IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2006;47:794-801.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0292
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fuchshofer, R.
Right arrow Articles by Birke, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fuchshofer, R.
Right arrow Articles by Birke, M.

Biochemical and Morphological Analysis of Basement Membrane Component Expression in Corneoscleral and Cribriform Human Trabecular Meshwork Cells

Rudolf Fuchshofer,1 Ulrich Welge-Lüssen,2 Elke Lütjen-Drecoll,3 and Marco Birke3

1From the Department of Human Anatomy, University Regensburg, Regensburg, Germany; the 2Department of Ophthalmology, Maximilians-University, Munich, Germany; and the 3Department of Anatomy II, Friedrich Alexander University, Erlangen, Germany.


   Abstract
Top
 Abstract
Material and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine whether cells of the cribriform trabecular meshwork (TM) express basement membrane (BM) components similar to corneoscleral TM cells and to determine whether cribriform cells are connected to the elastic tendon net of the TM.

METHODS. TM cells of the corneoscleral and the cribriform regions were cultured from 10 eyes of 10 donors, aged 20 to 87 years. Cell types were classified by {alpha}-smooth muscle actin (smA), desmin, and {alpha}B-crystallin staining. Expression of collagen type IV (ColIV) chains {alpha}1 to 6; collagen type VIII (ColVIII) {alpha}1; laminin subunits {alpha}1 to 5, ß1 to 3, and {gamma}1 to 3; and nidogen 1 and 2 was tested in both cell types by semiquantitative RT-PCR (sqPCR). Expression of ColIV{alpha}2, ColVIII{alpha}1, laminin ß2, and nidogen 1 was quantified by Northern blot analysis. The response to transforming growth factor (TGF)-ß2 treatment was investigated. Serial tangential and sagittal TM sections of 16 eyes from 10 donors (aged 12–90 years) were used for electron- and immunoelectron microscopy.

RESULTS. Both TM cell types expressed ColIV chains {alpha}1, {alpha}2, {alpha}4, {alpha}5, and {alpha}6; ColVIII {alpha}1; laminin subunits {alpha}3, {alpha}4, ß1, ß2, ß3, {gamma}1, and {gamma}2; and nidogen 1, as determined by Northern blot analysis and sqPCR. ColIV {alpha}3; laminin subunits {alpha}1, {alpha}2, and {gamma}3; and nidogen 2 were not detectable by PCR. Responses to TGF-ß2 treatment did not differ between cell types. In vivo, all cribriform cells were in contact with ColIV containing BM material and were found to connect to the cribriform elastic network.

CONCLUSIONS. Cribriform and corneoscleral TM cells show no differences in expression of BM components and response to TGF-ß2. The direct connection of cribriform cells to the elastic tendon network suggests that they are under mechanical tension. This could explain previous findings of {alpha}B-crystallin expression in the cribriform region.

The trabecular meshwork (TM) of primate eyes consists of two distinct portions that differ in morphology and function. The inner TM is formed by connective tissue lamellae, which are completely covered by trabecular cells. These cells produce a continuous basement membrane (BM) around the central lamellae. Abutting this region lies the cribriform or juxtacanalicular meshwork, formed by star-shaped cribriform cells embedded in extracellular matrix (ECM). These cells are connected to each other and also to the inner wall of Schlemm’s canal (SC).1 Experimental and comparative physiological studies indicate that approximately 90% of the aqueous humor (AH) outflow resistance of the TM is located within the cribriform region.2 The aqueous passages through the corneoscleral lamellae are too large to account for outflow resistance; instead, this region serves as a biological filter.3 4

Trabecular cells in the corneoscleral and cribriform regions may have functional differences. Cells cultured from both regions stain for {alpha}-smooth muscle actin ({alpha}-smA) and vimentin, but only cribriform cells stain for the stress protein {alpha}B-crystallin.5 This difference led us to conduct studies with cribriform and corneoscleral cells separately, a laborious process. Of interest, however, we found that the expression pattern of corneoscleral and cribriform cells was similar for the ECM components metalloproteinases and their inhibitors, as well as the cells’ reaction to some exogenous factors like transforming growth factor (TGF) ß or dexamethasone (DEX).6 7 8 Why the two trabecular cell types have similar expression patterns for many molecules, but not {alpha}B-crystallin, is unknown. In intact TM, morphologic differences between cribriform and corneoscleral cells exist in their shape, location, and BM. Understanding the in situ milieu of the cells may explain their different molecular characteristics.

To investigate the potential similarity or difference of these cells, we compared BM production in cultured cribriform and corneoscleral cells. In addition, we used electron microscopy to determine whether BM material is morphologically associated with cribriform cells. We also examined their potential connection to the cribriform elastic network.


   Material and Methods
Top
 Abstract
 Material and Methods
Results
 Discussion
 References
 
Tissue Culture
TM cell cultures were prepared from eyes of 10 human donors and grown as described previously.5 6 The age of the donors ranged from 20 to 87 years. Cells derived from the cribriform and outer corneoscleral meshwork were termed cribriform cells, cells derived from the inner lamellar and uveal portion were termed corneoscleral TM cells. Both TM cell types were distinguished from each other and adjacent scleral spur or ciliary muscle cells by their morphology and immunohistochemical staining, as described previously.6 In brief, third-passage cells were kept confluent for 7 days and stained with antibodies against {alpha}-smA, desmin, and {alpha}B-crystallin (dilutions and suppliers are given in Table 1 ). For desmin and {alpha}-smA staining, cells were fixed with methanol; for {alpha}B-crystallin staining, cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature (RT). After two washes with phosphate-buffered saline (PBS; pH 7.2/0.1% vol/vol) Triton X-100, antibodies were added in PBS and 2% (wt/vol) bovine serum albumin (BSA) at specific dilutions (see Table 1 ) and incubated overnight at 4°C. After they were washed for 5 minutes in PBS, the cells were incubated with cognate Alexa488-conjugated secondary antibodies (Table 1) for 1 hour at RT. Slides were mounted in Kaiser’s jelly and viewed and photographed with a microscope (Aristoplan; Leitz, Wetzlar, Germany). Methods of securing human tissue were humane, included proper consent and approval, and complied with the Declaration of Helsinki.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Antibodies Used for Immunohistochemistry and Immunoelectron Microscopy

 
TGF-ß2 Treatment
Third-passage corneoscleral and cribriform TM cells were kept confluent for 7 days. Before treatment, cells were preincubated in serum-free medium for 24 hours. TGF-ß2 (1 ng/mL, active form; Roche, Basel, Switzerland) was then added for 24 hours. This concentration was chosen as it resembles the mean average of TGF-ß2 levels measured in the AH of patients with POAG. Corresponding control cells were treated equally, but did not receive TGF-ß2. Cell viability was tested before and at the end of treatment and did not reveal any signs of increased cell death in TGF-ß2-treated cells.

RNA Isolation and Polymerase Chain Reaction
TM cells were harvested from 35-mm Petri dishes, and total RNA was extracted with an RNA isolation kit (Stratagene, Heidelberg, Germany). Structural integrity of the total RNA samples was confirmed by electrophoresis in 1% (wt/vol) agarose gels. RNA yield and purity were determined photometrically. First-strand complementary DNA (cDNA) for PCR was prepared from total RNA by using Moloney murine leukemia virus (M-MuLV) reverse transcriptase and oligo[dT]-17 primer. The PCR was performed with the temperature profile as follows: 36 cycles of 1 minute melting at 94 °C, 1 minute of annealing, and 2 minutes’ extension at 72°C followed by an end extension step for 10 minutes at 72°C after the last cycle. All PCR primers were purchased from Invitrogen (Darmstadt, Germany) and span exon–intron boundaries. Primer sequences, positions, annealing temperatures, and PCR product sizes are given in Table 2 . Functionality of primer pairs was tested before to exclude false-negative results. Sizes of the PCR products were estimated from the migration of a DNA size marker run concurrently (100-bp DNA ladder; Promega, Madison, WI). RNA that was not reverse transcribed served as the negative control for PCR and showed no amplified products. To allow semiquantification, PCRs for the housekeeping-gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were performed on the cDNAs of corneoscleral and cribriform TM cells to estimate the amount of cDNA used for the subsequent BM component PCRs. Band intensities were quantified (BioDocAnalyze image analysis software; Whatman-Biometra, Göttingen, Germany) and differences in GAPDH were considered. Mean averages of corneoscleral TM cells are set to 100%, and values of cribriform cells are given as mean average (MA) ± SD percentage of corneoscleral expression.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Primers Used for PCR Amplification and Semiquantitative RT-PCR

 
Northern Blot Analysis
Total RNA (15 µg) was size fractionated by gel electrophoresis in 1% (wt/vol) agarose gels with 2.2 M formaldehyde, subsequently transferred onto a nylon membrane (Roche, Basel, Switzerland) by vacuum blotting, and cross-linked at 1600 µJ (Stratalinker; Stratagene). Membranes were prehybridized in solution (Dig Easy Hyb; Roche Diagnostics, Indianapolis, IN) for 1 hour at 68°C. Hybridizations were performed at 68°C overnight in the hybridization solution (Dig Easy Hyb; Roche) containing 50 ng/mL of a specific antisense probe. Riboprobe synthesis has been described previously.9 After hybridization, the membranes were washed twice in 2x SSC and 0.1% (vol/vol) sodium dodecyl sulfate (SDS) for 10 minutes at RT, then either twice in 0.1% SDS for 10 minutes at RT or 15 minutes at 68°C and once for 5 minutes in washing buffer (100 mM maleic acid, 150 mM NaCl [pH 7.5], and 0.3% [vol/vol] Tween-20). Membranes were then blocked in blocking solution (100 mM maleic acid, 150 mM NaCl [pH 7.5], 1% [vol/vol]) blocking reagent [Roche]). Alkaline-phosphatase-conjugated anti-digoxygenin antibody ({alpha}-DIG-AP; Roche Diagnostics) was added at a dilution of 1:10000 in blocking solution and allowed to react for 30 minutes at RT. After incubation, the membranes were washed four times for 15 minutes each in washing buffer at RT and equilibrated in detection buffer (100 mM Tris-HCl, 100 mM NaCl [pH 9.5]) for 10 minutes at RT. For chemiluminescence detection, membranes were incubated for 5 minutes with chemiluminescent substrate (CDP-Star; Roche Diagnostics) diluted 1:100 in detection buffer at RT. Chemiluminescence was detected (Lumi-Imager workstation; Roche Diagnostics, Mannheim, Germany), with exposure times ranging between 2 minutes and 1 hour. Amount and quality of the RNA for quantification was tested afterward by methylene blue staining of the 28S and 18S rRNA bands. Quantification was performed on the workstation (LumiAnalyst Imaging software; Roche Diagnostics).

Histologic Processing
Sixteen eyes of 10 donors with no history of ocular disease were studied. The age of these donors ranged from 12 to 90 years. The eyes were bisected at the equator and fixed by immersion in a mixture of glutaraldehyde-formaldehyde. Mean postmortem time until fixation was 15 ± 3 hours. Anterior segments of the eyes were cut into quadrants, and wedges of 2-mm width were dissected from each quadrant, containing the TM and adjacent cornea, sclera, and ciliary body. Specimens were rinsed in cacodylate buffer, postfixed in 1% osmium tetroxide (OsO4), dehydrated in an ascending series of alcohol, and embedded in Epon according to standard methods. Sagittal and serial tangential ultrathin sections parallel to the inner wall of SC from the chamber angle were cut and mounted on coated slotted grids (Pioloform; Plano, Marburg, Germany). Sections were stained with uranyl acetate and lead citrate and viewed by electron microscope (EM902; Carl Zeiss Meditec, Inc., Oberkochen, Germany).

Immunoelectron Microscopy
Specimens of the chamber angle were prepared as just described and fixed in 4% (vol/vol) PFA and 0.1% (vol/vol) glutaraldehyde in PBS for 2 hours at 4°C. To prepare frozen sections, we soaked pieces of fixed tissues in 4% (wt/vol) sucrose in PBS for at least 24 hours and froze them in liquid nitrogen. For pre-embedding immunocytochemistry, 25-µm cryostat sections were cut and mounted on coverslips (Thermanox; Nunc, Wiesbaden, Germany). Cryosections were blocked in dry milk solution (Blotto; Santa Cruz Biotechnology, Santa Cruz, CA) for 30 minutes. Anti-ColIV or i-ColVI antibodies were applied in PBS and 0.2% (wt/vol) BSA to the sections and incubated overnight at 4°C (for dilutions and suppliers, see Table 1 ). After six rinses in PBS and 2% (wt/vol) BSA, sections were incubated overnight at 4°C with ultrasmall gold–conjugated anti-rabbit or ant-mouse F(ab')2 in PBS and 0.2% (wt/vol) BSA. Sections were then rinsed five times in PBS and 2% (wt/vol) BSA and two times in PBS and fixed with 2.5% (vol/vol) glutaraldehyde in PBS for 2 hours at 4°C. Silver enhancement was then performed (R-Gent SE-EM; Aurion, Wageningen, The Netherlands) for 1.5 hours in darkness. Sections were postfixed with 0.5% (wt/vol) OsO4 in PBS and embedded in Epon. Ultrathin sections of the specimens were cut and examined by electron microscope (EM 902; Carl Zeiss Meditec, Inc.). Controls sections were treated the same way but without primary antibodies. No control sections showed labeling with gold particles.


   Results
Top
 Abstract
 Material and Methods
 Results
Discussion
 References
 
Classification of Cultured Cribriform and Corneoscleral TM Cells
All 10 monolayers of the cells from the cribriform and corneoscleral TM consisted of small, elongated cells that were almost all positive for {alpha}-smA staining (Figs. 1A 1D) . None of the cells stained for desmin, a typical marker for contamination with ciliary muscle cells (Figs. 1B 1E) . The cribriform TM cells showed intense staining for {alpha}-B crystallin (Fig. 1F) , whereas the corneoscleral TM cells did not stain, or only faintly stained, for that small stress protein (Fig. 1C) .


Figure 1
View larger version (73K):
[in this window]
[in a new window]
  		FIGURE 1. Immunohistochemical staining of corneoscleral and cribriform TM cell monolayers. Monolayer cultures of corneoscleral and cribriform TM cells of the third passage were kept confluent for 7 days. All 10 cultures of each TM cell type showed the same staining pattern. Corneoscleral TM cells stained positive for {alpha}-smA (A), negative for {alpha}-desmin (B), and very weak for {alpha}B-crystallin (C). Cribriform TM cells were also positive for {alpha}-smA (D) and negative for {alpha}-desmin (E), but were positive for {alpha}B-crystallin (F). Magnification, x200.

 
In Vitro Expression Analysis of BM Components in Corneoscleral and Cribriform TM Cells
BM component expression was assessed in all 10 cribriform and corneoscleral cell lines. Type IV and VIII collagens, laminins, nidogens, and their subunits were analyzed with sqRT-PCR. The results of all 10 corneoscleral TM cells were identical. We obtained specific products of the ColIV chains {alpha}1, {alpha}2, {alpha}4, {alpha}5, and {alpha}6 (Fig. 2A) , whereas the {alpha}3 chain was not detectable (not shown). Type VIII collagen was also studied, as it is a component of Descemet’s membrane, and the BM of the inner TM lamellae appears to be the continuation of Descemet’s membrane under electron microscopy. ColVIII showed a specific signal for the {alpha}1 chain (Fig. 2A) . Analysis of laminin subunits revealed positive signals for laminin {alpha}3, {alpha}4, ß1, ß2, ß3, {gamma}1, and {gamma}2 (Fig. 2A) , but not for the subunits {alpha}1, {alpha}2, and {gamma}3 (not shown). Nidogen 1 (Fig. 2A) , but not nidogen 2 (not shown), expression was detectable. The expression patterns of all 10 cribriform TM cell lines was identical with the corneoscleral cells, even for the expression of type VIII collagen. Figure 2A shows a single representative experiment. Semiquantification of PCR band signal intensities showed no differences in the expression levels of the BM components (Fig. 2B) . Mean averages of signal intensities obtained from corneoscleral cells are set to 100%, and the values of cribriform cells are given as MA percentage ± SD (n = 10).


Figure 2
View larger version (80K):
[in this window]
[in a new window]
  		FIGURE 2. Expression of different BM component subunits in corneoscleral and cribriform TM cells. (A) Representative semiquantitative RT-PCR experiment. Corneoscleral (cc) and cribriform (cr) TM cells qualitatively expressed the same BM components. (B) Quantification of signal intensities did not show differences in expression levels. MAs of corneoscleral cells are set to 100%, and levels in cribriform cells are given as MA percentage ± SD (n = 10).

 
To assess slight quantitative differences in the expression of the BM components between the cell lines, Northern blot analysis, as a more sensitive method, was conducted. As we were limited by the amount of RNA we could extract from the single cultures, we examined only one member of each BM component group that gave an explicit positive signal in the PCR (Fig. 3A) . The quantification of the Northern blot data did not reveal any differences in the mRNA amounts of ColIV{alpha}2, ColVIII{alpha}1, laminin ß2, and nidogen 1 between the two cell lines. Signal intensities were quantified by adjustment to the signals of the 28S and 18S rRNA bands. Mean averages of the corneoscleral TM cells are set to one, and the expression in cribriform cells is given as x-fold (MA ± SD; n = 3; Fig. 3C , Table 3 ).


Figure 3
View larger version (70K):
[in this window]
[in a new window]
  		FIGURE 3. Northern blot analysis of ColIV{alpha}2, ColVIII{alpha}1, Laminin ß2, and nidogen 1 expression in corneoscleral and cribriform TM cells and TGF-ß2 response. (A) ColIV{alpha}2, ColVIII{alpha}1, laminin ß1, and nidogen-1 were equally expressed in corneoscleral (cc) and cribriform (cr) human TM cells. (B) Treatment with 1 ng/mL TGF-ß2 had no effect on the expression of these genes, and both cell types responded identically, shown exemplarily for nidogen-1. (C) Quantification plot of the Northern blot data. Values are normalized to untreated corneoscleral TM cells and given as MA x-fold expression ± SD (n = 3).

 

View this table:
[in this window]
[in a new window]
  		TABLE 3. Quantitative Evaluation of BM Component Expression in Untreated and 24-Hour TGF-ß2-Treated Corneoscleral and Cribriform TM Cells

 
Response to TGF-ß2
TGF-ß2 did not alter expression levels of nidogen 1 (Figs. 3B 3C) , ColIV{alpha}2, ColVIII{alpha}1, and laminin ß2 (Fig. 3C) for either corneoscleral or cribriform cells (Figs. 3B 3C) . For quantification, values of the untreated corneoscleral cells are set to one, and signal intensities of TGF-ß2-treated corneoscleral and cribriform TM cells are given as x-fold (MA ± SD; n = 3). Signals of the 28S and 18S rRNAs served as the control for equal loading and were considered for quantification (Figs. 3B 3C ; Table 3 ).

Electron Microscopy
Serial sagittal and especially tangential sections through the cribriform region revealed BM-like material adjacent to all cribriform cells. Fine fibrils branching off the sheath of the cribriform elastic fiber network inserted into this BM material (Fig. 4A) . The distribution of the material varied in amount and localization among different cells. Some single cells had almost half of the circumference of the cell surface in contact with BM-like material, whereas other cells had the material only in small regions of the cell surface (Fig. 4B) . These differences in amount of BM-like material were dependent on the position of the cell within the TM and its environment. In places where a cribriform cell bordered optically empty pathways, the cell surface was completely free of BM-like material (Fig. 4B) . In contrast, those parts of the cell surface facing fibrils of extracellular material showed a clearly distinguishable line of BM material (Fig. 4A) . In these places, connecting fibrils originating from the cribriform elastic network were in direct contact with the BM-material. The connecting fibrils showed a typical cross-banding where they branched off the sheath of the elastic fibers. Toward the cribriform cells this cross-banding was no longer visible, and the connecting fibrils split into bundles of fine fibrils that attached to the BM-like material of the cribriform cells (Fig. 4A) . In these areas, the cell membrane of the cribriform cells showed densifications and small extensions toward the region of attachment. The cell membrane undulated in the attachment areas. Existence of BM material at the cribriform cells was independent of the functional state of the cells. Both cribriform cells filled with phagolysosomes, and cells containing numerous Golgi membranes and rough endoplasmic reticulum, but without phagosomes, showed BM material at their surface (Fig. 4B) .


Figure 4
View larger version (78K):
[in this window]
[in a new window]
  		FIGURE 4. Electron micrographs of tangential sections through the cribriform TM region. (A) The cribriform cell (CR) was attached to BM-like material (BM) at places where the cribriform elastic fibers (EL) were connected to the cell by cross-banded connecting fibrils (CFs; arrows). The cell membrane was undulated and thick-ened at these positions. At that part of the circumference where the cell faces optically empty spaces (OE), no BM-like material was visible. (B) The phagolysosome containing cribriform cells (CR) was mainly surrounded by optically empty spaces (OE). BM-like material was only visible at the small cytoplasmic extensions where the cell is in contact with fibrillar material (arrows). Magnification: (A) x5100; (B) x3600.

 
Collagen type IV specific immunogold labeling confirmed that the BM-like material adjacent to the trabecular cells of the lamellae contained this collagen (Fig. 5A) . A similar staining was present within the BM-like material of the cribriform cells (Fig. 5B) . Labeling for collagen type VI was found at the fine fibrils splitting off the cross-banded connecting fibrils and inserting into the BM-like material. The cross-banded fibrils themselves were not labeled (Fig. 5C) . Collagen type IV (not shown) and type VI (Fig. 5D) staining control sections did not show labeling within the BM-like material.


Figure 5
View larger version (115K):
[in this window]
[in a new window]
  		FIGURE 5. Electron micrographs of silver-enhanced collagen type IV and VI immunogold-stained sections through the lamellar meshwork and the cribriform region. (A) In this sagittal section, through a corneoscleral trabecular lamellae, type IV collagen immunoreactivity (gold particles, arrows) was present in the basement membrane-like material (BM) within the lamellae. TE, trabecular cell. (B) Sagittal section through the cribriform region stained for type IV collagen. Immunoreactivity (gold particles, arrows) was present in the BM-like material adjacent to the cell where connecting fibrils (CF) attached. TE, trabecular cell; EL, elastic fibers. (C) In this oblique section through the cribriform region, collagen type VI reactivity (gold particles, arrows) was present at connecting fibrils (CF), which diverged from the sheaths of the elastic fibers (EL) and were connected to the BM-like material of the cribriform TM cell (CR). (D) Collagen type VI staining control.

 

   Discussion
Top
 Abstract
 Material and Methods
 Results
 Discussion
References
 
In the present work we studied whether two cell types in the TM, cribriform and corneoscleral cells, can be distinguished by differences in the expression of BM components. To answer this, we focused on three distinct questions: (1) which BM components are expressed by TM cells, (2) what are the differences in the expression profiles between corneoscleral and cribriform TM cells, and (3) do these two cell types respond differently to TGF-ß2? We examined four components of BM: collagen IV, laminin, nidogen, and collagen VIII. Collagen type IV chains {alpha}1 and {alpha}2 are ubiquitous BM components forming a cross-linked scaffold in mature basal laminae.10 11 12 In contrast, the incorporation of {alpha}3, {alpha}4, {alpha}5, or {alpha}6 chains is variable and apparently linked to functional features of specialized BMs.11 12 13 14 Mutations in the genes coding for these chains correlate with genetic diseases like Alport syndrome, Goodpasture syndrome, and leiomyomatosis, causing severe organ- or tissue-specific dysfunction.11 15 16 17 18 19 20 21 Pathogenic mutations in just one of these genes may lead to the absence of the other isoforms within the BM, suggesting dependency of expression among them.16 17 In contrast, however, in the {alpha}3 knockout mouse, expression of the {alpha}4, {alpha}5, and {alpha}6 chains was detectable, which contradicts a coupled expression mechanism.13 20 In our study, cribriform and corneoscleral TM cells expressed the {alpha}1, {alpha}2, {alpha}4, {alpha}5, and {alpha}6 chains but not the {alpha}3 chain, supporting the knockout mouse data. The functional relevance of the {alpha}3 chain, however, is not known. In the anterior eye segment, {alpha}3 expression and deposition was shown in the specialized BMs of Descemet’s membrane and the lens capsule.15 19 Of interest, at the transition from cornea to TM, {alpha}3 expression ends, and the tissue loses its transparency.

Laminins are the most abundant linking proteins in BMs.14 22 23 We found expression of laminin subunits {alpha}3, {alpha}4, ß1, ß2, ß3, {gamma}1, and {gamma}2 but not of {alpha}1, {alpha}2, and {gamma}3 in cribriform and in corneoscleral TM cells. Laminins are heterotrimers of one {alpha}, ß, and {gamma} chain, whereas the {alpha} chains seem to be crucial for specific function and tissue distribution.14 22 23 Laminin {alpha}1 expression was described only in early stages of epithelial cell BM development,23 and {alpha}2 seems to be restricted to skeletal and cardiac muscle cells and the peripheral nervous system.14 22 23 The {alpha}3 chain is typically found in stratified epithelium, like in skin, which is of ectodermal origin, and {alpha}4 is characteristic for mesenchyme-derived cells.14 22 23 Because both the {alpha}3 and {alpha}4 chain are expressed in corneoscleral and cribriform cells, this finding does not help to clarify the question of whether the TM is of neuroectodermal origin or invades the eye with the vasculature, and would thus be of mesenchymal origin. The absence of the {gamma}3-chain was not surprising, as this chain is not BM associated and is found in apical epithelial surfaces and the peripheral nervous system.24 Nidogens function as bridging proteins between collagens and laminins.14 22 23 We detected expression of nidogen-1 in both trabecular cell types, whereas nidogen-2 was not detectable. Nidogen-2 is not essential for BM formation as shown in the knock-out (–/–) mouse,25 and therefore expression of one nidogen seems to be sufficient for correct BM formation.

The fourth BM component we investigated, type VIII collagen, is predominantly synthesized by endothelial cells such as vascular endothelium cells.26 27 It is the major component of the wide-spaced collagen of Descemet’s membrane.28 29 30 31 32 In Descemet’s membrane, type VIII collagen may stabilize the cornea against IOP and outside influences.26 32 The peripheral regions of Descemet’s membrane are the regions where the elastic fibers of the TM as well as the tendons of the ciliary muscle insert.33 34 Shuttleworth26 noted that type VIII collagen is associated with microfibrils of elastic tissues. Based on this, ColVIII expression in the TM cells could contribute to stability against fluctuations of IOP and the mechanical tension of the ciliary body tendons, which insert to the elastic network of fibers in the TM. This network connects to the BM by connecting fibrils.

The second question we investigated was the difference in expression profiles of BM components between corneoscleral and cribriform cells. To our surprise, despite the initially described morphologic differences,4 5 35 our biochemical investigations showed that the selected BM components were expressed qualitatively and quantitatively equally. The answer to the third question, response to TGF-ß2, also revealed similar expression profiles for both cell types. Of note, we did not find any changes in collagen type IV expression after treatment with TGF-ß2 in concentrations that resembled those found in the aqueous of patients with POAG. In contrast, astrocytes of the optic nerve head respond to TGF-B2 with an increase in collagen type IV expression.36 In keeping with this, increased amounts of BM material under the inner wall of SC are not found in the TM of patients with POAG, whereas in the lamina cribrosa region of the optic nerve, type IV collagen is significantly increased.37

Our ultrastructural investigations show that all cells of the cribriform region have adjacent BM-like material and that this BM-like material is connected to the elastic cribriform network. The cribriform cells are thus indirectly connected to the elastic tendons of the ciliary muscle. In regions of "optically empty" spaces, no BM was visible. "Optically empty" in this context does not necessarily mean that there is no ECM in these regions; however, this material is probably lost during the embedding procedure.38 At places where the elastic fibers were attached to the BM-like material of the cribriform TM cells, the cell membrane was undulated and thickened, whereas the corneoscleral TM cells had a smooth cell membrane. This finding could indicate that the cribriform cells are exposed to a higher mechanical tension than the cells of the corneoscleral region. In the corneoscleral region, ciliary muscle tendons insert into the connective tissue core of the lamellae and not to the BM of the cells. This difference in mechanical tension caused by different tendon insertions could explain why cribriform cells show increased expression of the stress protein {alpha}B-crystallin. This protein is the only differentiating factor we have found between the two cell types.5


   Acknowledgements
 
The authors thank Julia Mausolf, Heide Wiederschein, and Marco Gösswein for expert technical assistance.


   Footnotes
 
Supported by SFB 539 (Glaukome) of the Deutsche Forschungsgemeinschaft, Bonn, Germany.

Submitted for publication March 8, 2005; revised August 4 and November 28, 2005; accepted January 19, 2006.

Disclosure: R. Fuchshofer, None; U. Welge-Lüssen, None; E. Lütjen-Drecoll, None; M. Birke, 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: Marco Birke, Anatomisches Institut II, Universitätsstr. 19, D-91054 Erlangen, Germany; marco.birke{at}anatomie2.med.uni-erlangen.de.


   References
Top
 Abstract
 Material and Methods
 Results
 Discussion
 References
 

  1. Lütjen-Drecoll E, Futa R, Rohen JW. Ultrahistochemical studies on tangential sections of the trabecular meshwork in normal and glaucomatous eyes. Invest Ophthalmol Vis Sci. 1981;21:563–573.[Abstract/Free Full Text]
  2. Maepea O, Bill A. Pressures in the juxtacanalicular tissue and Schlemm’s canal in monkeys. Exp Eye Res. 1992;54:879–883.[CrossRef][ISI][Medline][Order article via Infotrieve]
  3. Rohen JW, van der Zypen E. The phagocytic activity of the trabecular meshwork endothelium: an electron-microscopic study of the vervet (Cercopithecus aethiops). Albrecht Von Graefes Arch Klin Exp Ophthalmol. 1968;175:143–160.[CrossRef][Medline][Order article via Infotrieve]
  4. Rohen JW, Lütjen-Drecoll E. Biology of the Trabecular Meshwork. 1982; Schattauer Verlag New York.
  5. Siegner A, May CA, Welge-Lussen UW, Bloemendal H, Lütjen-Drecoll E. Alpha B-crystallin in the primate ciliary muscle and trabecular meshwork. Eur J Cell Biol. 1996;71:165–169.[ISI][Medline][Order article via Infotrieve]
  6. Welge-Lussen U, Eichhorn M, Bloemendal H, Lütjen-Drecoll E. Classification of human scleral spur cells in monolayer culture. Eur J Cell Biol. 1998;75:78–84.[ISI][Medline][Order article via Infotrieve]
  7. Welge-Lussen U, May CA, Lütjen-Drecoll E. Induction of tissue transglutaminase in the trabecular meshwork by TGF-beta1 and TGF-beta2. Invest Ophthalmol Vis Sci. 2000;41:2229–2238.[Abstract/Free Full Text]
  8. Fuchshofer R, Welge-Lussen U, Lütjen-Drecoll E. The effect of TGF-beta2 on human trabecular meshwork extracellular proteolytic system. Exp Eye Res. 2003;77:757–765.[CrossRef][ISI][Medline][Order article via Infotrieve]
  9. Welge-Lussen 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]
  10. Aumailley M, Timpl R. Attachment of cells to basement membrane collagen type IV. J Cell Biol. 1986;103:1569–1575.[Abstract/Free Full Text]
  11. Kalluri R, Cosgrove D. Assembly of type IV collagen: insights from alpha3(IV) collagen-deficient mice. J Biol Chem. 2000;275:12719–12724.[Abstract/Free Full Text]
  12. Kuhn K. Basement membrane (type IV) collagen. Matrix Biol. 1995;14:439–445.[CrossRef][ISI][Medline][Order article via Infotrieve]
  13. Kleppel M, Fan W, Cheong H, Michael A. Evidence for separate networks of classical and novel basement membrane collagen: characterization of alpha 3(IV)-alport antigen heterodimer. J Biol Chem. 1992;267:4137–4142.[Abstract/Free Full Text]
  14. Yurchenco PD, Amenta PS, Patton BL. Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol. 2004;22:521–538.[CrossRef][ISI][Medline][Order article via Infotrieve]
  15. Colville DJ, Savige J. Alport syndrome: a review of the ocular manifestations. Ophthalmic Genet. 1997;18:161–173.[ISI][Medline][Order article via Infotrieve]
  16. Gunwar S, Ballester F, Noelken ME, Sado Y, Ninomiya Y, Hudson BG. Glomerular basement membrane: identification of a novel disulfide cross-linked network of alpha 3, alpha 4, and alpha 5 chains of type IV collagen and its implications for the pathogenesis of alport syndrome. J Biol Chem. 1998;273:8767–8775.[Abstract/Free Full Text]
  17. Kahsai TZ, Enders GC, Gunwar S, et al. Seminiferous tubule basement membrane: composition and organization of type IV collagen chains, and the linkage of alpha 3(IV) and alpha 5(IV) chains. J Biol Chem. 1997;272:17023–17032.[Abstract/Free Full Text]
  18. Kashtan CE. Clinical and molecular diagnosis of Alport syndrome. Proc Assoc Am Physicians. 1995;107:306–313.[Medline][Order article via Infotrieve]
  19. Kashtan CE. Alport syndrome: an inherited disorder of renal, ocular, and cochlear basement membranes. Medicine (Baltimore). 1999;78:338–360.[CrossRef][Medline][Order article via Infotrieve]
  20. Kashtan CE. Animal models of Alport syndrome. Nephrol Dial Transplant. 2002;17:1359–1362.[Free Full Text]
  21. Gregory MC. Alport syndrome and thin basement membrane nephropathy: Unraveling the tangled strands of type IV collagen. Kidney Int. 2004;65:1109–1110.[CrossRef][Medline][Order article via Infotrieve]
  22. Sasaki T, Fassler R, Hohenester E. Laminin: the crux of basement membrane assembly. J Cell Biol. 2004;164:959–963.[Abstract/Free Full Text]
  23. Ekblom P, Lonai P, Talts JF. Expression and biological role of laminin-1. Matrix Biol. 2003;22:35–47.[CrossRef][ISI][Medline][Order article via Infotrieve]
  24. Koch M, Olson PF, Albus A, et al. Characterization and expression of the laminin gamma3 chain: a novel, non-basement membrane-associated, laminin chain. J Cell Biol. 1999;145:605–618.[Abstract/Free Full Text]
  25. Schymeinsky J, Nedbal S, Miosge N, et al. Gene structure and functional analysis of the mouse nidogen-2 gene: nidogen-2 is not essential for basement membrane formation in mice. Mol Cell Biol. 2002;22:6820–6830.[Abstract/Free Full Text]
  26. Shuttleworth CA. Type VIII collagen. Int J Biochem Cell Biol. 1997;29:1145–1148.[CrossRef][ISI][Medline][Order article via Infotrieve]
  27. Sage H, Trueb B, Bornstein P. Biosynthetic and structural properties of endothelial cell type VIII collagen. J Biol Chem. 1983;258:13391–13401.[Abstract/Free Full Text]
  28. Jakus MA. Studies on the cornea. II. The fine structure of Descemet’s membrane. J Biophys Biochem Cytol. 1956;2:243–252.
  29. Levy SG, McCartney AC, Sawada H, Dopping-Hepenstal PJ, Alexander RA, Moss J. Descemet’s membrane in the iridocorneal-endothelial syndrome: morphology and composition. Exp Eye Res. 1995;61:323–333.[CrossRef][Medline][Order article via Infotrieve]
  30. Levy SG, Moss J, Sawada H, Dopping-Hepenstal PJ, McCartney AC. The composition of wide-spaced collagen in normal and diseased Descemet’s membrane. Curr Eye Res. 1996;15:45–52.[ISI][Medline][Order article via Infotrieve]
  31. Sawada H. The fine structure of the bovine Descemet’s membrane with special reference to biochemical nature. Cell Tissue Res. 1982;226:241–255.[CrossRef][Medline][Order article via Infotrieve]
  32. Sawada H, Konomi H, Hirosawa K. Characterization of the collagen in the hexagonal lattice of Descemet’s membrane: its relation to type VIII collagen. J Cell Biol. 1990;110:219–227.[Abstract/Free Full Text]
  33. Rohen JW, Lutjen E, Barany E. The relation between the ciliary muscle and the trabecular meshwork and its importance for the effect of miotics on aqueous outflow resistance: a study in two contrasting monkey species, Macaca irus and Cercopithecus aethiops. [in German]Albrecht Von Graefes Arch Klin Exp Ophthalmol. 1967;172:23–47.[Medline][Order article via Infotrieve]
  34. Lütjen E. Histometric studies on the ciliary muscle in primates. [in German]Albrecht Von Graefes Arch Klin Exp Ophthalmol. 1966;171:121–133.[Medline][Order article via Infotrieve]
  35. Flügel C, Tamm E, Lütjen-Drecoll E. Different cell populations in bovine trabecular meshwork: an ultrastructural and immunocytochemical study. Exp Eye Res. 1991;52:681–690.[CrossRef][ISI][Medline][Order article via Infotrieve]
  36. Fuchshofer R, Birke M, Welge-Lussen U, Kook D, Lütjen-Drecoll E. Transforming growth factor-ß2 modulated extracellular matrix component expression in cultured human optic nerve head astrocytes. Invest Ophthalmol Vis Sci. 2005;46:568–578.[Abstract/Free Full Text]
  37. Hernandez MR, Ye H, Roy S. Collagen Type IV Gene expression in human optic nerve heads with primary open angle glaucoma. Exp Eye Res. 1994;59:41–52.[CrossRef][ISI][Medline][Order article via Infotrieve]
  38. Lütjen-Drecoll E. Functional morphology of the trabecular meshwork in primate eyes. Prog Retinal Eye Res. 1999;18:91–119.[CrossRef][ISI][Medline][Order article via Infotrieve]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
W. M. Dismuke, C. C. Mbadugha, and D. Z. Ellis
NO-induced regulation of human trabecular meshwork cell volume and aqueous humor outflow facility involve the BKCa ion channel
Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1378 - C1386.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
K. E. Keller, M. J. Kelley, and T. S. Acott
Extracellular Matrix Gene Alternative Splicing by Trabecular Meshwork Cells in Response to Mechanical Stretching
Invest. Ophthalmol. Vis. Sci., March 1, 2007; 48(3): 1164 - 1172.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
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.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fuchshofer, R.
Right arrow Articles by Birke, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fuchshofer, R.
Right arrow Articles by Birke, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS