Changes in HNK-1 epitope and collagen type IX in the aqueous humour of patients with pseudoexfoliation syndrome.

PURPOSE: To investigate alterations in the proteoglycan (PG) and glycosaminoglycan (GAG) content of the aqueous humour in patients with pseudoexfoliation syndrome (PEX). MATERIALS AND METHODS: Aqueous humor samples were obtained during cataract surgery from nineteen patients bearing PEX features and twenty-three age-matched normal controls. Protein and IgG were quantified densitometrically after their electrophoretic separation. Collagen type IX, 3-sulphoglucuronic acid (HNK-1 epitope), biglycan and heparan sulphate proteoglycans were detected in Western and dot blots by using specific monoclonal antibodies (MAbs). The immunochemical analysis was performed in native aqueous humour or after degradation of the glycosaminoglycans with chondroitinases. RESULTS: Degradation of the samples with chondroitinases ABC, AC and B revealed that, in the aqueous humour from PEX eyes, collagen type IX and biglycan had a more dermatan sulphate than did normal eyes. In addition, more HNK-1 epitope was observed in PEX eyes, which after similar enzymatic treatment was found to be located mainly in dermatan sulphate sequences. 3-sulphoglucuronic acid was a constituent of the GAG chains of the collagen type IX. We found that the electrophoretic mobility of the bands of collagen type IX and HNK-1 epitope was exactly the same in the aqueous humour of normal and PEX samples; both migrated as four bands at 120, 113, 92.6 and 56 kDa. The PGs bearing heparan sulphate were found only in normal samples. Other PGs were not detected. CONCLUSIONS: Because no significant difference was observed in the concentration of albumin and IgG in PEX and normal samples, the blood-aqueous barrier was probably not significantly compromised in PEX patients with cataract but without open-angle glaucoma. The results support the hypothesis that the pathogenesis of PEX can be linked to disturbed metabolism of GAGs and PGs.

An extensive system of extravascular smooth muscle cells exists in the choroid of the rabbit eye.

We investigated the structural organization of the choroid especially with regard to the presence of extravascular smooth muscle (EVSM) cells in albino rabbits. The eyes were fixed by intracardiac perfusion and processed for light, confocal, and electron microscopy. An unlabeled monoclonal antibody against alpha-actin of smooth muscle and a horseradish-peroxidase-conjugated secondary antibody were used for immunodetection of smooth muscle actin by light microscopy. For confocal microscopy, whole mount choroids were immunostained with a fluorescein isothiocyanate-conjugated (FITC) antibody. Our investigations revealed that the choroidal vessels are enveloped by bundles of EVSM. In contrast to the circular orientation of the smooth muscle cells of the tunica media of the choroidal vessels, the cells of the EVSM system were oriented longitudinally along the external surface of the vessel wall. The EVSM cells were strongly immunopositive for smooth muscle alpha-actin and exhibited a green fluorescence of the FITC-labeled anti-alpha-actin antibody. Individual cells were elongated and spindle-shaped, had the usual ultrastructural features of smooth muscle cells and, in places, were organized as 20 layers. EVSM cells were present throughout the thickness of the choroid, but not between the fenestrated endothelial lining of the choriocapillaris and Bruch's membrane, and extended from the optic nerve to the ciliary body where they merged with the ciliary muscles. Based on the three dimensional organization, immunoreactivity, and cellular and subcellular features of the EVSM system as well as information in the literature, we hypothesize that, functionally, this system, in conjunction with the choroidal vasculature, contributes to the myogenic control of choroidal blood flow and tissue volume, and also affects the intraocular pressure as well as the refractive and accommodative state of the eye.

Modulation of pre-mRNA splicing and protein production of fibronectin by TGF-beta2 in porcine trabecular cells.

PURPOSE: To determine the effect of transforming growth factor (TGF)-beta2 on the pre-mRNA splicing pattern of fibronectin, as well as on the synthesis and secretion of this glycoprotein by porcine trabecular cells. METHODS: First-passage porcine trabecular cells were rendered quiescent and incubated in culture medium containing 15% newborn calf serum, in serum-free culture medium containing either activated TGF-beta2 (concentration range: 0.2-2.7 ng/ml) or activated TGF-beta1 (1 ng/ml), or in serum-free medium alone (untreated control samples). For investigation of alternative splicing, total RNA was extracted, and reverse transcription-polymerase chain reaction (RT-PCR) was performed with primer pairs located in exons flanking the exon (extra domain [ED]A, or EDB) that undergoes alternative splicing. The polymerase chain reaction (PCR) products were verified by Southern hybridization and quantified by using laser densitometry. The percentage of EDA-positive (+) isoforms was compared with that of the EDB+ isoforms among the groups. To study the effect of TGF-beta2 on the synthesis and secretion of fibronectin, total protein was extracted from both cultured cells and conditioned medium, Western blot analysis was performed using an anti-fibronectin antibody, and the products were quantified by laser densitometry. Immunocytochemical analysis was also performed on cultured trabecular cells to detect fibronectin. RESULTS: Fibronectin mRNA that was detected in untreated serum-starved control cells was EDA and EDB negative. Incubation of trabecular cells in medium containing 1 ng/ml TGF-beta2, 1 ng/ml TGF-beta1, or 15% newborn calf serum induced the expression of EDA+ and EDB+ mRNA to varying degrees. At concentrations of 0.2, 0.5, 1.5, and 2.7 ng/ml, TGF-beta2 increased the concentration of fibronectin by 2-, 3-, 3.8-, and 5-fold in the conditioned medium, and by 3-, 3.7-, 4-, and 4.3-fold in the cell extracts, respectively. The trabecular cells treated with TGF-beta2 exhibited strong immunoreaction for fibronectin, whereas the cells incubated in serum-free medium showed only minimal immunoreactivity. CONCLUSIONS: Our results demonstrate that TGF-beta2 and TGF-beta1 modified the alternative splicing pattern of fibronectin pre-mRNA and enhanced the synthesis and secretion of this extracellular matrix molecule by trabecular cells in a dose-dependent fashion. These findings indicate a mechanism whereby TGF-beta2, the concentration of which is elevated in aqueous humor of patients with primary open-angle glaucoma, contributes to the increased deposition of extracellular matrix molecules in the outflow pathway.

Corticosteroids and glaucoma risk.

Corticosteroids (glucocorticoids), used frequently as potent anti-inflammatory agents, increase the risk of glaucoma by raising the intraocular pressure (IOP) when administered exogenously (topically, periocularly or systemically) and in certain conditions of increased endogenous production (e.g. Cushing's syndrome). Approximately 18 to 36% of the general population are corticosteroid responders. This response is increased to 46 to 92% in patients with primary open-angle glaucoma (POAG). Patients over 40 years of age and with certain systemic diseases (e.g. diabetes mellitus, high myopia) as well as relatives of patients with POAG are more vulnerable to corticosteroid-induced glaucoma. The association of corticosteroid-induced ocular hypertension in other conditions which are considered as risk factors for glaucoma (racial origins, hypertension, migraine, vasospasm) is likely but not fully established. The proposed mechanism of corticosteroid-induced glaucoma includes morphological and functional changes in the trabecular meshwork system and is similar to the pathogenesis of POAG. Trabecular cells exposed to corticosteroids in vitro show endoreplication of nuclei, an increase in cell size and excessive production of an approximately 56kD glycoprotein, identified as myocilin and transcribed by the GLC1A gene. Induction of ocular hypertension after corticosteroid administration depends on the specific drug, the dose, the frequency of administration and the corticosteroid responsiveness of the patient. The risk of corticosteroid-induced glaucoma can be minimised with judicious use of corticosteroids, as well as education of patients and medical practitioners. New treatment modalities include modified steroids and nonsteroidal anti-inflammatory agents that will have less effect on the elevation of IOP.

Increased level of vascular endothelial growth factor in aqueous humor of patients with neovascular glaucoma.

PURPOSE: This study aimed to quantitate and compare the concentration of vascular endothelial growth factor (VEGF) in aqueous humor samples from patients with neovascular glaucoma (NVG), primary open-angle glaucoma (POAG), and cataract, as well as in serum samples of healthy human subjects. METHODS: The authors collected aqueous humor samples by using their previously published technique of limbal paracentesis. The authors determined the concentration of VEGF by using a competitive enzyme immunoassay system and four-parameter logistic curve fitting and performed statistical analysis by using the Mann-Whitney-Wilcoxon test. RESULTS: The authors detected VEGF in 12 of 12 samples from patients with NVG (mean +/- standard error of the mean, 29.267 +/- 7.350 ng/ml), 15 of 28 samples from patients with POAG (0.726 +/- 0.204 ng/ml), 4 of 20 aqueous humor samples from patients with cataract (0.257 +/- 0.043 ng/ml), and 16 of 16 human serum samples (20.246 +/- 1.568 ng/ml). The mean concentration of VEGF in aqueous humor of patients with NVG was 40- and 113-fold higher than that in patients with POAG and cataract, respectively, and the difference was statistically significant (P < 0.01). The VEGF level in patients with POAG was elevated compared with that in patients with cataract (P < 0.05). Although the mean concentration of VEGF in aqueous humor of patients with NVG was approximately 1.45-fold higher than that in serum, the difference was not significant (P > 0.05). CONCLUSION: The authors' findings show that patients with NVG had a significantly increased level of VEGF in the aqueous humor and implicate VEGF as an important factor in the pathogenesis of intraocular neovascularization in these patients. The authors discuss the possible role of the ciliary epithelium, in addition to retina, in the production of VEGF and the complementary function of basic fibroblast growth factor and other growth factors.

Quantitative characterization of high- and low-affinity binding sites for basic fibroblast growth factor on trabecular cells of the eye.

By radioligand binding followed by Scatchard analysis, we characterized and quantitated the specific binding sites for bFGF on cultured trabecular meshwork cells obtained from freshly enucleated porcine eyes. We detected two binding sites: 1.67 x 10(4) +/- 5.75 x 10(2) high-affinity receptors per cell with a Kd of 33.4 +/- 7.90 pM, and 1.70 x 10(4) +/- 7.57 x 10(5) low-affinity binding sites per cell with a Kd of 3.84 +/- 1.41 nM. At low concentrations of 125I-bFGF (< 1.50 ng ml-1), binding was primarily determined by the high-affinity receptors and, at high concentrations (> 2.50 ng ml-1), binding was dependent on the low-affinity binding sites. By phase-contrast time-lapse video micrography and sequential photomicrography, we demonstrated that at a concentration of 1 ng ml-1, bFGF significantly stimulated the rate of mitosis of the trabecular meshwork cells in G0-phase compared with control cultures maintained in serum-free medium alone. Treatment with higher concentrations of bFGF did not reveal more potent effects on these cells. Our findings demonstrate that trabecular meshwork cells do possess low- and high-affinity receptors for bFGF and that bFGF induces these cells in vitro to re-enter the cell cycle. Because the low-affinity interactions of 125I-bFGF were reduced by 75% following pretreatment of the trabecular meshwork cells with heparinase, these sites represent cell-associated heparin-like molecules and heparan sulfate proteoglycans, and may control the bioavailability of bFGF to ocular tissues. Heparinase treatment also resulted in a 30% reduction in high-affinity binding, which may be secondary to the decreased low-affinity binding. This finding agrees with the well-established scheme for bFGF-receptor interaction. We conclude that bFGF at the concentration present in aqueous humor is capable of stimulating the mitotic activity of trabecular meshwork cells in vitro, suggesting a possible paracrine role of aqueous humour bFGF in vivo. The results obtained in this study, together with our previous findings on bFGF mRNA expression by trabecular meshwork cells and protein deposition in this tissue, also indicates that trabecular cells of the eye may utilize bFGF by an autocrine mechanism.

Age-related changes in trabecular cells in vitro.

The changes in the morphology and the amounts of selected extracellular matrix proteins in serially passaged porcine trabecular cells cultured from young animals (age range, 4-6 months) were investigated. After 4 weeks incubation, trabecular cells in primary confluent cultures had a flattened elongated profile, whereas the cells in secondary or tertiary cultures, after an additional 4 to 8 weeks incubation, respectively, were epithelioid in shape. Time-lapse micrography of the passaged cells revealed membrane ruffling at the cell periphery, but sparse cell movement. No mitotic events took place in either the secondary or tertiary cultures for 24 hr prior to harvesting the cells. No remarkable differences were observed in the total polypeptide profile of primary, secondary or tertiary cells analysed by SDS gel electrophoresis and silver staining. However, laser densitometry of immunoblots treated with antibodies against type VI collagen, thrombospondin, fibronectin, and laminin demonstrated that, compared to primary cultures of trabecular cells, the amount of type VI collagen was elevated 20.5-fold in tertiary cultures, thrombospondin 6-fold, and fibronectin 5-fold, but laminin was not detectable. Because aging trabecular cells in vitro exhibit specific biochemical characteristics that are comparable to those known to occur in the human trabecular meshwork in vivo, the present method provides a model for the investigation of age-related changes in this tissue at the cellular level under defined and controlled conditions.

Transforming growth factor-beta 1 and -beta 2 positively regulate TGF-beta 1 mRNA expression in trabecular cells.

PURPOSE: To determine whether transforming growth factor (TGF)-beta 1 and -beta 2 and basic fibroblast growth factor (bFGF) induce the gene expression of TGF-beta 1 in the first-passage trabecular meshwork cells of the eye. METHODS: Trabecular meshwork cells were cultured from fresh porcine eyes and treated with 1 ng/ml of TGF-beta 1, TGF-beta 2, or bFGF for 1 hour. Cells maintained in serum-free medium were used as controls. Total cellular RNA was extracted, and the first-strand cDNA was synthesized. Multiplex polymerase chain reaction (PCR) and competitive PCR were performed on aliquots of the cDNAs by using either endogenous (glyceraldehyde-3-phosphate dehydrogenase [G3PDH]) or exogenous sequence (PCR mimic for TGF-beta 1) as internal standards, respectively. The obtained products were quantitated by laser densitometry, and statistical analysis was performed. RESULTS: The findings show that trabecular cells in vitro express the TGF-beta 1 messenger RNA constitutively. Both the techniques of multiplex PCR and competitive PCR demonstrated that the addition of either TGF-beta 1 or TGF-beta 2 at a concentration present in normal aqueous humor increased the mRNA levels of TGF-beta 1 by 2.82-to 3.07-fold over the controls, and these results were statistically significant (P < 0.01). Basic fibroblast growth factor did not have an effect on TGF-beta 1 expression (P < 0.05). CONCLUSIONS: Transforming growth factor-beta 1 activates its own gene expression in trabecular cells. Considering the multifunctional property of this cytokine, which includes increased deposition of extracellular matrix material and growth inhibition of trabecular cells, a change in its concentration within the eye would have a profound effect because of this autoinductive activity. Transforming growth factor-beta 2 treatment of trabecular cells also increased their expression of the TGF-beta 1 gene. The authors previously showed that the level of TGF-beta 2 in the aqueous humor of glaucomatous eyes is significantly higher than that of age-matched nonglaucomatous controls. The current finding suggests that this growth modulator may exert its effects directly on the trabecular cells or that it may act indirectly through upregulating the production of TGF-beta 1.

Expression of growth factor mRNAs by human Tenon's capsule fibroblasts.

By using the techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern hybridization, we demonstrated that human Tenon's capsule fibroblasts in primary cultures, express the messenger RNA (mRNA) transcripts encoding transforming growth factor-beta 1 (TGF-beta 1), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF), but not those coding for aFGF and TGF-beta 2. Two PCR products were obtained for TGF-beta 1: a major fragment of 161 base pairs that corresponded to the expected size, and a minor sequence of 400 base pairs. An antisense oligonucleotide probe specific for TGF-beta 1 detected only the band of 161 base pair of PCR-amplified sequence. For bFGF, PDGF-A and PDGF-B, we obtained only a single PCR product with the anticipated length of 222, 225, and 217 base pairs, respectively. Southern blotting experiments revealed that these PCR-amplified fragments were specific for the respective growth factors. The negative control experiments without template did not reveal any amplification. No product for aFGF or TGF-beta 2 was detected. By radioimmunoassay, TGF-beta 1 protein was detected at the level of 24-30 pg ml-1 per 2 million Tenon's fibroblasts during a 24-hour period in acid-activated conditioned medium. These results indicate that human Tenon's capsule fibroblasts in primary cultures express TGF-beta 1 at the translational as well as at the transcriptional levels, and they also have the capacity to synthesize bFGF and PDGF. Considering the significant effects of bFGF, TGF-beta 1, and PDGF in wound healing response, these growth factors are implicated in tissue repair process after glaucoma filtration surgery that contributes to the failure of the procedure.

Aqueous humor in glaucomatous eyes contains an increased level of TGF-beta 2.

By using the highly sensitive and specific technique of enzyme-linked immunosorbent assay, we investigated the presence and amount of transforming growth factor-beta 2 (TGF-beta 2) in samples of aqueous humor obtained from 15 patients who had a clinically established diagnosis of advanced primary open-angle glaucoma (POAG), as well as from ten age-matched normal human subjects undergoing cataract surgery. The total amount of TGF-beta 2 in the samples of normal aqueous humor ranged from 0.41 to 2.24 ng ml-1 (mean +/- S.D.: 1.48 +/- 0.68 ng ml-1) of which 4.88 to 37.05% (11.99 +/- 9.95%) was intrinsically active. Compared with normal subjects, the aqueous humor from POAG patients had a statistically significantly greater amount of total TGF-beta 2 (2.70 +/- 0.76 ng ml-1, P < 0.01), as well as a higher level of intrinsically active TGF-beta 2 (0.45 +/- 0.28 ng ml-1, P < 0.05) which corresponded to 1.09 to 60.84% (18.33 +/- 15.50%) of the total amount. No linear correlation was found between the age of the subjects and the protein concentration of the aqueous humor from either normal or glaucomatous eyes, nor between the age of the patient and the total amount of TGF-beta 2. The negligible amount of TGF-beta 2 present in serum argues against its influx into the aqueous humor after breakdown of the blood-aqueous barrier that is known to occur in glaucomatous eyes; rather, our present findings support the concept of the intraocular derivation of this cytokine.(ABSTRACT TRUNCATED AT 250 WORDS)

Surgical removal of congenital pupillary-iris-lens membrane.

A pupillary membrane in a case of congenital pupillary-iris-lens membrane with goniodysgenesis was surgically peeled from the lens without causing cataract formation. Histopathology revealed ectopic iris. The ectopic iris found in this condition differentiates congenital pupillary-iris-lens membrane with goniodysgenesis as an entity from persistent pupillary membrane, hereditary goniodysgenesis, and Rieger's anomaly. We suggest that congenital pupillary-iris-lens membrane with goniodysgenesis is a neurocristopathy. The finding of ectopic iris muscle is consistent with avian chimera experiments that have suggested that iris sphincter muscle is derived from the neural crest, not neural ectoderm. Membranes in this condition can be successfully removed when they cause vision loss and amblyopia.

Trabecular cells express the TGF-beta 2 gene and secrete the cytokine.

We investigated whether trabecular cells contain TGF-beta 2 mRNA and whether they synthesize this cytokine. By using a primer pair for TGF-beta 2 and the highly specific and sensitive techniques of reverse transcriptase-polymerase chain reaction, a single amplified sequence was obtained from cultured trabecular cells and tissue samples of trabecular meshwork, corneal epithelium, iris and ciliary body ex vivo from fresh porcine eyes. Regression analysis with standard DNA size markers indicated that the products were of the anticipated size of 310 base pairs. Southern hybridization further confirmed that all of the products were specific for TGF-beta 2. Enzyme-linked immunosorbent assay (ELISA) with a specific antibody against TGF-beta 2 revealed that this cytokine was secreted by cultured trabecular cells in the conditioned medium. The TGF-beta 2 produced by trabecular cells was in a latent form, and no intrinsically active protein was detected. The results suggest that TGF-beta 2 has an autocrine and/or paracrine action in the trabecular meshwork system, and that trabecular cells could contribute to the presence of TGF-beta 2 in their microenvironment as well as in aqueous humor. It is hypothesized that abnormal changes in the production of TGF-beta s by trabecular cells and/or in the activation of these growth modulators contribute to the excess accumulation of extracellular matrix components in the aqueous outflow system as observed in aging and glaucomatous eyes.

Identification of type VI collagen in the trabecular meshwork and expression of its mRNA by trabecular cells.

To investigate the nature of the 140 kDa glycoprotein in the trabecular meshwork, polypeptides were extracted with either urea/sodium dodecyl sulfate (SDS)/beta-2-mercaptoethanol (BME) or guanidine hydrochloride followed by pepsin digestion. After electrophoresis and immunoblotting with anti-type-VI-collagen antibodies, a single fraction of molecular weight 140 kDa was identified in the urea/SDS/BME extracts. Pepsin solubilization revealed two immunoreactive fractions (molecular weights 75 and 85 kDa) that comigrated with purified, pepsin-solubilized type VI collagen. By using the polymerase chain reaction (PCR) and primers specific for the alpha 2(VI) chain of type VI collagen, a single PCR product was obtained, which corresponded to the expected size of 137 base pairs, from the total RNA extracted from the trabecular meshwork ex vivo. Southern hybridization with the antisense oligonucleotide probe of the alpha 2(VI) chain confirmed that the amplified sequence was specific. The results show that the trabecular meshwork contains a significant amount of type VI collagen and that trabecular cells express the mRNA coding for the alpha 2(VI) chain of this glycoprotein. The presence of type VI collagen in the trabecular meshwork is implicated in cell-extracellular matrix interactions at this site, and its abnormal accumulation in glaucomatous and aging eyes probably signifies a defect in the function of the trabecular cells in these eyes.

Fuchs' endothelial dystrophy of the cornea.

Fuchs' endothelial dystrophy of the cornea is a significant cause of corneal blindness in the United States. The disease is characterized by a slow, continuous loss of morphologically and physiologically altered endothelial cells, eventually leading to corneal edema. The endothelial cells synthesize a thickened Descemet's membrane with focal excrescences of altered basement membrane material (guttae). This review details the epidemiological, clinical, and laboratory data that have accumulated on Fuchs' dystrophy. Several hypotheses regarding the pathogenesis of Fuchs' dystrophy are discussed, including the possible influences of aberrant embryogenesis, hormones, and injury on the development of the disease. The current state of medical and surgical management is summarized, along with the future prospects for treatment.

Trabecular cells of the eye express messenger RNA for transforming growth factor-beta 1 and secrete this cytokine.

PURPOSE: To determine whether trabecular tissue in vivo and cultured trabecular cells have the messenger RNA transcript for transforming growth factor-beta 1 (TGF-beta 1), and to examine whether these cells synthesize and secrete TGF-beta 1 in vitro. METHODS: Total RNA was isolated from the trabecular meshwork, iris, and ciliary body freshly excised from porcine eyes as well as from cultured trabecular cells, and the reverse transcriptase-polymerase chain reaction and Southern hybridization were used for detection of TGF-beta 1 messenger RNA. The amount of TGF-beta 1 secreted by trabecular cells in culture was determined by radioimmunoassay. RESULTS: Excised whole trabecular tissue, iris, and ciliary body, as well as cultured trabecular cells expressed messenger RNA transcripts for TGF-beta 1. On the ethidium bromide-stained agarose gel, two PCR-amplified products (161 and 400 base pairs) were found in the total RNA isolated from cultured trabecular cells. The oligonucleotide probe specific for TGF-beta 1 detected only one band with the expected length of 161 base pairs. The secretion of TGF-beta 1 into conditioned medium was at the level of 16.7-20 pg/ml per 2 million trabecular cells during a 24-hr period. CONCLUSIONS: These investigations show that the trabecular meshwork, iris, and ciliary body in vivo express the messenger RNA transcript for TGF-beta 1, and that trabecular cells in vitro synthesize and secrete this cytokine. The TGF-beta 1 present in normal aqueous humor may be derived locally, at least in part, from the cells of the trabecular meshwork, iris, and ciliary body. Abnormal synthesis, secretion, activation, and clearance of TGF-beta 1 may contribute to the pathogenesis of many ocular disorders, including primary open-angle glaucoma.