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


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data Not in Print
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 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 Google Scholar
Google Scholar
Right arrow Articles by Wirtz, M. K.
Right arrow Articles by Acott, T. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wirtz, M. K.
Right arrow Articles by Acott, T. S.
(Investigative Ophthalmology and Visual Science. 2002;43:3698-3704.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Expression Profile and Genome Location of cDNA Clones from an Infant Human Trabecular Meshwork Cell Library

Mary K. Wirtz, John R. Samples, Hong Xu, Tesa Severson and Ted S. Acott

From the Department of Ophthalmology, Casey Eye Institute, Oregon Health and Sciences University, Portland, Oregon.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To delineate the profile of genes expressed in infant human trabecular meshwork and identify candidate genes for glaucoma.

METHODS. Human trabecular meshwork cell cultures were established from six young donors. A cDNA library was made from the combined trabecular meshwork mRNA. The end-sequence of random clones was determined by direct sequencing. These sequences were then analyzed by a National Center for Biotechnology Information (NCBI, Bethesda, MD) database search. Nucleotide searches were performed using the BLASTN (ver. 2.1.3; against the nonredundant nucleic acid sequence) and dbEST databases (both provided by NCBI in the public domain at www.ncbi.nlm.nih.gov).

RESULTS. Sequences from 1118 clones from this nonamplified trabecular meshwork cDNA library were categorized. Of these, 877 expressed sequence tags (ESTs) (78.7%) were known genes. One hundred thirty-nine ESTs (12.5%) showed close identity to EST sequences reported in the public domain database (dbEST). Thirteen ESTs (1.2%) showed no significant similarity to known genes or ESTs in the public databases and were thus defined as novel ESTs. The most abundant genes expressed by the human trabecular meshwork included ferritin H, eukaryotic translation elongation factor 1-{alpha}, ferritin L, fibronectin, and TIMP-1. Ferritin H was the most abundant transcript, making up more than 4% of the genes expressed by the human trabecular meshwork. Extracellular matrix proteins were also highly expressed. The chromosome location of the trabecular meshwork ESTs is reported.

CONCLUSIONS. A profile of genes expressed by human trabecular meshwork is presented. Thirteen novel ESTs were identified. The combined information obtained from expression analysis and chromosomal localization of trabecular meshwork cDNAs should be valuable in identifying candidate genes for glaucoma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Regulation of intraocular pressure is critical for maintaining the correct shape of the eye for vision.1 Abnormal elevation of intraocular pressure often results in damage to the optic nerve and ultimately to glaucoma.2 The human trabecular meshwork is a unique ocular tissue that controls intraocular pressure by regulating the outflow of aqueous humor from the anterior chamber into the venous system.3 Perturbation of the trabecular meshwork and its ability to regulate intraocular pressure may result in glaucoma. The trabecular meshwork is a tiny tissue weighing only 100 to 150 µg and containing approximately 200,000 to 300,000 cells per eye.4 Consequently, molecular analysis of the trabecular meshwork has been limited. Identification of genes active in the trabecular meshwork is central to further understanding of the unique functions of this tissue in normal and glaucomatous eyes.

Primary open-angle glaucoma (POAG) is one of the leading causes of blindness in the industrialized world.5 A disease of the elderly, POAG will have a major impact on the quality of life of a population that is rapidly aging. The pathophysiology of POAG is not well understood. Six POAG genes have been mapped6 7 8 9 10 11 12 and two of these have been identified, MYOC (GLC1A) and OPTN (GLC1E).13 14 Developing better therapeutic strategies for this major blinding disease depends on breakthroughs in understanding the pathophysiology of POAG.

To identify the molecules expressed specifically in the human trabecular meshwork, we used a method called expression profiling of active genes. This method entails sequencing randomly selected clones from a 3'-directed cDNA library that faithfully represents the original composition of mRNA species.15 The active genes are then identified by their sequences, and the relative abundance of each transcript can be estimated by the frequency of the corresponding EST in the cDNA library. The resultant gene expression profile provides a quantitative estimate of the profile of the relative transcript abundance in human trabecular meshwork. This approach is very useful in the identification of cell tissue-type–specific genes by comparing the expression profiles obtained with those from other cell and tissue types.16 17

This is the first analysis of a profile of gene expression in a human trabecular meshwork cell line to be reported. In this study, human trabecular meshwork cell lines from infant donors (age range, 0–2 years) were combined to prepare a cDNA library. An expression profile of the human trabecular meshwork was obtained. Identification of these human trabecular meshwork genes will help in developing a framework for characterizing gene expression in the normal trabecular meshwork. Candidate POAG genes (GLC1) were identified.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of the cDNA Library
All eyes used for cell culture were from patients without a history of ocular disease or surgery, diabetes, liver failure, sepsis, immune disease, or neurologic disease. Eyes were obtained from the Oregon Lions Sight and Hearing Foundation (Portland, OR), and protocols were approved by the Institutional Human Subjects Committee and conformed to the guidelines set forth in the Declaration of Helsinki and by the National Institutes of Health.

The human cDNA library was made from mRNA isolated from trabecular meshwork cells established from eyes of six individuals, aged 2 weeks to 2 years. These cell cultures were initiated and maintained in medium with 10% fetal calf serum, as described earlier.18 Confluent cells were harvested at passages 3 through 6. The cells were trypsinized, neutralized with media, flash frozen in liquid N2 and stored at -70°C. The frozen cells (~96 x 106) were shipped on dry ice to Invitrogen (San Diego, CA) where a unidirectional cDNA library was constructed by using a pcDNA3 vector and TOP10F' host cells. The isolated mRNA was reverse transcribed with an oligo-dT primer. To make the library unidirectional, the inserts were cut with the restriction enzymes BstXI for the 5' end and NotI for the 3' end. The size of the inserts ranged from 0.5 to 1.6 kb, with the average size being 0.85 kb.

Template Preparation and Sequencing
Plasmid purifications were performed with one of three kits (Mini-prep 24 deom McConnell Research, San Diego, CA; the QIAwell 8 ultra plasmid kit from Qiagen, Valencia, CA; or the Perfectprep Plasmid Mini Kit from Eppendorf, Westbury, NY). The purified plasmid preps were then sequenced on an automated sequencer (model 377; Applied Biosystems, Foster City, CA) at the Molecular Microbiology and Immunology (MMI) Research Core Facility, Oregon Health and Sciences University, or the Veterans Administration Medical Center Molecular Biology Core Laboratory (Portland, OR) using the T7 primer. Because the trabecular meshwork cDNA library is unidirectional, all the ESTs were sequenced from the 5' end of the cDNA clones.

Sequence Analysis
Nucleotide searches were first performed using BLASTN (ver. 2.1.3; unless otherwise noted databases used as resources in this study are provided in the public domain by the National Center for Biotechnology Information [NCBI], Bethesda, MD, and are available at the Internet addresses shown, in this case: http://www.ncbi.nlm.nih.gov/BLAST/) against the nonredundant nucleic acid sequence database. Matching sequences with an Expect (E)-value equaling zero were identified as known genes. Sequences with E-values close to zero, were manually analyzed for sequencing errors, alternative splicing, chimeric clones or contamination with vector sequences.

DNA sequences with no matches in the nonredundant GenBank database (http://www.ncbi.nlm.nih.gov/GenBank/) were subjected to a second search with BLASTN against the dbEST (http://www.ncbi.nlm.nih.gov/dbEST/) database. The dbESTs identified were searched for in the Unique Human Gene Sequence Collection database (UniGene; http://www.ncbi.nlm.nih.gov/UniGene/) to determine whether they had been placed in a gene cluster. BLAST searches were performed between September 2000 and September 2001. All the clones that had been identified as ESTs or unknown genes were BLASTed between August and September 2001. GeneRIF (http://www.ncbi.nlm.nih.gov/LocusLink/) was used to identify the functional role of each gene. Some genes had more than one function, and these were placed in one functional classification to avoid confusion.

The sequences of the 1118 cDNA clones were each mapped by BLASTing the sequences with BLASTN against the human genome database. Chromosome locations were also identified in UniGene, and more than 99% agreement was found between the two methods. Sequence data from this article has been deposited with the European Molecular Biology Laboratory (EMBL) and GenBank Data Libraries (the European Molecular Biology Laboratory library is available in the public domain at http://www.embl-heidelberg.de/).

Semiquantitative RT-PCR Analysis of IGFBP-5, -6, and -7
Human trabecular meshwork cDNA was harvested from infant human trabecular meshwork cell cultures containing 10% fetal calf serum. Isolation of mRNA and reverse transcription of the mRNA was performed, as described previously.19 PCR amplification was performed in a final volume of 25 µL containing 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.5 mM MgCl2; 0.001% gelatin; 10 pmol of each primer; 200 µM of dATP, dCTP, dGTP, dTTP; 5 ng of trabecular meshwork cDNA; and 1.0 U Taq polymerase (REDTaq; Sigma, St. Louis, MO) using a commercial PCR system (model 9700; Applied Biosystems) with initial denaturation of 94°C for 10 seconds, 70°C to 60°C for 20 seconds, and 72°C for 40 seconds; 23 cycles of 94°C for 10 seconds, 55°C for 20 seconds, and 72°C for 40 seconds; and a final incubation at 72°C for 5 minutes. The PCR primers used to amplify each of the specific IGFBP mRNAs are shown in Table 1 .


View this table:
[in this window]
[in a new window]
  		Table 1. IGFBP Primers Used to RT-PCR the IGFBP cDNA

 
Touchdown amplification was used to eliminate nonspecific annealing. Different template concentrations and cycle numbers were used to make certain that reactions were in the linear phase of amplification. Different template concentrations, including 0.05, 0.5, 5, and 25 ng were used, whereas other parameters were kept constant. Five nanograms of template was the optimal concentration, with reactions in the linear phase. To further refine the reaction, different numbers of cycles from 20 to 25 were tested after 11 cycles of touchdown amplification. Optimal results within the linear range were established, with 23 cycles used for all three IGFBP PCR products.

Identification of Glaucoma Candidate Genes
The genomic locations of the trabecular meshwork cDNA clones were identified as described above. If a clone mapped to chromosomes 2, 3, 8, or 7 (GLC1B, GLC1C, GLC1D, or GLC1F, respectively), the contig containing that sequence was noted in our database. The initial Human Genome Database sequencing the entire human genome was completed after the GLC1 genes were mapped.20 21 With this new sequence data, the flanking markers of each of the GLC1 loci were identified in their respective contigs and the contigs encompassing each GLC1 locus were aligned. The contigs containing sequences from the trabecular meshwork clones were then compared with the GLC1 contigs. If a contig containing a gene identified by a trabecular meshwork clone was also in a GLC1 contig, then the gene was considered to be a glaucoma candidate gene.

A second method used to determine whether a trabecular meshwork EST was a glaucoma candidate gene was to identify the GLC1 cytogenetic location using the Online Mendelian Inheritance in Man (OMIM) Morbid Map (http://www.ncbi.nlm.nih.gov/Omim/searchmorbid/). LocusLink was then queried with the gene’s accession number to determine whether the gene was within the GLC1 region as specified by the OMIM Morbid Map.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
cDNA Library Analysis
A cDNA library was constructed from human trabecular meshwork cells, as has been described. A listing of the genes in the library is available online (see "cDNA Library of Trabecular Meshwork Genes" at http://www.iovs.org/cgi/content/full/43/12/3698/DC1). No amplification of the mRNA was required, and the library was not screened or subtracted. Therefore, the recurrence of specific genes should represent the actual gene expression level in the human trabecular meshwork. A total of 1118 ESTs were categorized into five classes to determine the fidelity of the library (Table 2) . Class I (883 ESTs, 79.6%) showed identity with the sequences of the nonredundant GenBank database (using BLASTN and E values of zero). These ESTs were labeled as known genes. Class II (137 ESTs, 12.3%) showed close identity to EST sequences reported in the public-domain database (dbEST). These ESTs were named known ESTs-unknown genes. Class III (13 ESTs, 1.2%) showed no significant similarity to known genes or known ESTs in the public databases and were thus defined as novel ESTs. Class IV (43 ESTs, 3.8%) contained transcripts of mitochondrial origin. Class V (42 ESTs, 3.8%) included vector, ribosomal RNA (three sequences), or repetitive elements. The low number of class IV and V elements indicates that genomic and mitochondrial contamination of the cDNA library was low.


View this table:
[in this window]
[in a new window]
  		Table 2. Summary of ESTs Found in the HTM Library

 
The 883 known genes expressed by the human trabecular meshwork cell lines were categorized into eight subgroups based on their functions, according to the format of Adams et al.22 The 883 clones representing known genes are listed online at the Internet address provided in the prior paragraph with their RefSeq accession numbers, how many times they were observed (frequency), and their chromosome location. With the near completion of the Human Genome Project, more than 95% of the genes have been mapped.

Unknown Human Trabecular Meshwork Genes
Of the 1118 clones sequenced, 137 (12.3%) produced sequences matching ESTs from unknown genes previously reported in the dbEST databank. A listing of these ESTs is also available online (see "ESTs from Unknown Genes in the Trabecular Meshwork Library" at http://www.iovs.org/cgi/content/full/43/12/3698/DC1). UniGene clusters have been established for 122 of these ESTs. The number of ESTs in each of these clusters is shown in the online listing of ESTs from unknown genes. The ESTs are sorted by their abundance in UniGene. The ESTs not matching UniGene clusters are shown at the bottom of the table. Sixteen of these clones matched an EST in dbEST but have no UniGene cluster. The remaining 13 clones were unique sequences not found in BLASTN, dbEST, or UniGene, which implies that these genes are expressed only in trabecular meshwork or are very rare transcripts that have not been found in previous EST projects. All the unknown genes were relatively rare trabecular meshwork transcripts, with only one or two clones being found. The majority (91.5%) of the unknown human trabecular meshwork sequences have been mapped. Thus, ESTs that were GLC candidate genes were established.

Highly Expressed ESTs
The ESTs more highly represented (by six or more clones) in the human trabecular meshwork library are summarized in a listing of highly expressed genes (Table 3) . The three most abundant transcripts are ferritin H, eukaryotic translation elongation factor 1-{alpha}, and ferritin L chain. Ferritin H comprises 4.28% of the clones in the human trabecular meshwork library. ESTs shared with the organ culture human trabecular meshwork cDNA library23 are indicated by asterisks. Of the 17 highly expressed genes, 7 were also present in the organ culture trabecular meshwork cDNA library.23


View this table:
[in this window]
[in a new window]
  		Table 3. Highly Expressed Genes in TM cDNA Library

 
Interleukin-1ß, which may mediate the response of trabecular meshwork cells to laser trabeculoplasty as shown by our group,24 was expressed in the trabecular meshwork library (See Trabecular Meshwork Gene listing online).

Insulin-like Growth Factor–Binding Proteins
Most of the known insulin-like growth factor binding proteins (IGFBPs)25 were expressed in the human trabecular meshwork cDNA library. Two of the IGFBPs were highly expressed: IGFBP7 and IGFBP2 (Table 3) . Lower amounts of IGFBP4, IGFBP5, IGFBP6, and IGFBP8 were present. To determine whether the number of clones reflects the abundance of mRNA in the human trabecular meshwork, RT-PCR of IGFBP7, IGFBP6, and IGFBP5 was performed. All three IGFBP messages were present (Fig. 1) . The quantity of IGFBP7 mRNA appeared to be at least two to four times higher than IGFBP6 and IGBP5 mRNAs, reflecting the fourfold increase in IGFBP7 found in the trabecular meshwork cDNA library.



View larger version (84K):
[in this window]
[in a new window]
  		Figure 1. RT-PCR of IGFBP5, IGFBP6, and IGFBP7 from cDNA isolated from confluent human infant trabecular meshwork cells grown in 10% fetal calf serum.

 
Candidate Glaucoma Genes Expressed in the Human Trabecular Meshwork Library
Six loci for POAG have been mapped.7 8 9 10 11 12 The genes for GLC1A and GLC1E have been identified, MYOC and OPTN, respectively.13 14 The genomic location of all 1118 ESTs in the human trabecular meshwork library was established to determine whether any of these were glaucoma candidate genes for the four remaining GLC1 loci. As shown in Table 4 , 10 of the ESTs mapped to the GLC loci. Two different methods were used to determine whether the ESTs from the human trabecular meshwork library were GLC1 candidate genes. Agreement between the two methods was found for the GLC1C, GLC1D, and GLC1F candidate genes (see Table 4 , OMIM Morbid Map versus MapView; http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/map_search). However, discrepancy between the two methods was observed for three of the GLC1B candidate genes. This most likely is due to the refined mapping of the flanking marker, D2S2161, which, according to the Human Genome Database, is within NT_015805, which maps to 2p12-p11.1. Originally, D2S2161 was mapped to 2qcen.11 GLC1B had the most candidate genes including endobrevin, DKFzp586M0122, a homologue of mouse DNA-directed RNA polymerase I, ribosomal protein L31, and thymosin-ß.10 The gene for GLC1A, myocilin, was not present in the trabecular meshwork library, however, previous reports have shown that myocilin is expressed at low levels in trabecular meshwork cell lines unless the cells are stressed or exposed to glucocorticoids.26 27 28 29 30 OPTN, the GLC1E gene, was also not present in the screened library.14 Ten candidate genes for glaucoma have been identified in this human trabecular meshwork library. In addition, PITX2, which causes Reiger syndrome type I (RIEG1) was identified in the library.31 32 33 However, CYP1B1, which is associated with congenital glaucoma was not found.34


View this table:
[in this window]
[in a new window]
  		Table 4. Candidate GLC1 Genes Expressed in the Trabecular Meshwork cDNA Library

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Composition of Human Trabecular Meshwork cDNA Library
The majority (78.9%) of ESTs identified in the human trabecular meshwork library are known genes. With each succeeding cDNA library added to the public database, a greater number of genes are being identified. Thus, the high number of known genes is to be expected. Conversely, fewer (1.2%) unique genes were identified in our human trabecular meshwork library, again reflecting the growing database. However, these unique genes are probably of specific importance to trabecular meshwork function. The low number of unique genes also illustrates that this library reflects the total transcript level within the trabecular meshwork, because it is not subtracted. The low number (3.8%) of mitochondrial ESTs as well as of repetitive sequences, vector or ribosomal RNA (3.8%) shows that this library primarily consists of cDNA transcripts.

Characterization of ESTs Present in Normal Human Trabecular Meshwork Cell Lines
The trabecular meshwork morphology is still developing in the period after birth up to 4 years of age.35 Thus, the cell lines, which were established from individuals aged 2 weeks to 2 years may represent a transitional stage with some fundamental differences in gene expression compared with adult cells. Of note, the two major transcripts in our cDNA library were also the two major transcripts in a mouse blastocyst cDNA library.36

Glaucoma Genes
The transcript for myocilin, the GLC1A gene, was not observed in the sequenced clones. This is not unexpected, because myocilin is expressed at low levels in trabecular meshwork cell lines, unless they have been stressed or induced with glucocorticoids.26 27 28 29 30 Candidate genes for GLC1B, GLC1C, GLC1D, and GLC1F were identified. Hepatocellular carcinoma-associated antigen 112 has been analyzed in affected members of GLC1F; however, no disease-causing mutations have been found. We are in the process of analyzing the remaining GLC1C and GLC1F candidate genes in affected family members. PITX2, the RIEG1 gene, was expressed in our library.31 32 33

Abundant Human Trabecular Meshwork Transcripts
The most abundantly expressed transcript in the human trabecular meshwork library was ferritin H, representing 4.28% of the library clones sequenced. The ferritin L gene was the third highest expressed in the trabecular meshwork library. Ferritin is the major iron storage protein of eukaryotes and appears to be ubiquitously expressed.37 The ferritin protein is a spherical shell composed of 24 subunits of varying proportions of H and L chains, depending on the tissue type.38 H-rich ferritin predominates in erythropoietic tissues, where iron availability is required both for heme synthesis and host defense. Ferritin H accumulates and releases iron faster than L-rich ferritin.38 For long-term iron storage in the liver and spleen, L-rich ferritin is the preferred form. The human trabecular meshwork cells were cultured in 10% fetal calf serum, which is rich in iron. The levels of ferritin H transcripts may result from exposure of the cells to a high concentration of iron and/or serum, because both are known to induce synthesis of ferritin H.39 Potential functions for ferritin in the anterior chamber may include providing iron to apoenzymes, boosting host defense and neutralization of oxidative stress by the trabecular meshwork. Myocilin (GLC1A) may be a heat shock or oxidative stress protein. The similar role of ferritin H in macrophages during oxidative stress suggests that, under certain environmental stresses, ferritin H and myocilin may both be important in protecting the trabecular meshwork from reactive radicals. Numerous heat shock proteins, including {alpha}B crystallin, HSPB1, HSPA5, HSPCA, and chaperonin were found in the trabecular meshwork library in relatively abundant numbers.

Ferritin was not as highly expressed in the human trabecular meshwork library synthesized from organ culture message,23 although ferritin L was an abundant transcript. This difference between the two human trabecular meshwork libraries could either reflect cell culture versus organ culture, the age difference between the samples, or serum versus serum-free conditions. The ratio of H to L chains varies during development.38 40 Our cDNA library is from cells from infants in whom the trabecular meshwork is still maturing, which may be why this ratio is observed.

Another gene, known for its role in protecting motoneurons from iron toxicity, macrophage inhibitory cytokine-1, was expressed in the trabecular meshwork library.41

IGFBP7 and IGFBP2 were highly expressed in the trabecular meshwork cDNA library, with eight and six clones, respectively. Lower expression of IGFBP5, IGFBP6, IGFBP8, and IGFBP4 was observed. In an earlier article, we described the expression of IGFBP5 in trabecular meshwork.42 In the organ culture human trabecular meshwork, IGFBP3 and IGFBP5 were expressed.23 Thus, all the known IGFBPs with the exception of IGFBP1 have been shown to be expressed in human trabecular meshwork.

Gene Expression and Mechanisms of Trabecular Meshwork Outflow
The trabecular meshwork has been implicated as the site of obstruction of aqueous outflow, which in turn leads to increased intraocular pressure and potentially to glaucoma.4 43 44 Several of the genes expressed in our trabecular meshwork library have been explored by us and others as possible modifiers of aqueous outflow through the trabecular meshwork. Interleukin-1ß, which appears to mediate the response of trabecular meshwork cells to laser trabeculoplasty as shown by our group,24 was expressed in the trabecular meshwork library (see Trabecular Meshwork Gene listing online). Endogenous production of interleukin-1ß by glaucomatous trabecular meshwork cells also appears to protect the cells from responding to oxidative stress by induction of apoptosis.45

The cytoskeletal proteins may also affect aqueous outflow. Several pharmacologic agents that cause changes in cytoskeletal structure in trabecular meshwork cells are used clinically to increase outflow facility.46 47 Several cytoskeleton proteins, including {alpha}-smooth muscle actin, {gamma}1 actin, three isoforms of myosin, and vinculin were expressed in our trabecular meshwork library. Expression of {alpha}-smooth muscle actin has previously been demonstrated in trabecular meshwork organ culture and cells.48 49 50

Fibronectin, also highly expressed in our library, has been known to be a major component of trabecular meshwork extracellular matrix.51 52 Increased fibronectin deposition in the aqueous outflow pathway has been shown to occur in elderly and glaucomatous eyes,51 although a recent study found no differences between normal and glaucomatous eyes.53 The potential for fibronectin to influence the flow of aqueous humor through the juxtacanalicular trabecular meshwork and the inner walls of Schlemm’s canal has intrigued many glaucoma researchers over the years.51 52 53 54 55 56 57

Another abundant gene expressed in the trabecular meshwork was GAPDH, which is known for its role in glycolysis and oxidative phosphorylation. Considering that glycolysis is the major energy pathway for the trabecular meshwork,58 expression of the glycolytic enzymes would be expected. However, although high levels of GAPDH transcript were found, other genes for glycolytic enzymes were expressed at lower levels: glucose-6-phosphate dehydrogenase and enolase 1, each of which had two clones compared with seven clones for GAPDH. Once considered a housekeeping protein, many new functions for GAPDH have been ascribed, including membrane, cytoplasmic and nuclear functions in endocytosis, mRNA regulation, tRNA export, DNA replication, and DNA repair.59 Thus, the presence of GAPDH in our cDNA library may reflect additional roles for GAPDH in the trabecular meshwork in addition to glycolysis.

Comparison of Genes Highly Represented in Cell Line and Organ Culture Human Trabecular Meshwork cDNA Libraries
Four genes were highly expressed in both the organ culture library reported by Gonzalez et al.23 and our cell culture human trabecular meshwork cDNA library. These include ferritin L, translation elongation factor 1-{alpha}, GAPDH, and TIMP1. Approximately 25% of the transcripts were shared between the two libraries. Glycolysis provides the major source of energy to the trabecular meshwork; thus, high levels of GAPDH would be expected in both human trabecular meshwork cDNA libraries.58 The high levels of translation elongation factor 1-{alpha} in both cDNA libraries suggests that translational control may be an important method for regulating gene expression in the trabecular meshwork.

Primary cultured cells and organ cultures each have their unique advantages and disadvantages. One advantage of using primary cultured cells is that more cells can be obtained, thus allowing RNA work without need for amplification. In addition, the cells can be frozen for storage and thus are readily available for experimentation, whereas experiments with organ cultures are dependent on the supply of eyes. Because the trabecular meshwork cells have been isolated, changes occurring within these cells are solely a result of the trabecular meshwork’s physiology and are not influenced by adjacent tissues. However, some limitations of the cell culture system do exist. One of these is that mechanical forces acting on the outflow pathway tissue are not present in traditional cell culture. In addition, changes in gene expression may occur in cultured cells that do not reflect in vivo conditions. Organ cultures are probably a closer approximation of what is actually happening in the eye, because this model preserves the architecture of the outflow tissue. However, a limited amount of tissue necessitates amplification of the isolated message. Thus, the resultant library may not accurately reflect actual levels of specific messages, because some may be amplified better than others. Both primary cultured cells and organ cultures have unique advantages and disadvantages that must be taken into consideration in analysis of resultant data. Analysis of the two systems will give a much fuller picture of what is actually occurring in vivo, then either one alone could do.

Additional research is needed to generate a comprehensive profile of genes expressed in the trabecular meshwork during development and aging. The study reported herein and another recent publication23 provide a comprehensive reference point and an important resource of cloned "trabecular meshwork-expressed genes" that can be used for biological and functional investigations. Expression and chromosomal localization of trabecular meshwork ESTs should facilitate identification of mutations that result in or contribute to glaucoma.


   Acknowledgements
 
The authors thank the Oregon Lions Sight and Hearing Foundation for providing eyes and the MMI Research Core Facility at Oregon Health and Sciences University and the Portland VAMC Molecular Biology Core Laboratory for sequencing.


   Footnotes
 
Supported by National Eye Institute Grants R01 EY10555, R01 EY11650, R01 EY03279, R01 EY08247, and R01 EY10572, Research to Prevent Blindness, and Alcon Laboratories.

Submitted for publication March 5, 2002; revised July 12, 2002; accepted July 23, 2002.

Commercial relationships policy: F.

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: Mary K. Wirtz, Department of Ophthalmology, Casey Eye Institute, Oregon Health and Sciences University, 3375 SW Terwilliger Boulevard, Portland, OR 97201-4197; wirtzm{at}ohsu.edu.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Civan, MM. (1998) Transport components of net secretion of the aqueous humor and their integrated regulation Civan, MM eds. The Eye’s Aqueous Humor 45,1-18 Academic Press San Diego.
  2. Hitchings, R, Tan, J. (2001) Target pressure J Glaucoma 10(suppl 1),568-570
  3. Lütjen-Drecoll, E, Gabelt, BT, Tian, B, Kaufman, PL. (2001) Outflow of aqueous humor J Glaucoma 10(suppl 1),S42-S44[Medline][Order article via Infotrieve]
  4. Acott, TS, Wirtz, MK. (1996) Biochemistry of aqueous outflow Ritch, R Shields, MB Krupin, T eds. The Glaucomas 1,281-306 Mosby St. Louis.
  5. Quigley, HA. (1996) Number of people with glaucoma worldwide Br J Ophthalmol 80,389-393[Abstract/Free Full Text]
  6. Raymond, V. (1997) Molecular genetics of the glaucomas: mapping of the first five "GLC" loci Am J Hum Genet 60,272-277[Medline][Order article via Infotrieve]
  7. Wirtz, MK, Samples, JR, Kramer, PL, et al (1997) Mapping a gene for adult-onset primary open-angle glaucoma to chromosome 3q Am J Hum Genet 60,296-304[Medline][Order article via Infotrieve]
  8. Wirtz, MK, Samples, JR, Rust, K, et al (1999) GLC1F, a new primary open-angle glaucoma locus, maps to 7q35–q36 Arch Ophthalmol 117,237-241[Abstract/Free Full Text]
  9. Sarfarazi, M, Child, A, Stoilova, D, et al (1998) Localization of the fourth locus (GLC1E) for adult-onset primary open-angle glaucoma to the 10p15–p14 region Am J Hum Genet 62,641-652[Medline][Order article via Infotrieve]
  10. Trifan, OC, Traboulsi, EI, Stoilova, D, et al (1998) A third locus (GLC1D) for adult-onset primary open-angle glaucoma maps to the 8q23 region Am J Ophthalmol 126,17-28[Medline][Order article via Infotrieve]
  11. Stoilova, D, Child, A, Trifan, OC, et al (1996) Localization of a locus (GLC1B) for adult-onset primary open angle glaucoma to the 2cen-q13 region Genomics 36,142-150[Medline][Order article via Infotrieve]
  12. Sheffield, VC, Stone, EM, Alward, WLM, et al (1993) Genetic linkage of familial open angle glaucoma to chromosome 1q21–q31 Nat Genet 4,47-50[Medline][Order article via Infotrieve]
  13. Stone, EM, Fingert, JH, Alward, WLM, et al (1997) Identification of a gene that causes primary open angle glaucoma Science 275,668-670[Abstract/Free Full Text]
  14. Rezaie, T, Child, A, Hitchings, R, et al (2002) Adult-onset primary open-angle glaucoma caused by mutations in optineurin Science 295,1077-1079[Abstract/Free Full Text]
  15. Okubo, K, Hori, N, Matoba, R, et al (1992) Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression Nat Genet 2,173-179[Medline][Order article via Infotrieve]
  16. Shimizu-Matsumoto, A, Adachi, W, Mizuno, K, et al (1997) An expression profile of genes in human retina and isolation of a complementary DNA for a novel rod photoreceptor protein Invest Ophthalmol Vis Sci 38,2576-2585[Abstract/Free Full Text]
  17. Yamada, S, Murakami, S, Matoba, R, et al (2001) Expression profile of active genes in human periodontal ligament and isolation of PLAP-1, a novel SLRP family gene Gene 275,279-286[Medline][Order article via Infotrieve]
  18. Alexander, JP, Samples, JR, Van Buskirk, EM, Acott, TS. (1991) Expression of matrix metalloproteinases and inhibitor by human trabecular meshwork Invest Ophthalmol Vis Sci 32,172-180[Abstract/Free Full Text]
  19. Xu, H, Acott, TS, Wirtz, MK. (2000) Identification and expression of a novel type I procollagen C-proteinase enhancer protein gene from the glaucoma candidate region on 3q21-24 Genomics 66,264-273[Medline][Order article via Infotrieve]
  20. Lander, ES, Linton, LM, Birren, B, et al (2001) Initial sequencing and analysis of the human genome Nature 409,860-921[Medline][Order article via Infotrieve]
  21. Venter, JC, Adams, MD, Myers, EW, et al (2001) The sequence of the human genome Science 291,1304-1351[Abstract/Free Full Text]
  22. Adams, MD, Kerlavage, AR, Fleischman, RD, et al (1995) Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence Nature 28(suppl),33-174
  23. Gonzalez, P, Epstein, DL, Borras, T. (2000) Characterization of gene expression in human trabecular meshwork using single-pass sequencing of 1060 clones Invest Ophthalmol Vis Sci 41,3678-3693[Abstract/Free Full Text]
  24. Bradley, JMB, Anderssohn, AM, Colvis, CM, et al (2000) Mediation of laser trabeculoplasty-induced matrix metalloproteinase expression by Il-1ß and TNF{alpha} Invest Ophthalmol Vis Sci 41,422-430[Abstract/Free Full Text]
  25. Baxter, RC. (2000) Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities Am J Physiol Endocrinol Metab 278,E967-E976[Abstract/Free Full Text]
  26. Polansky, JR, Fauss, DJ, Chen, P, et al (1997) Cellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product Ophthalmologica 211,126-139[Medline][Order article via Infotrieve]
  27. Clark, AF, Steely, HT, Dickerson, JEJ, et al (2001) Glucocorticoid induction of the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues Invest Ophthalmol Vis Sci 42,1769-1780[Abstract/Free Full Text]
  28. Wentz-Hunter, K, Ueda, J, Shimizu, N, Yue, BYJT. (2002) Myocilin is associated with mitochondria in human trabecular meshwork cells J Cell Physiol 190,46-53[Medline][Order article via Infotrieve]
  29. O’Brien, ET, Metheney, CD, Polansky, JR. (1999) Immunofluorescence method for quantifying the trabecular meshwork glucocorticoid response (TIGR) protein in trabecular meshwork and Schlemm’s canal cells Curr Eye Res 19,517-524[Medline][Order article via Infotrieve]
  30. Stamer, WD, Roberts, BC, Howell, DN, Epstein, DL. (1998) Isolation, culture, and characterization of endothelial cells from Schlemm’s canal Invest Ophthalmol Vis Sci 39,1804-1812[Abstract/Free Full Text]
  31. Alward, WL, Semina, EV, Kalenak, JW, et al (1998) Autosomal dominant iris hypoplasia is caused by a mutation in the Rieger syndrome (RIEG/PITX2) gene Am J Ophthalmol 125,98-100[Medline][Order article via Infotrieve]
  32. Kulak, SC, Kozlowski, K, Semina, EV, Pearce, WG, Walter, MA. (1998) Mutation in the RIEG1 gene in patients with iridogoniodysgenesis syndrome Hum Mol Genet 7,1113-1117[Abstract/Free Full Text]
  33. Amendt, BA, Sutherland, LB, Semina, EV, Russo, AF. (1998) The molecular basis of Rieger syndrome: analysis of Pitx2 homeodomain protein activities J Biol Chem 273,20066-20072[Abstract/Free Full Text]
  34. Stoilov, I, Akarsu, AN, Sarfarazi, M. (1997) Identification of three different truncating mutations in cytochrome P4501B1 (CYP1B1) as the principal cause of primary congenital glaucoma (buphthalmos) in families linked to the GLC3A locus on chromosome 2p21 Hum Mol Genet 6,641-647[Abstract/Free Full Text]
  35. Tripathi, BJ, Tripathi, RC, Wisdom, JE. (1996) Embryology of the anterior segment of the human eye Ritch, R Shields, MB Krupin, T eds. The Glaucomas 1,3-38 Mosby St. Louis.
  36. Sasaki, N, Nagaoka, S, Itoh, M, et al (1998) Characterization of gene expression in mouse blastocyst using single-pass sequencing of 3995 clones Genomics 49,167-179[Medline][Order article via Infotrieve]
  37. Boyd, D, Vecoli, C, Belcher, DM, Jain, SK, Drysdale, JW. (1985) Structural and functional relationships of human ferritin H and L chains deduced from cDNA clones J Biol Chem 260,11755-11761[Abstract/Free Full Text]
  38. Beard, JL. (2001) Iron biology in immune function, muscle metabolism and neuronal functioning J Nutr 131,568S-580S[Abstract/Free Full Text]
  39. Gdaniec, Z, Sierzputowska-Gracz, H, Theil, EC. (1998) Iron regulatory element and internal loop/bulge structure for ferritin mRNA studied by cobalt(III) hexamine binding, molecular modeling, and NMR spectroscopy Biochemistry 37,1505-1512[Medline][Order article via Infotrieve]
  40. Connor, JR, Menzies, SL. (1996) Relationship of iron to oligodendrocytes and myelination Glia 17,83-93[Medline][Order article via Infotrieve]
  41. Strelau, J, Sullivan, A, Bottner, M, et al (2000) Growth/differentiation factor-15/macrophage inhibitory cytokine-1 is a novel trophic factor for midbrain dopaminergic neurons in vivo J Neurosci 20,8597-8603[Abstract/Free Full Text]
  42. Wirtz, MK, Xu, H, Rust, K, Alexander, JP, Acott, TS. (1998) Insulin-like growth factor binding protein-5 expression by human trabecular meshwork Invest Ophthalmol Vis Sci 39,45-53[Abstract/Free Full Text]
  43. Hoskins, HD, Kass, MA. (1989) Aqueous humor outflow Diagnosis and Therapy of the Glaucomas ,41-66 CV Mosby Co. St. Louis.
  44. Shields, MB. (1992) Aqueous humor dynamics. I. Anatomy and physiology Shields, MB eds. Textbook of Glaucoma ,5-36 Williams & Wilkins Baltimore.
  45. Wang, N, Chintala, SK, Fini, ME, Schuman, JS. (2001) Activation of a tissue-specific stress response in the aqueous outflow pathway of the eye defines the glaucoma disease phenotype Nat Med 7,304-309[Medline][Order article via Infotrieve]
  46. Cai, S, Liu, X, Glasser, A, et al (2000) Effect of latrunculin-A on morphology and actin-associated adhesions of cultured human trabecular meshwork cells Mol Vis 6,132-143[Medline][Order article via Infotrieve]
  47. Tripathi, BJ, Tripathi, RC. (1984) Effect of epinephrine in vitro on the morphology, phagocytosis, and mitotic activity of human trabecular endothelium Exp Eye Res 39,731-744[Medline][Order article via Infotrieve]
  48. de Kater, AW, Shahsafaei, A, Epstein, DL. (1992) Localization of smooth muscle and nonmuscle actin isoforms in the human aqueous outflow pathway Invest Ophthalmol Vis Sci 33,424-429[Abstract/Free Full Text]
  49. Gibson, IK, Anderson, RA. (1979) Actin filaments in cells of human trabecular meshwork and Schlemm’s canal Invest Ophthalmol Vis Sci 18,547-561[Abstract/Free Full Text]
  50. Tamm, ER, Siegner, A, Baur, A, Lütjen-Drecoll, E. (1996) Transforming growth factor-beta 1 induces alpha-smooth muscle-actin expression in cultured human and monkey trabecular meshwork Exp Eye Res 62,389-397[Medline][Order article via Infotrieve]
  51. Floyd, BB, Cleveland, PH, Worthen, DM. (1985) Fibronectin in human trabecular drainage channels Invest Ophthalmol Vis Sci 26,797-804[Abstract/Free Full Text]
  52. Sato, T, Roy, S. (2002) Effect of high glucose on fibronectin expression and cell proliferation in trabecular meshwork cells Invest Ophthalmol Vis Sci 43,170-175[Abstract/Free Full Text]
  53. Hann, CR, Springett, MJ, Wang, X, Johnson, DH. (2001) Ultrastructural localization of collagen IV, fibronectin, and laminin in the trabecular meshwork of normal and glaucomatous eyes Ophthalmic Res 33,314-324[Medline][Order article via Infotrieve]
  54. Filla, MS, Liu, X, Nguyen, TD, et al (2002) In vitro localization of TIGR/MYOC in trabecular meshwork extracellular matrix and binding to fibronectin Invest Ophthalmol Vis Sci 43,151-161[Abstract/Free Full Text]
  55. Babizhayev, MA, Brodskaya, MW. (1989) Fibronectin detection in drainage outflow system of human eyes in ageing and progression of open-angle glaucoma Mech Ageing Dev 47,145-157[Medline][Order article via Infotrieve]
  56. Zhou, L, Yuhong, L, Yue, BYJT. (1999) Oxidative stress affects cytoskeletal structure and cell-matrix interactions in cells from an ocular tissue: the trabecular meshwork J Cell Physiol 180,182-189[Medline][Order article via Infotrieve]
  57. Li, J, Tripathi, BJ, Tripathi, RC. (2000) Modulation of pre-mRNA splicing and protein production of fibronectin by TGF-beta2 in porcine trabecular cells Invest Ophthalmol Vis Sci 41,3437-3443[Abstract/Free Full Text]
  58. Anderson, PJ, Wang, J, Epstein, DL. (1980) Metabolism of calf trabecular (reticular) meshwork Invest Ophthalmol Vis Sci 19,13-20[Abstract/Free Full Text]
  59. Sirover, MA. (1997) Role of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in normal cell function and in cell pathology J Cell Biochem 66,133-140[Medline][Order article via Infotrieve]



This article has been cited by other articles:


Home page
 IOVSHome page
I. Michael, M. Shmoish, D. S. Walton, and S. Levenberg
Interactions between Trabecular Meshwork Cells and Lens Epithelial Cells: A Possible Mechanism in Infantile Aphakic Glaucoma
Invest. Ophthalmol. Vis. Sci., September 1, 2008; 49(9): 3981 - 3987.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
W. Xue, N. Comes, and T. Borras
Presence of an Established Calcification Marker in Trabecular Meshwork Tissue of Glaucoma Donors
Invest. Ophthalmol. Vis. Sci., July 1, 2007; 48(7): 3184 - 3194.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
W. Xue, R. Wallin, E. A. Olmsted-Davis, and T. Borras
Matrix GLA Protein Function in Human Trabecular Meshwork Cells: Inhibition of BMP2-Induced Calcification Process.
Invest. Ophthalmol. Vis. Sci., March 1, 2006; 47(3): 997 - 1007.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
M. P. Fautsch, K. G. Howell, A. M. Vrabel, M. C. Charlesworth, D. C. Muddiman, and D. H. Johnson
Primary Trabecular Meshwork Cells Incubated in Human Aqueous Humor Differ from Cells Incubated in Serum Supplements
Invest. Ophthalmol. Vis. Sci., August 1, 2005; 46(8): 2848 - 2856.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
P. B. Liton, X. Liu, W. D. Stamer, P. Challa, D. L. Epstein, and P. Gonzalez
Specific Targeting of Gene Expression to a Subset of Human Trabecular Meshwork Cells Using the Chitinase 3-Like 1 Promoter
Invest. Ophthalmol. Vis. Sci., January 1, 2005; 46(1): 183 - 190.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
P. Gonzalez, M. Caballero, P. B. Liton, W. D. Stamer, and D. L. Epstein
Expression Analysis of the Matrix GLA Protein and VE-Cadherin Gene Promoters in the Outflow Pathway
Invest. Ophthalmol. Vis. Sci., May 1, 2004; 45(5): 1389 - 1395.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data Not in Print
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 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 Google Scholar
Google Scholar
Right arrow Articles by Wirtz, M. K.
Right arrow Articles by Acott, T. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wirtz, M. K.
Right arrow Articles by Acott, T. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS