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(Investigative Ophthalmology and Visual Science. 2004;45:153-158.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0796
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Effects of TGF-ß2 in Perfused Human Eyes

Johannes Gottanka,1 Darren Chan,2 Michael Eichhorn,1 Elke Lütjen-Drecoll,1 and C. Ross Ethier2,3

1From the Department of Anatomy 2, University of Erlangen-Nuremburg, Erlangen, Germany; and the 2Department of Mechanical and Industrial Engineering and the 3Department of Ophthalmology, University of Toronto, Toronto, Ontario, Canada.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. TGF-ß2 is known to be present at elevated levels in the aqueous humor of patients with primary open-angle glaucoma (POAG). Studies have shown that TGF-ß2 influences cultured trabecular meshwork (TM) cells, but the effects of this cytokine on intact TM and outflow facility have not been studied. The purpose of this study was to investigate whether TGF-ß2 treatment induces changes in outflow facility and morphologic changes in the TM tissue and whether these changes are comparable to those previously recorded in glaucomatous eyes.

METHODS. Baseline facility was measured in paired human eyes (n = 8 pairs), with a constant-flow anterior segment culture system. Medium perfusing experimental eyes was then supplemented with activated human recombinant TGF-ß2 (3.0 ng/mL, comparable to or slightly greater than measured aqueous humor levels in patients with POAG), and facility was measured for at least 8 days. At the conclusion of the perfusion, eyes were fixed and processed for light microscopy, transmission electron microscopy, and immunolabeling studies.

RESULTS. TGF-ß2 perfusion reduced outflow facility by 27% (P = 0.03) and promoted focal accumulation of fine fibrillar extracellular material in multilayered structures under the inner wall of Schlemm’s canal. In treated eyes, Schlemm’s canal was 27% shorter (P = 0.02), and the length of the inner wall apparently available for fluid flow was 33% less (P = 0.001), both compared with paired control eyes.

CONCLUSIONS. TGF-ß2 reduces outflow facility when perfused into cultured human anterior segments. Furthermore, TGF-ß2 affects the extracellular matrix of the trabecular meshwork in a manner that is consistent with the observed reduction in outflow facility. Although the distribution of accumulated fibrillar material was different in these perfused eyes than that in POAG, the difference could be due to variation in biomechanical environment for TM cells in cultured anterior segments compared with the living eye. Overall, these results support the hypothesis that elevated TGF-ß2 levels in the aqueous humor play a role in the pathogenesis of the ocular hypertension in POAG.

The pathophysiology of ocular hypertension remains poorly understood, and its elucidation is a major goal of glaucoma research. Of interest is the finding that TGF-ß2 concentrations are increased in the aqueous humor of 50% of patients with primary open-angle glaucoma (POAG). Tripathi et al.1 have reported that the mean level of TGF-ß2 in aqueous humor from patients with POAG was 2.7 ng/mL versus 1.48 ng/mL in aqueous humor from normal subjects. Furthermore, active levels of TGF-ß2 were higher in POAG: 0.45 ng/mL versus 0.2 ng/mL. Picht et al.2 also measured higher levels of TGF-ß2 in the aqueous humor of patients with POAG or juvenile glaucoma, but not in patients with pseudoexfoliation glaucoma. Other investigators have obtained similar results.3 4

An increasing body of evidence suggests that TGF-ß2 has the potential to play an important role in the pathogenesis of POAG. First, TGF-ß2 can alter the production, degradation, and composition of the extracellular matrix (ECM) produced by trabecular meshwork (TM) cells, which plays a role in determining aqueous outflow resistance. For example, TGF-ß2 increases the expression of both fibronectin and the cross-linking enzyme tissue transglutaminase, leading to an increase in the amount of irreversibly cross-linked fibronectin in TM cell cultures.5 There are also indications that TGF-ß2 influences turnover of ECM components by TM cells. For example, it increases expression of tissue inhibitor of metalloproteinase (TIMP)-1 in porcine TM cell culture.6 Second, TGF-ß2 increases mRNA and protein levels of {alpha}B-crystallin in TM cells,5 whereas TGF-ß1 increases levels of myocilin mRNA.7 Both {alpha}B-crystallin and myocilin have been observed to be increased in the TM of POAG eyes. However, all these studies were performed in TM cell culture models, and thus we do not know whether these changes would occur in situ and affect outflow facility.

We studied the effects of TGF-ß2 in normal perfused human eyes in an anterior segment organ culture model. Our goals were twofold. First, we wanted to determine whether TGF-ß2 affects outflow facility in this experimental model. Second, we wanted to determine the effects of TGF on the TM in situ (i.e., within the perfused, intact TM), rather than under cell culture conditions. In view of previous findings, we focused on possible alterations of extracellular matrix components in the TM.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Perfusion Studies
Paired, ostensibly normal human donor eyes were obtained from the Eye Bank of Canada (Ontario Division; Toronto) and the National Disease Research Interchange (NDRI; Philadelphia, PA), in accordance with the provisions of the Declaration of Helsinki for research involving human tissue. Eyes were free of any known ocular disease and were stored in moistened chambers at 4°C until use. The perfusion protocol was similar to that described by Johnson et al.,8 and Johnson and Tschumper,9 10 with several modifications. The first modification was that, in addition to removing the iris, lens, vitreous body, choroid, and retina, we carefully dissected the ciliary processes, leaving in place the longitudinal portion of the ciliary muscle. The second modification was that we added 1% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO) to the perfusion medium, consisting of Dulbecco’s modified Eagle’s medium (DMEM; catalog no. 21885025; Gibco, Toronto, Ontario, Canada) to which antibiotics (0.17 mg/mL gentamicin, 0.25 µg/mL amphotericin-B, 100 U/mL penicillin, and 100 µg/mL streptomycin; all from Sigma-Aldrich) were added. These modifications were made for several reasons. First, retention of the longitudinal ciliary muscle bundles maintained some tension on the TM between ciliary muscle, scleral spur, and cornea, better approximating the in vivo situation. Second, removal of the ciliary processes prevented TM cells from being overloaded with pigment originating from the degenerating pigmented cells of the ciliary epithelium. Third, the ciliary processes were a potential source of growth factors or other agents that could modify the TM cell biology in an uncontrolled fashion, and it therefore seemed prudent not to include them in these studies. In contrast, in the absence of ciliary epithelium, TM cell survival was markedly enhanced by the addition of 1% FBS. To quantify the basal levels of TGF-ß1 and -ß2 due to the presence of FBS in the perfusion media, we measured these cytokines by an ELISA assay (R&D Systems, Minneapolis, MN) in three batches of perfusion media.

After dissection, the anterior segments were extensively washed with media. After mounting in culture dishes, the anterior segments were perfused at a constant flow rate of 2.5 µL/min, and intraocular pressure was continuously measured. After several days of perfusion, when a stable baseline facility was reached, fluid in the eyes and supply syringes was exchanged under approximately 10 mm Hg constant pressure with medium containing 3.0 ng/mL human recombinant TGF-ß2 (hrTGF-ß2; R&D Systems). Contralateral control eyes received a sham exchange with perfusion media only. The hrTGF-ß2 was made up as a 1-µg/mL stock solution from which aliquots were taken and frozen. Aliquots were thawed and used to make up solutions containing fresh medium and TGF-ß2 every 2 to 3 days. This fresh solution was then used to fill the supply syringes in the organ culture system. It is important to note that the recombinant TGF-ß2 was manufactured in its activated form—that is, it was nominally 100% activated as supplied. We perfused the eyes with hrTGF-ß2 for 8 days, except for pair 523/524 which was perfused for 15 days (Table 1) . This pair was not qualitatively or quantitatively different from eyes perfused for 8 days, and we therefore included it in the analysis. Only eyes in which the trabecular cells were still covering the beams and appeared well-preserved were included in this study. This was the case for eight pairs of eyes (Table 1) , which was approximately half of the total number of pairs of eyes perfused.


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  		TABLE 1. Summary of Characteristics of Eyes and Results of Morphometric Measurements

 
We present the facility data in two ways. The first is by simple averaging of facility data between eyes, and the resultant values are reported as the mean ± SEM. The second is by normalizing the raw facility data, thereby allowing comparison of relative facility changes between pairs of eyes. Normalization involved dividing the measured facility for a given eye at each time point by the average facility reading for that eye in the 24-hour period before anterior chamber exchange with TGF-ß2. The net percentage facility change due to TGF-ß2 perfusion was then computed as 100 x (Cnorm,TGF - Cnorm, cont), where Cnorm, TGF and Cnorm, cont were the normalized facilities for the treated and control eyes, respectively. The statistical significance of facility changes was computed from a paired two-tailed Student’s t-test using the net percentage facility change as the statistic of interest.

Morphology and Immunolabeling
After perfusion with hrTGF-ß2, fluid in the eyes was exchanged at approximately 10 mm Hg constant pressure with 3% paraformaldehyde (PFA), which was perfused at 2.5 µL/min for 1 hour. Eyes were then removed from the culture dishes, and 1- to 2-mm wide wedges from each quadrant containing outflow tissues were rapidly cut and immersed overnight in universal fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M Sörensen buffer, pH 7.3), and sent to Erlangen for processing for light and electron microscopy. Wedges were rinsed in cacodylate buffer, postfixed in 1% osmium tetroxide, dehydrated in an ascending series of alcohols, and embedded in epoxy resin, according to standard methods. Sagittally oriented semithin sections (1 µm) of the chamber angle region were cut and stained with toluidine blue. Ultrathin sections (60 nm) of the trabecular meshwork (TM) were cut and transferred to polyvinylbutyral (Pioloform)-coated slotted grids (Plano, Marburg, Germany), stained with uranyl acetate and lead citrate, and viewed with an electron microscope (model EM 902; Carl Zeiss Meditec, Oberkochen, Germany).

Morphometric analysis was performed on one sagittally oriented ultrathin section from each quadrant of each eye using a computer-based morphometric system (Quantimet 500; Leica, Cambridge, UK). All measurements were performed in a masked fashion by the same person (JG) at a magnification of 1100x. Three parameters were measured (Fig. 1) :

  1. The anterior–posterior length of the inner wall (IW) of Schlemm’s canal (SC). Only SC regions with a well-defined inner and outer wall were measured—that is, excluding septae (Fig. 1 , distance 1 plus distance 2).
  2. The cumulative length of the IW regions that were in direct contact with subendothelial fibrillar material (Fig. 1 , distance 2).
  3. The diameters of five thickest connecting fibrils in each section (Fig. 1 , distance 3).



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  		FIGURE 1. Electron micrograph of a sagittal section through the cribriform and IW region of SC of a TGF-ß2–treated eye, showing the parameters measured. 1, length of optically empty pathways under the IW endothelium; 2, length of IW in contact with fibrillar material; 3, diameter of a connecting fibril; E, endothelium of SC; CF, connecting fibrils. Scale bar, 2 µm.

 
From these measurements we then computed the free filtering length of SC (representing the length of SC that appeared available for fluid drainage) as quantity 1 minus quantity 2 (Fig. 1 , distance 1). In addition to the above analysis, the number of vacuoles was counted on each semithin section.

For immunolabeling studies, the remaining PFA-fixed tissue was further fixed in 3% PFA overnight, placed in 1% PFA, and sent to Erlangen. Frozen sagittal and serial tangential sections were cut parallel to the IW of SC. For immunohistology, the sections were blocked with nonfat dry milk solution (Blotto; Santa Cruz Biotechnology, Santa Cruz, CA), washed, and incubated with primary antibody overnight. They were then washed in DPBS and incubated with the fluorescent secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG; Molecular Probes/MoBiTec, Göttingen, Germany) diluted 1:2000 in DPBS for 75 minutes at 4°C. Primary antibodies used were: rabbit anti-{alpha}B-crystallin (kindly provided by Hans Bloemendal, Department of Biochemistry, University of Nijmegen, Nijmegen, The Netherlands) diluted 1:400 and rabbit anti-human fibronectin diluted 1:200 (Dako, Hamburg, Germany).

ELISA Assay
To verify that TGF-ß2 was delivered to the anterior segments and did not adhere to the tubing of the perfusion system, media samples were withdrawn immediately before fixation from the tubing system, immediately upstream of the culture dishes. For these experiments FBS was not added to the perfusion medium. These fluid samples were frozen and stored, and TGF-ß2 concentrations were later measured with an ELISA kit (R&D Systems). This kit has a sensitivity of 7 pg/mL. The samples were not activated before the ELISA.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
TGF-ß2 ELISA Assays
TGF-ß2 concentrations in the fluid collected from the tubing just as it entered the culture dish were 1.33 ± 0.044 ng/mL (mean ± SD), which was approximately half of the nominally infused concentration of 3.0 ng/mL. This indicates that there was some binding of TGF-ß2 to tubing and other flow system components. The mean total TGF-ß2 concentration in three samples of culture medium was 0.198 ± 0.086 ng/mL (individual concentrations, 0.108, 0.280, and 0.207 ng/mL). It is unclear how much TGF-ß2 is activated within the meshwork, and thus what the effective active concentration is within the TM of the living eye. However, we can state that the total concentration of TGF-ß2 perfused into experimental eyes was of the same order as total levels of TGF-ß2 measured in the aqueous humor of POAG eyes.1 2 In control eyes, the total perfused TGF-ß2 concentration was somewhat less than that in normal aqueous humor and 7 to 15 times less than that perfused into experimental eyes.

Facility Studies
The donor age was 68 ± 12 years (mean ± SD), and the time since death (to beginning of perfusion) was 31 ± 6 hours (Table 1) . The starting (baseline) facility in control eyes was 0.35 ± 0.22 µL/min per mm Hg, which declined slightly to 0.34 ± 0.30 µL/min per mm Hg, 8 days after exchange with control solution. The starting facility for experimental eyes was 0.36 ± 0.19 µL/min per mm Hg, which was not significantly different from the starting facility of the contralateral control eyes (P = 0.91). After 8 days of TGF-ß2 perfusion, the facility decreased to 0.23 ± 0.22 µL/min per mm Hg. The time-course of the facility decrease was notable: it began within 1 to 2 days after administration of TGF-ß2 and continued in a gradual, time-dependent manner for approximately 100 hours, after which time the facility seemed to stabilize at a lower level (Fig. 2) . When expressed as a percentage change, the net facility decrease was 27% (P = 0.03).



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  		FIGURE 2. A plot of normalized outflow facility versus time shows that facility decreased within 1 to 2 days after initiation of perfusion with 3.0 ng/mL hrTGF-ß2, gradually decreasing for approximately 100 hours until reaching a steady state value approximately 30% lower than that in the contralateral control eyes. These curves are an average of data from eight pairs of human eyes, scaled so that the time of anterior chamber exchange with hrTGF-ß2 is zero in all eyes. Normalized facility is instantaneous facility divided by the facility in the 24-hour period before exchange. The inset shows mean values of facility in eyes just before anterior chamber exchange (Baseline) and 8 days later. Error bars, SEM.

 
Histologic Findings
In all eight pairs of eyes, the trabecular lamellae showed normal architecture, with open spaces between beams and no evidence of TM collapse (Fig. 3) . Cells covering the trabecular beams were larger than those observed in fresh human donor eyes and had enlarged nuclei and one or two nucleoli.



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  		FIGURE 3. Sagittal sections through the TM of a control eye (A) and an eye treated with TGF-ß2 for 8 days (B). Horizontal lines: length of SC facing an open lumen. Arrows: extracellular material in the IW region. TM, trabecular meshwork; L, tips of the longitudinal portion of the ciliary muscle. Scale bar, 50 µm.

 
The morphology of the inner uveal and the corneoscleral portions of the TM was similar in treated and untreated eyes. However, there were profound differences between TGF-ß2–treated and control eyes in the cribriform region and in the IW of SC. Specifically, the TGF-ß2–treated eyes frequently showed regions with greatly increased amounts of amorphous extracellular material, separation of the IW of SC from the fibrils connecting the IW endothelium with the elastic network of the cribriform meshwork (connecting fibrils), and local collapse of SC. In such locations, there were multiple layers of thin cells within the extracellular material between the IW endothelium and the tips of the connecting fibrils (Fig. 3B) .

The IW of SC was remarkable for having very few vacuoles present in the TGF-ß2–perfused eyes. However, in isolated regions, the IW appeared attenuated and bulged into the lumen of the canal. Usually the extracellular spaces of the cribriform region under such locations were optically empty, and it appeared that these locations were preferred fluid drainage routes. They were often in the posterior portion of SC, but in some sections were seen in other parts of the canal.

Electron Microscopic Findings in TGF-ß2–Treated Eyes
In all perfused eyes, cells on TM beams showed, in addition to the enlarged nuclei seen by light microscopy, larger-than-normal endoplasmic reticulum and Golgi apparatus. Overall, TM cells were clearly in an activated state, consistent with increased levels of protein synthesis.

The amorphous extracellular material under the IW of SC consisted of fine fibrils and appeared qualitatively different from the previously described type I plaques of Rohen. Some of these fibrils were in direct contact with the IW (Fig. 4) . Generally, these fibrils formed multiple parallel layers with interspersed elongated cells. The extracellular material appeared to attach to these cells, and at such attachment points, the cells demonstrated dense peripheral bodies. Although the amount of ECM material was variable between eyes, and within quadrants of a given eye, all TGF-ß2–treated eyes showed these multilayered ECM cellular structures. The tips of connecting fibrils were never seen to penetrate to the interior of these structures.



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  		FIGURE 4. Electron micrograph of the IW of SC of a TGF-ß2–treated eye. The connecting fibrils (CF) are separated from the IW endothelium (E) by multilayered fibrils (F) and interspersed flat, elongated cells connected to each other (arrows). Scale bar, 2 µm.

 
These structures were frequently present at locations where SC was observed by light microscopy to be collapsed. In these regions, no inner and outer wall endothelium could be identified, and it seemed that the region between the cribriform region and the sclera had been remodeled and filled with a mixture of ECM and these elongated cells (Fig. 5) . Even though the morphology of these multilayered regions differed profoundly from the appearance of the normal TM, the cells in these regions had characteristics of corneoscleral meshwork cells; for example, they laid down a basement membrane, the innermost layer of these membranes being connected to the tips of the connecting fibrils. Further, the cells within these layers appeared to be in focal contact with each other.



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  		FIGURE 5. Electron micrograph of a remodeled region of SC from a TGF-ß2–treated eye. At places where the lumen of SC was collapsed, the endothelial lining was absent. Arrow: connecting fibril of the former outer wall of SC; arrowheads: connecting fibrils of the former IW of SC. Scale bar, 2 µm.

 
Measurements to quantify the qualitative changes were hampered by the large variability between and within sections, especially by the presence of the apparent preferential flow pathways seen by light microscopy. Nonetheless, there was a 27% shortening of SC in TGF-ß2–treated eyes (157 ± 44 µm vs. 220 ± 51 µm; Table 1 ) which was statistically significant (P = 0.02). Further, a greater proportion of the IW was in contact with fibrillar material in TGF-ß2–treated eyes, so that the free filtering length in these eyes was reduced by 33% (104 ± 28 µm vs. 162 ± 28 µm), which was statistically significant (P = 0.001). Measurements of connecting fibril (plaque) diameter showed little difference between TGF-ß2 and control eyes.

Electron Microscopic Findings in Control Eyes
The length of SC in control eyes was slightly less than that reported for normal, freshly fixed (nonperfused) human eyes (264 ± 55 µm).11 Further, there was occasional disconnection of connecting fibrils from the IW of SC, as well as occasional accumulation of ECM in the subendothelial space and the formation of multilayered structures. However, the frequency of these alterations, as well as their magnitude, were much less than that in TGF-ß2–treated eyes. Vacuoles were present in control eyes, but less frequently than typically found in nonperfused human eyes. Nonetheless, the number of vacuoles in control eyes was greater than that in TGF-ß2–treated eyes (1.16 ± 0.93 vs. 0.31 ± 0.29 vacuoles/section; P = 0.027).

Immunolabeling Findings
In contrast to the situation in normal, freshly fixed human eyes, all perfused eyes showed intense fluorescent labeling for {alpha}B-crystallin throughout the TM (Fig. 6) . There was no difference between control and TGF-ß2–treated eyes. The staining and distribution of fibronectin was according to that described in normal eyes in the literature12 13 (not shown). Histologically, there was no difference in staining between TGF-ß2–treated and control eyes.



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  		FIGURE 6. Sagittal section through the trabecular meshwork (TM) of a TGF-ß2–treated eye immunolabeled for {alpha}B-crystallin. Extensive staining appears throughout the trabecular meshwork (TM). Scale bar, 50 µm.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Our results clearly demonstrate that TGF-ß2 reduces outflow facility when perfused in the human anterior segment organ culture model. Furthermore, TGF-ß2 affects the ECM of the TM in a manner that is consistent with the observed reduction in outflow facility.

Changes in the TM induced by TGF-ß2 included a decrease in the filtering length of SC, concomitant with increased amounts of ECM in multilayered structures at focal locations along the canal. The ECM within these regions was probably produced by TM cells, either by being washed into the subendothelial region from upstream within the TM or by being locally produced. The cells within these regions were connected to each other and to connecting fibrils of the cribriform region. One can therefore assume that the multilayered cells consist of remodeled TM cells. In extreme cases, the lumen of SC was obliterated by this remodeling process, and inner and outer wall endothelia were lost. The net result was a relatively impermeable-appearing cribriform region (with associated reduced filtration length of SC and few vacuoles) interspersed with a few isolated regions that appeared to be preferred fluid pathways.

Some of these changes were also present in control eyes, although to a much smaller extent than in TGF-ß2–perfused eyes. It is clear from these observations that the organ culture system does not replicate precisely the environment within the living eye. For example, TM cells in all perfused eyes expressed higher-than-normal levels of {alpha}B-crystallin, indicative of a stress response. Disconnection of the connecting fibrils was seen focally in all eyes, due perhaps to the unavoidable dissection process in donor tissue. This disconnection led to some perfusion-induced local collapse of SC in control eyes, consistent with the slightly shorter length of SC in these eyes compared with unperfused, normal human eyes. Despite these drawbacks, the organ culture system is still a valuable model that allowed us to demonstrate that TGF-ß2 perfusion clearly induced increased formation of ECM in the TM in situ, concomitant with decreased outflow facility.

These results have implications for the pathogenesis of POAG. It seems clear that TGF-ß2 promotes accumulation of ECM in the TM of human eyes in situ. Why then is the pattern of ECM deposition that we saw in the organ culture model different from that in POAG? The difference could be due in part to the acute nature of our perfusions compared with the long-term development of ocular hypertension and in part to the absence of unknown factors in the perfusion media that are present in aqueous humor. In addition, it could be due to the absence of varying ciliary muscle tension over time in our model, as well as the disconnection of the connecting fibrils. These factors mean that TM cells are not subject to variance in stretching over time, due to forces originating in the ciliary muscle, such as occurs in the living eye. Stretching is an important mechanical stimulus that apparently promotes meshwork remodeling14 15 16 and may prevent the establishment of only a few semipermanent preferential flow pathways, such as we observed in perfused eyes. When this stimulus is absent (such as in the organ culture model) ECM material can accumulate in multilayer structures. In the living eye this material would instead be more evenly distributed throughout the meshwork and may tend to accumulate around the intact connecting fibrils, producing the well-known plaques in POAG eyes.

The net facility reduction we measured in the presence of TGF-ß2 corresponds to an IOP increase of approximately 3 mm Hg, which, acting in isolation in a normotensive eye, does not generate a glaucomatous IOP. What then is the long-term significance of such a facility reduction? We can only speculate about this: Perhaps pathologic levels of IOP require an exposure to TGF-ß2 measured in months or years, instead of the 1- to 2-week time scale that we were able to use in our experiments. Alternatively, perhaps clinically significant ocular hypertension requires a confluence of pressure-elevating factors, and elevated TGF-ß2 levels in the aqueous humor are only one such factor. Despite these uncertainties, the IOP elevation and TM morphology changes caused by TGF-ß2 are consistent with a role for this cytokine in the pathogenesis of ocular hypertension. Obtaining a better understanding of the effects of TGF-ß on TM function seems to be a fruitful area for further research.


   Acknowledgements
 
The authors thank the Eye Bank of Canada (Ontario Division) for supplying eyes, and Anke Fischer, Angelika Hauser, and Marco Gösswein for expert technical assistance.


   Footnotes
 
Supported by the von Humboldt Foundation, Canadian Institutes of Health Research Grant MA-10051 (CRE), the Glaucoma Research Society of Canada (CRE), and Deutsche Forschungsgemeinschaft Grant SFB 539 (EL-D).

Submitted for publication July 27, 2003; revised September 9, 2003; accepted September 26, 2003.

Disclosure: J. Gottanka, None; D. Chan, None; M. Eichhorn, None; E. Lütjen-Drecoll, None; C.R. Ethier, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: C. Ross Ethier, Department of Mechanical and Industrial Engineering, University of Toronto, 5 King’s College Road, Toronto, Ontario M5S 3G8, Canada; ethier{at}mie.utoronto.ca.


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Tripathi RC, Li J, Chan WF, Tripathi BJ. Aqueous humor in glaucomatous eyes contains an increased level of TGF-beta 2. Exp Eye Res. 1994;59:723–727.[CrossRef][ISI][Medline][Order article via Infotrieve]
  2. Picht G, Welge-Luessen U, Grehn F, Lutjen-Drecoll E. Transforming growth factor beta 2 levels in the aqueous humor in different types of glaucoma and the relation to filtering bleb development. Graefes Arch Clin Exp Ophthalmol. 2001;239:199–207.[ISI][Medline][Order article via Infotrieve]
  3. Inatani M, Tanihara H, Katsuta H, Honjo M, Kido N, Honda Y. Transforming growth factor-beta 2 levels in aqueous humor of glaucomatous eyes. Graefes Arch Clin Exp Ophthalmol. 2001;239:109–113.[ISI][Medline][Order article via Infotrieve]
  4. Ochiai Y, Ochiai H. Higher concentration of transforming growth factor-beta in aqueous humor of glaucomatous eyes and diabetic eyes. Jpn J Ophthalmol. 2002;46:249–253.[CrossRef][Medline][Order article via Infotrieve]
  5. Welge-Lüssen U, May CA, Eichhorn M, Bloemendal H, Lütjen-Drecoll E. {alpha}B-crystallin in the trabecular meshwork is inducible by transforming growth factor-beta. Invest Ophthalmol Vis Sci. 1999;40:2235–2241.[Abstract/Free Full Text]
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  11. Allingham RR, deKater AW, Ethier CR. Schlemm’s canal in primary open angle glaucoma: correlation between Schlemm’s canal dimensions and outflow facility. Exp Eye Res. 1996;62:101–109.[CrossRef][ISI][Medline][Order article via Infotrieve]
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J. Kuchtey, M. E. Kallberg, K. N. Gelatt, T. Rinkoski, A. M. Komaromy, and R. W. Kuchtey
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G. Schlunck, H. Han, T. Wecker, D. Kampik, T. Meyer-ter-Vehn, and F. Grehn
Substrate Rigidity Modulates Cell Matrix Interactions and Protein Expression in Human Trabecular Meshwork Cells
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G. S. Zode, A. F. Clark, and R. J. Wordinger
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Z. Wan, D. F. Woodward, C. L. Cornell, H. G. Fliri, J. L. Martos, S. N. Pettit, J. W. Wang, A. B. Kharlamb, L. A. Wheeler, M. E. Garst, et al.
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W. Xue, N. Comes, and T. Borras
Presence of an Established Calcification Marker in Trabecular Meshwork Tissue of Glaucoma Donors
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R. J. Wordinger, D. L. Fleenor, P. E. Hellberg, I.-H. Pang, T. O. Tovar, G. S. Zode, J. A. Fuller, and A. F. Clark
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R. Fuchshofer, A. H. L. Yu, U. Welge-Lussen, and E. R. Tamm
Bone Morphogenetic Protein-7 Is an Antagonist of Transforming Growth Factor-{beta}2 in Human Trabecular Meshwork Cells
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M. P. Fautsch, D. H. Johnson, and the Second ARVO/Pfizer Research Institute Working
Aqueous humor outflow: what do we know? Where will it lead us?
Invest. Ophthalmol. Vis. Sci., October 1, 2006; 47(10): 4181 - 4187.
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B. Bachmann, M. Birke, D. Kook, M. Eichhorn, and E. Lutjen-Drecoll
Ultrastructural and Biochemical Evaluation of the Porcine Anterior Chamber Perfusion Model
Invest. Ophthalmol. Vis. Sci., May 1, 2006; 47(5): 2011 - 2020.
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D. L. Fleenor, A. R. Shepard, P. E. Hellberg, N. Jacobson, I.-H. Pang, and A. F. Clark
TGF{beta}2-Induced Changes in Human Trabecular Meshwork: Implications for Intraocular Pressure
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S. Sugita, T. F. Ng, P. J. Lucas, R. E. Gress, and J. W. Streilein
B7+ Iris Pigment Epithelium Induce CD8+ T Regulatory Cells; Both Suppress CTLA-4+ T Cells
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J. Gottanka, A. Kuhlmann, M. Scholz, D. H. Johnson, and E. Lutjen-Drecoll
Pathophysiologic Changes in the Optic Nerves of Eyes with Primary Open Angle and Pseudoexfoliation Glaucoma
Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 4170 - 4181.
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R. Fuchshofer, M. Birke, U. Welge-Lussen, D. Kook, and E. Lutjen-Drecoll
Transforming Growth Factor-{beta}2 Modulated Extracellular Matrix Component Expression in Cultured Human Optic Nerve Head Astrocytes
Invest. Ophthalmol. Vis. Sci., February 1, 2005; 46(2): 568 - 578.
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X. Zhao, K. E. Ramsey, D. A. Stephan, and P. Russell
Gene and Protein Expression Changes in Human Trabecular Meshwork Cells Treated with Transforming Growth Factor-{beta}
Invest. Ophthalmol. Vis. Sci., November 1, 2004; 45(11): 4023 - 4034.
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C. R. Ethier, S. Wada, D. Chan, and W. D. Stamer
Experimental and Numerical Studies of Adenovirus Delivery to Outflow Tissues of Perfused Human Anterior Segments
Invest. Ophthalmol. Vis. Sci., June 1, 2004; 45(6): 1863 - 1870.
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