quercetin, an inhibitor of ATP dependent Ca++-transport. Changes in the concentrations of Ca (1-10mM) greatly influenced the activity of the cytoplasmic matrix. The most intense activity was obtained at 1mM Ca. These findings suggest that Ca++-ATPase of mitochondria and sarcoplasmic reticulum is related to the active uptake of Ca++ by these structures, and the reaction within actin filament bundles may reflect actomyosin ATPase activity. The function of cAMP and actomyosin ATPase reaction may be to provide contractility of the trabecular meshwork resulting in an alteration of outflow resistance of aqueous humor drainage.

Animals↗
PubMed1989

Experimental formation of 100 nm periodic fibrils in the mouse corneal stroma and trabecular meshwork.

The so-called long-spacing collagen that exhibits approximately 100 nm periodicity has been observed in the interstitial connective tissue of various organs under normal and pathological conditions. Although we, as well as many other investigators, have reported an increasing amount of these structures with age in the human trabecular meshwork, the pathological significance and the mechanism of its formation are still unknown. We incubated mouse ocular tissues in culture medium containing 20 mM ATP and prepared them for electron microscopic observation according to the method of Bruns et al (J Cell Biol 103:393, 1986). After the incubation, abundant 100 nm periodic fibrils were observed in the corneal stroma and the region of the trabecular meshwork, both of which show no structure of 100 nm periodicity under normal conditions. For the experimental formation of 100 nm periodic fibrils, ATP, acidic condition and temperature around 37 degrees C are necessary. The 100 nm periodic fibrils observed in our experiment were very similar to long-spacing collagen, in that the dark transverse bands have 100 nm intervals and very fine filaments of 6-7 nm diameter axially connect the bands. Long-spacing collagen is not usually observed in the human cornea, even in aged persons. The results of our study suggest that the occurrence of long-spacing collagen is related to special conditions developing in the trabecular meshwork with age but not in the corneal stroma. Experimental studies of 100 nm periodic fibril formation in mice offer a useful model for the age-related increase of long-spacing collagen in the trabecular meshwork of the human eye.

Adenosine Triphosphate↗
PubMed1998

Glycoproteins of trabecular meshwork, cornea and sclera.

PURPOSE: To analyse high-molecular-weight matrix glycoproteins in trabecular meshwork, cornea and sclera using SDS/PAGE and immuno- and lectin blotting. METHOD: Extracts of normal trabecular meshwork (TM), cornea and sclera were analysed under reducing conditions on SDS/ PAGE. Western blots were stained for total protein, and major high-molecular-weight components were identified by immunoblotting with antibodies to fibronectin (FN) and type VI collagen. Lectin blotting with PSA, MPA and DSA identified some of the glycoprotein glycans. RESULTS: FN antibody bound to the 240 kDa band in TM, cornea and sclera. Type VI collagen antibody bound more strongly to one band and less so to two other bands at approximately 200 kDA in normal TM and to a ladder of bands in cornea and sclera. PSA and DSA bound at 240, 200 and 140 kDa in TM, cornea and sclera. MPA bound at 240, 200 and 140 kDa in TM and at 240, 200 and approximately 120 kDA in cornea and sclera. CONCLUSIONS: FN is a component of the band at 240 kDA in TM, cornea and sclera. Normal TM was found to contain relatively more of one of the isoforms of the alpha 3 (VI) chain whilst cornea and sclera contained all the alpha 3 (VI) isoforms. Complex N-linked bi/tri-antennary glycans were localised in FN and the alpha 1, alpha 2 and alpha 3 (VI) chains in TM, cornea and sclera. O-linked glycans (identified by MPA binding) were located in FN and alpha 3 (VI) chains of TM, cornea and sclera.

Aged↗
PubMed2005

Cationic ferritin and segmental flow through the trabecular meshwork.

PURPOSE: To determine whether segmental labeling by the tracer molecule cationic ferritin (CF) is indicative of preferential patterns of fluid flow in the trabecular meshwork or of differences in cell and extracellular matrix properties. Nonlabeled regions could indicate no fluid entering that area, insufficient perfusion time, or that the cells and extracellular matrix differ in that region and cannot bind CF. METHODS: Six whole eyes (three normal and three with pseudoexfoliation [PEX]) syndrome were perfused with CF for 30 minutes to 4 hours. Wedges of trabecular meshwork were dissected and some wedges immediately fixed. Adjacent wedges were placed in a CF bath before fixation. Transmission electron microscopy was used to analyze CF labeling. RESULTS: CF increased in the trabecular meshwork with increasing perfusion time. At 30 minutes, CF labeled mainly the uveal and corneoscleral regions. By 4 hours, CF was found diffusely through the meshwork, although a few isolated nonlabeled areas were still present. Wedges immersed in the CF bath showed fewer nonlabeled regions at all time points. Clumps of PEX material labeled more heavily in the periphery than the center, suggesting the clumps were less permeable than surrounding regions. PEX eyes otherwise had similar labeling patterns. CONCLUSIONS: Segmental labeling with CF implies regions of preferential flow exist in the meshwork. With increasing perfusion time, there were fewer nonlabeled regions. CF labeling of most regions of bath-immersed tissue suggests that nonlabeled regions do not differ in the characteristics of the cells, but rather that CF does not reach these regions.

Aged↗
PubMed1986

Prostaglandin synthesis and release from cultured human trabecular-meshwork cells and scleral fibroblasts.

Human trabecular-meshwork (HTM) cells in culture convert arachidonic acid to two products: PGE2 and 6-keto PGF1 alpha. Prostaglandin PGE2 was the major product of arachidonic-acid metabolism. The synthesis and release of PGE2 and 6-keto PGF1 alpha was inhibited by a 15-min pre-treatment with indomethacin (5 X 10(-6) M) or a 4-24-hr incubation with 10(-7) M dexamethasone. The effects of dexamethasone could be prevented by cycloheximide (0.5 micrograms ml-1), or actinomycin D (2 micrograms ml-1). Prostaglandin E2 synthesis and release by these HTM cells from two different individuals could be stimulated by bradykinin, arachidonic acid and the calcium ionophore, A23187.

6-Ketoprostaglandin F1 alpha↗
PubMed1998

Glucocorticoid effects on extracellular matrix proteins and integrins in bovine trabecular meshwork cells in relation to glaucoma.

The trabecular meshwork (TM) is a specialized eye tissue essential for regulation of the aqueous humor outflow and control of the intraocular pressure. Disturbances of TM cells may lead to elevated intraocular pressure and glaucoma. This study assessed the dexamethasone effects on levels of extracellular matrix proteins and their integrin receptors in bovine TM cells. Instillation of glucocorticoids such as dexamethasone is known to result in ocular hypertension. The histologic changes induced resemble those seen in glaucoma. Examination of the effects of glucocorticoid therefore may provide insights into the pathogenesis of glaucoma. TM cells in either tissue culture or organ cultures were treated with 0 (control), 0.1, or 1 microM of dexamethasone for 72 h. Immunostaining, Western, Northern and dot blot analyses showed that dexamethasone caused an increase in levels of fibronectin and collagen type IV in tissue-cultured TM cells. Increased focal contacts were also observed but the levels of laminin and collagen type I were unaffected. The dexamethasone effect was similarly demonstrated in organ cultures, with the exception that collagen type I also was enhanced. These results suggest that dexamethasone modulates extracellular matrices in the TM. Glucocorticoid may exert its effect through such a modulation in the development of steroid glaucoma.

Animals↗
PubMed2001

Involvement of phosphorylation of myosin phosphatase by ROCK in trabecular meshwork and ciliary muscle contraction.

The control of smooth muscle contraction is an important factor in maintaining normal intraocular pressure. However, the specific factors causing changes in control by phosphorylation/dephosphorylation schemes in the eye are not well-defined. The purposes of this experiment were to (i) determine the localization of ROCK (Rho-associated, coiled coil-forming kinase) in monkey and rabbit eye tissues and (ii) measure phosphorylation of ROCK substrate during trabecular meshwork or ciliary muscle contraction induced by carbachol. We found that mRNAs for both ROCK I and II were expressed in most eye tissues from rabbit and monkey. Proteins for ROCK I and II were present in all eye tissues studied except lens. When trabecular meshwork or ciliary muscle were incubated with carbachol to induce contraction, phosphorylation of the myosin-binding subunit (MBS) of myosin phosphatase, a substrate for ROCK, started within 1 min and continued for at least 1 h. This phosphorylation was well correlated with contraction of trabecular meshwork or ciliary muscle. These results suggested that ROCK might regulate contraction of trabecular meshwork or ciliary muscle through phosphorylation of MBS of myosin phosphatase.

Animals↗
PubMed2003

Electrophysiological response of cultured trabecular meshwork cells to synthetic ion channels.

The response of living cells of the trabecular meshwork to synthetic ion channels is described. The THF-gramicidin hybrids THF-gram and THF-gram-TBDPS as well as a linked gA-TBDPS and gramicidin A were applied to cultured ocular trabecular meshwork cells. THF-gram application (minimal concentration, 10(-8) M; saturation, 10(-7) M) led to an additional conductance which displayed characteristics of weak Eisenman-I-selective cation channels, no cell destruction, an asymmetric change of the inward/outward currents, and higher current densities using Cs(+) as charge carrier compared to Na(+) and K(+). Linked-gA-TBDPS showed at 10(-12) M increases of the membrane conductance comparable to gA at 10(-7) M and a much faster response of the cells. Thus, THF-gramicidin hybrids form a basis for the use of synthetic ion channels in biological systems, which eventually may lead to new therapeutic approaches.

Animals↗
PubMed1998

Expression of CD44 isoforms in cultured human trabecular meshwork cells.

PURPOSE: To determine the expression of CD44 isoforms in cultured human trabecular meshwork (HTM) and to discuss their possible relationship with outflow facility. METHODS: CD44 isoform expression in cultured HTM was qualitatively examined using immunohistochemistry and RT-PCR analysis. RESULTS: Immunohistochemistry of cultured HTM showed intense staining with a CD44s antibody, and with antibodies against CD44 exon 7, exon 11-12 and exon 14. By RT-PCR, at least three isoforms of CD44 were expressed in HTM: CD44s, CD44v-III and CD44v-I. CONCLUSIONS: At least three isoforms of CD44 are expressed in the HTM. CD44 may play a role in binding and turnover of hyaluronic acid in the trabecular meshwork, thereby regulating outflow facility.

Cells, Cultured↗
PubMed2005

Melanin in the trabecular meshwork is associated with age, POAG but not Latanoprost treatment. A masked morphometric study.

We wished to conduct a light and electron microscopic investigation of pigmentation within the trabecular meshwork of normals and primary open angle glaucoma (POAG) patients. In particular we wished to get a precise determination of whether there was a relationship between pigmentation and age. In addition we wanted to know if there was a difference between normals and POAGs and whether trabecular meshwork hyperpigmentation was associated with topical latanoprost medication. A total of 25 sham trabeculectomies conducted on post mortem donor eyes provided the age-matched normals and there were 62 trabeculectomy specimens from POAG patients. These were masked and the meshwork subjected to qualitative and quantitative morphological investigation. Light and electron microscopy confirmed that most of the trabecular meshwork melanin was phagocytosed and within meshwork cells. The granules were measured and found to be of the large iris epithelial type. Light microscopic morphometric analysis showed that the number of meshwork cell profiles that contained melanin increased both in normals and POAGs with age. However there was nearly three times more pigmented meshwork cells in the POAGs than the normals. The POAGs were divided into three groups of (1) minimal or no medication prior to surgery, (2) maximal medical therapy and (3) maximum medical therapy including latanoprost (12 specimens). All groups were significantly greater that the normals but of the three it was the maximal medical therapy group (without latanoprost) that had the highest pigmentation. We concluded that pigmentation of the meshwork is age-related and it is elevated in POAG by mechanisms unknown. The melanin accumulation seems to be partly due to the disease process, partly as a consequence of chronic antiglaucoma medication but interestingly not due to latanoprost even in patients where there is iris darkening (four specimens).

Administration, Topical↗
PubMed1984

Aqueous outflow in the beagle. II. Postnatal morphologic development of the iridocorneal angle: corneoscleral trabecular meshwork and angular aqueous plexus.

The morphology and postnatal ontogeny of the major pathway for aqueous outflow in the canine eye has been examined. Normal canine chamber angles of the Beagle at one day, one through ten weeks, three, four and six months, and one and two years of age were used. Angle tissue was prepared and examined by standard techniques for light and electron microscopy. At birth the corneoscleral trabecular meshwork largely consisted of spongy mesenchyme. By the end of the first month the corneoscleral trabecular meshwork developed into a series of longitudinally oriented sheets anteriorly covered by the peripheral extremity of the corneal endothelium. The angular aqueous plexus (trabecular veins), which was formed at birth, was continually separated from the corneoscleral meshwork by an endothelial lining. Transcellular channels interconnected the trabecular veins and the corneoscleral trabecular meshwork. The corneoscleral trabecular meshwork and angular aqueous plexus in the canine angle are similar topographically, structurally and ultrastructurally to that described in other nonprimate and primate angles.

Aging↗
PubMed1986

Glucose 6-phosphate dehydrogenase of calf trabecular meshwork.

Activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of calf trabecular meshwork were measured and found to be 0.23 and 0.47 mumole/min/g tissue, respectively. Glucose 6-phosphate dehydrogenase was purified 450-fold with a yield of 91% by anion exchange chromatography and 2',5'-ADP agarose affinity chromatography. It was activated by Ca2+, Mg2+, and Mn2+. It was deactivated by p-chloromecuribenzoate, p-chloromercuribenzene sulfonate, and iodoacetamide, but this deactivation could be prevented by pretreatment with cysteine or glutathione. Its rate was regulated by the NADPH/NADP+ ratio, being maximal at a ratio of 0, and negligible at a ratio of 10. At the physiological ratio of 5, its rate was approximately half maximal. On disc gel electrophoresis of both the crude and purified enzyme, seven bands of glucose 6-phosphate dehydrogenase activity could be seen. The isozyme pattern was similar to that of calf retina, but different from that of calf liver. These data suggest that trabecular meshwork is well supplied with the capacity to generate NADPH. Typical demands for NADPH may be to detoxify H2O2 and/or organic peroxides through the glutathione peroxidase/glutathione reductase system, and both generating and removing products of the "killing reaction" during phagocytosis.

Animals↗