Elke Lütjen-Drecoll

Cellular Pharmacology and Molecular Biology of the Trabecular Meshwork Inducible glucocorticoid Response Gene Product

Studies of the effects of glucocorticoid (GC) and oxidative stress stimuli in differentiated cultures of human trabecular meshwork (HTM) cells have provided the rationale for our studies of a major new gene termed TIGR (trabecular meshwork inducible GC response). The TIGR clone was isolated by differential library screening using selection criteria based on the induction pattern of a new protein/glycoprotein found in HTM cultures after prolonged but not brief exposure to GCs. This GC induction pattern matched the time course and dose response required for intraocular pressure elevation in patients receiving corticosteroids. The very large, progressive induction of TIGR combined with specific structural features of its cDNA suggested that TIGR should be considered a candidate gene for outflow obstruction in glaucoma. Among the properties of TIGR cDNA were a signal sequence for secretion, several structural features for interactions with glycosaminoglycans and other glycoproteins and putative sites for cell surface interactions. In addition, the leucine zippers in the structure were related to TIGR-TIGR oligomerization that was shown to occur with native and recombinant TIGR protein. The verification that TIGR was a major stress response protein in HTM cells following hydrogen peroxide (or phorbol esters) exposure provided a potential link between GC and oxidative mechanisms thought to be involved in glaucoma pathogenesis. Pharmacological evaluation showed that basic fibroblast growth factory and transforming growth factor beta decreased the GC induction of TIGR, and certain nonsteroidal anti-inflammatory drugs protected against both GC- and oxidation-induced stress responses in HTM cells. Our recent studies of TIGRs genomic structure have shown motifs in the promoter region that suggest a basis by which multiple hormonal/environmental stimuli can regulate TIGR production and by which putative genetic alterations could lead to an overexpression of the protein. Further application of cell biology/biochemistry, molecular biology, genetic and histological approaches will be helpful in understanding the role of TIGR in different glaucoma syndromes.

Transforming growth factor β2 levels in the aqueous humor in different types of glaucoma and the relation to filtering bleb development

Abstract. Purpose: To investigate the transforming growth factor β2 (TGF-β2) levels and total protein levels in the aqueous humor of eyes with different types of glaucoma [primary open-angle glaucoma (POAG), pseudoexfoliation glaucoma (PSX), juvenile glaucoma (JG)], and the relation to filtering bleb development after trabeculectomy. Methods: Aqueous humor was collected at the beginning of surgery from 52 eyes with glaucoma (29 POAG eyes, 17 PSX eyes, 6 JG eyes) and from 29 control eyes that underwent cataract operation. TGF-β2 levels (intrinsically activated and total TGF-β2 ) using ELISA methods as well as total protein concentrations of the aqueous humor were determined. All preoperative clinical data of the glaucoma eyes (age, gender, IOP, previous treatment, type of surgery) were compared with the TGF-β2 levels. In 40 of these eyes, the postoperative follow-up (filtering bleb development, need for intervention, IOP) was correlated to the preoperatively determined TGF-β2 levels. Results: TGF-β2 levels were increased in nearly half of the eyes with POAG and in most of the eyes with JG, but in eyes with PSX, TGF-β2 levels were within the normal range. No correlation between TGF-β2 levels and age, gender, IOP, previous treatment, or type of surgery, or between TGF-β2 levels and protein levels in aqueous humor, was found. Correlation between bleb formation and TGF-β2 levels revealed that all but two of the POAG eyes with good clinical outcome (type 1 bleb) had normal levels of activated TGF-β2. Of the 13 eyes that needed postoperative intervention (type 2 and type 3 bleb), 8 had high and 5 had normal TGF-β2 levels. Conclusions: PSX eyes differ from POAG and JG eyes not only by their clinical or biomicroscopic appearance, but also by their normal TGF-β2 levels in aqueous humor. The fact that most of the POAG eyes with favorable bleb development had normal TGF-β2 levels indicated that there might be some relationship between bleb formation and TGF-β2 levels. On the other hand, the fact that eyes with less favorable bleb development had both low and high TGF-β2 levels indicated that other factors are also involved in the scarring of the filtration bleb.

Quantitative analysis of ‘plaque material’ in the inner- and outer wall of Schlemm's canal in normal- and glaucomatous eyes

The amount of plaque material derived from the sheaths of the elastic-like fibers in the cribriform layer of the trabecular meshwork (sheath-derived or SD plaques) was quantitatively evaluated in normal eyes and in trabeculectomy specimens of chronic simple, intermittent angle closure, and pseudoexfoliation glaucoma. The amount of SD plaque material increases with age but no correlation between age and SD plaques was found in any of the glaucomas evaluated. In comparison to normal eyes, the amount of SD plaque material was significantly greater in cases of chronic simple glaucoma and in cases of intermittent narrow angle glaucoma, but not in pseudoexfoliation glaucoma. In the latter, number and distribution of SD plaques resembled those of normal eyes. SD plaques were also found in the outer wall of Schlemms canal. In normal eyes there was a correlation between outer-wall plaques and age as well as between inner- and outer-wall plaques. These correlations were not found in the different types of glaucoma. Our findings indicate that in addition to the age-related increase in plaque material, there is in glaucomatous eyes some plaque material which is distributed unevenly in the inner- and outer-wall of Schlemms canal. This additional material was not found in pseudoexfoliation glaucoma, indicating that elevated intraocular pressure alone can not be responsible for plaque formation.

Functional morphology of the trabecular meshwork in primate eyes.

The trabecular meshwork forms most of the resistance to aqueous humor outflow needed for maintenance of a pressure gradient between intraocular pressure of approximately 17 mmHg and venous pressure of approximately 10 mmHg. The composition of the extracellular material in the subendothelial or cribriform layer seems to be mainly responsible for outflow resistance. The aqueous humor pathways through the subendothelial layer can be influenced by ciliary muscle contraction and presumably also by contractile elements recently found both in trabecular meshwork and scleral spur. Pharmacologically induced disconnection of inner wall and cribriform cells leads to wash out of extracellular material through breaks of the endothelial lining of Schlemms canal and to increase of outflow facility. In glaucomatous eyes the resistance to aqueous humor outflow is increased due to an increase in different forms of extracellular material deposited within the cribriform layer. The amount of this newly developed extracellular material is correlated with loss of axons in the optic nerve, indicating that a common factor is responsible for both changes. To investigate the effect of various factors on the biology of trabecular cells monolayer cultures derived from cribriform and corneoscleral trabecular meshwork have been established. The two cell lines can be differentiated because cribriform cells in vivo as in vitro stain for alphabeta-crystallin whereas the corneoscleral cells remain unstained. The effect of TGFbeta, a growth factor increased in aqueous humor of glaucomatous eyes and glycocorticoids on trabecular meshwork cells show typical changes in formation of extracellular matrix components and of stress proteins. Dexamethasone and oxidative damage also lead to increase of trabecular meshwork inducible glucocorticoid response (TIGR) protein. A mutation of the TIGR-gene family has recently been found in families with juvenile and chronic simple glaucoma. Future research has to clarify the significance of these genetic factors for the pathophysiology of glaucoma and the role of trabecular cell activity in this respect.

Morphological study of the anterior segment of cynomolgus monkey eyes following treatment with prostaglandin F2α

Topically applied prostaglandin F2 alpha has been shown to lower intraocular pressure in cynomolgus monkeys. In this study the morphological changes, following topical treatment with 4-50 micrograms of prostaglandin F2 alpha for 4-8 days, were investigated. Semiquantitation of (1) accumulation of white blood cells as one sign of inflammation, (2) edema and (3) enlarged spaces between ciliary muscle cells were performed. Eighty sections per eye encompassing the whole circumference were investigated. No accumulation of white blood cells was seen in any of the eyes. Slight edema in the most anterior part of the ciliary processes occurred in most eyes, but only in part of the circumference. These changes could be either directly induced by the prostaglandin treatment or secondary to the decrease in intraocular pressure. The most pronounced change was the dilatation of the intramuscular spaces. These enlarged spaces could explain the physiologically shown increase in uveoscleral outflow.

The effect of TGF-β2 on human trabecular meshwork extracellular proteolytic system

Our study aimed to investigate whether transforming growth factor-beta2 (TGF-beta2), increased in the aqueous humor of eyes with primary open angle glaucoma (POAG), can affect factors responsible for the activity of matrix metalloproteinases (MMPs) in human trabecular cell cultures. With this goal in mind cultures of human trabecular meshwork (hTM) cells derived from 8 donors were treated with TGF-beta2 for 24, 36 and 48 hr. Influence of TGF-beta2 on expression of MMP-2, MMP-9, membrane type 1-MMP (MT1-MMP) and plasminogen activator inhibitor-1 (PAI-1) was examined using RT-PCR, Northern Blot, Western Blot and zymography. The influence of TGF-beta2 treatment on PAI-1 expression was also investigated using immunohistochemistry. It appeared that treatment with TGF-beta2 significantly increased expression of the proform of MMP-2, whereas the active form was not detectable. MMP-9 and MT1-MMP expression were not influenced by TGF-beta2 treatment. There was, however, a significant increase in PAI-1 expression. To investigate whether transformation of the proform of MMP-2 to the active form was inhibited by PAI-1, the influence of treatment with TGF-beta2 and a PAI-1 neutralizing antibody on MMP-2 was investigated using zymogram method. With this treatment protocol the active form of MMP-2 was clearly visible, indicating that TGF-beta2 enhancement of the PAI-1-expression decreases MMP activity. Inhibition of MMP activity through elevated levels of TGF-beta2 might contribute to the increase in ECM in the trabecular meshwork of glaucomatous eyes.

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 Schlemms 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.

Regulation of outflow rate and resistance in the perfused anterior segment of the bovine eye

Contractile properties of isolated trabecular meshwork strips have recently been described. In the present paper we characterize the regulation of the outflow pathway in the isolated perfused anterior segment of the bovine eye. Anterior segments of bovine eyes with detached iris, ciliary body and ciliary muscle were perfused at constant pressure of 8.8 mmHg. A constant outflow of approximately 6-8 microliters min-1 could be obtained for at least 3 hr. The calculated outflow resistance was in the range 1.1-1.4 mmHg min microliter-1. The relative outflow was significantly reduced after application of carbachol, reaching a maximal inhibition of 30%. EC50 for carbachol was 3 x 10(-8) mol l-1. Atropin completely blocked the effect of carbachol on outflow. Morphological examination of perfused anterior segments which were perfused with carbachol revealed an intact fine structure of the meshwork cells. Pilocarpine at 10(-5) mol l-1 reduced outflow by 15%. Epinephrine at 10(-5) mol l-1 reduced outflow, while epinephrine at 10(-6) mol l-1 slightly increased the outflow rate. This effect could be blocked by metipranolol. Endothelin-1 in concentrations of 2 x 10(-9) and 2 x 10(-8) mol l-1 inhibited relative outflow by > 30%. Carbachol, pilocarpine, endothelin and a high dose of epinephrine, which have been shown to induce contractions in isolated bovine trabecular meshwork and ciliary muscle strips, induced a reduction of outflow rate and an increase of outflow resistance of the anterior segment. Thus, at least in the bovine eye, the trabecular meshwork per se is directly involved in the regulation of aqueous humor outflow.

Carbonic anhydrase distribution in the human and monkey eye by light and electron microscopy

The distribution of carbonic anhydrase (CA) in the eye of man and three species of monkey was studied by light and electron microscopy using the histochemical method of Hansson. Carbonic anhydrase staining was found in the corneal endothelium. In monkeys the endothelial cells covering the inner surface of the operculum were also stained, whereas in the human the staining stopped at Schwalbes line. In the trabecular meshwork no cells exhibited CA staining. The iris dilator muscle showed some staining, while no clear staining was found in the pigment epithelium (PE). In the ciliary body both the PE and the non-pigmented epithelium (NPE) displayed CA staining, most prominently in the basal and lateral membranes but also discernible in the cytoplasm. The staining of the NPE (but not the PE) showed clear-cut regional differences, and the presence of CA coincided with morphological indicators of secretory activity. Heavy staining was found in the capillaries under the ciliary epithelium. In the pars plana many capillaries showed staining of only that part of the circumference of the vessel wall which faced the epithelium. Unstained segments were also found in many choroidal capillaries under the pigmented epithelium of the retina. In the retina itself CA stain was found in the pigmented epithelium, in the Müller cells and in capillaries supplying the inner retina (only monkey retina was studied). On the whole, the distribution of CA in the primate eye was found to be similar to that of other species. The main difference compared with the rabbit eye was that was CA staining in the capillaries of the primate eye much more prominent. The functional significance of this species difference is not clear at present.

Visualization of hyaluronic acid in the anterior segment of rabbit and monkey eyes

Hyaluronic acid was visualized using a new histochemical technique: the hyaluronic acid-binding region of the cartilage proteoglycan was isolated, linked with biotin and used in histological sections as a ligand for hyaluronic acid. Staining was performed using the avidin-biotin-peroxidase technique. The presence of hyaluronic acid in the anterior segments of rabbit and monkey eyes was studied in fresh-frozen, as well as in fixed paraffin sections; addition of cetyl-pyridinium chloride to the fixative was essential. In both species intense staining was seen in the stroma of the conjunctiva, the connective tissue of the ciliary processes, and on the apical membranes of the corneal endothelium. Species differences were observed in both conventional and unconventional outflow pathways. In rabbits, the tissue located directly adjacent to the aqueous plexus and the connective tissue surrounding the poorly developed ciliary muscle were stained. In monkeys, only the anterior, non-filtering part of the trabecular meshwork and the outer wall of Schlemms canal showed intense staining in paraffin sections. In frozen sections, the inner wall (cribriform region of the trabecular meshwork) was also stained. The spaces between the anterior part of the ciliary muscle bundles remained almost unstained.