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(Investigative Ophthalmology and Visual Science. 2005;46:2857-2868.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0075
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Changes in Gene Expression by Trabecular Meshwork Cells in Response to Mechanical Stretching

Vasavi Vittal, Anastasia Rose, Kate E. Gregory, Mary J. Kelley, and Ted S. Acott

From the Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Trabecular meshwork (TM) cells appear to sense changes in intraocular pressure (IOP) as mechanical stretching. In response, they make homeostatic corrections in the aqueous humor outflow resistance, partially by increasing extracellular matrix (ECM) turnover initiated by the matrix metalloproteinases. To understand this homeostatic adjustment process further, studies were conducted to evaluate changes in TM gene expression that occur in response to mechanical stretching.

METHODS. Porcine TM cells were subjected to sustained mechanical stretching, and RNA was isolated after 12, 24, or 48 hours. Changes in gene expression were evaluated with microarrays containing approximately 8000 cDNAs. Select mRNA changes were then compared by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). Western immunoblots were used to determine whether some of these changes were associated with changes in protein levels.

RESULTS. On the microarrays, 126 genes were significantly upregulated, and 29 genes were significantly downregulated at one or more time points, according to very conservative statistical and biological criteria. Of the genes that changed, several ECM regulatory genes, cytoskeletal-regulatory genes, signal-transduction genes, and stress-response genes were notable. These included several proteoglycans and matricellular ECM proteins composed of common repetitive binding domains. The results of analysis of mRNA changes in more than 20 selected genes by qRT-PCR supported the findings in the microarray analysis. Western immunoblots of several proteins demonstrated protein level changes associated with changes in the level of mRNA.

CONCLUSIONS. The expression of a variety of TM genes is significantly affected by mechanical stretching. These include several ECM proteins that contain multiple binding sites and may serve organizational roles in the TM. Several proteins that could contribute to the homeostatic modification of aqueous humor outflow resistance are also upregulated or downregulated.

The mechanisms that provide normal IOP homeostasis are only partially understood. A relatively effective homeostatic mechanism must exist, because only approximately 2% to 5% of people exhibit pathologic elevations in IOP with subsequent optic nerve damage, even at advanced ages.1 2 We have hypothesized that TM cells can adjust outflow resistance over a timescale of hours to days by modulating trabecular ECM turnover and subsequent biosynthetic replacement.3 4 5 6 Manipulation of the trabecular activity of a family of ECM turnover enzymes, the matrix metalloproteinases (MMPs), reversibly modulates outflow facility.7 Inhibition of the endogenous ECM turnover, which is initiated by these MMPs, increases the outflow resistance. Therefore, ongoing ECM turnover must be necessary for homeostatic maintenance of the IOP. In addition, laser trabeculoplasty, a common treatment for glaucoma, appears to owe its efficacy to producing relatively sustained trabecular MMP elevations, particularly within the juxtacanalicular region of the meshwork.8 9 10

Much of the aqueous humor outflow resistance appears to reside within the deepest portion of the TM.11 Consequently, this resistance is functionally stretched like a diaphragm across Schlemm’s canal. Alterations in the outflow resistance that are sufficient to affect IOP change the degree of stretching and distortion of the juxtacanalicular TM. A likely sensing mechanism in TM cells, indicating that an increase or decrease in outflow resistance is needed, is that of alterations in mechanical tension or stretching. This sensing could be mediated and transduced by integrin–ECM or similar types of interactions.12 13 We and others have found that trabecular cells can sense changes in IOP and mechanical stretching and produce several distinctive responses.14 15 16 17 18 19 20 21 22 23 24 In support of our overall hypothesis of IOP homeostasis, trabecular cells respond to pressure elevations in perfused organ culture or to mechanical stretching in cell culture by increasing MMP-2 (gelatinase A) and MMP-14 (membrane-type-1-MMP) activity or levels, while dramatically reducing levels of their primary tissue inhibitor, TIMP-2.14 Increased trabecular MMP activity results in reduced outflow resistance7 and thus should restore IOP to normal levels. Therefore, the components of a self-contained trabecular IOP homeostasis mechanism are present and functional. In perfused anterior segment organ culture, increased flow rates produce initial elevations of IOP, which return to normal over several days, even with sustained increases in perfusion rate.14 23

An important component of this putative IOP homeostasis mechanism is the nature of the changes in the ECM that adjust the outflow resistance. The ECM turnover process is initiated by secretion/activation of the MMPs, which partially degrades select ECM proteins. This phase is followed by the removal of proteolytic ECM fragments, presumably facilitated by TM cell endocytosis. New materials must then be synthesized by TM cells to replace degraded ECM components. To adjust the outflow resistance, changes in the amount, composition, or organization of this ECM are likely to be instituted. This process must occur within the active pathway of aqueous humor outflow without allowing excessive structural disorganization. To begin unraveling the details of this complex process, microarray analysis of TM gene expression changes occurring after sustained mechanical stretching were conducted.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Porcine eyes were obtained from Carlton Packing (Carlton, OR); proteinase inhibitor cocktail, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), and horseradish peroxidase-conjugated secondary antibodies from Sigma-Aldrich (St. Louis, MO); a DNA quantitation reagent (PicoGreen) from Molecular Probes (Eugene, OR); fibronectin antibody from Transduction Laboratories-BD Biosciences (San Diego, CA); tenascin C antibody from Santa Cruz Biotechnology (Santa Cruz, CA); Dulbecco’s modified Eagle’s medium (DMEM), antibiotics, and antimycotics from Invitrogen-Gibco (Grand Island, NY); fetal bovine serum from HyClone (Logan, UT); chemiluminescence detection kits (Super Signal) from Pierce (Rockford, IL); Falcon cell culture inserts (PET track-etched 3-µm pore membranes in six-well format) from BD Biosciences (Franklin Lakes, NJ); and RNA extraction kits (RNeasy) from Ambion (Austin, TX).

TM Cell Culture, Mechanical Stretching, Treatments, and Extractions
Porcine TM cells were cultured as previously described.5 25 26 27 By passages 3 to 5, cells were plated at a density of approximately 90% confluence onto cell culture insert membranes in six-well culture plates.14 28 When cells were densely confluent, 3 to 5 days later, they were placed in serum-free medium for 24 hours before and during stretching experiments. To apply mechanical stretch/distortion, a smooth, 5.25-mm glass bead was placed in the dish beneath the center of the insert membrane. A weight was then applied to the lid of the plate, which forced the insert down onto the bead.14 28 This process created a defined upward distortion of the membrane, which increased the surface area and produced mechanical stretching of the cells and their ECM. More than 20 different porcine cell lines, each comprising cells from 10 to 20 eyes, were studied. DNA analysis (PicoGreen; Molecular Probes) was conducted in parallel wells in some studies, to estimate cell density. Because the differences between wells were always less than 10%, the analysis was not conducted in all studies. At the indicated times after initiation of stretching, medium was collected and stored in aliquots frozen at –20°C until use. For cellular or ECM protein analysis, membranes were immediately rinsed with ice-cold phosphate-buffered saline and the cells lysed with a modified RIPA buffer29 30 31 (2 mM EDTA, 2 mM EGTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 2 mM DTT, 1 mM PMSF, proteinase inhibitor cocktail, and 50 mM Tris [pH 7.5]) on ice. For RNA analysis, the membranes were cut from the inserts after stretching and placed in denaturation buffer (RNeasy kit; Ambion). They were then vortexed and the lysate removed. The remaining steps were as described in the kit protocol, and total RNA was processed according the manufacturer’s instructions. The extracted RNA was subjected to DNase treatment with 20 units of RNase-free DNase (Promega, Madison, WI) for 20 minutes. This was followed by two phenol-chloroform extraction steps to obtain high-quality RNA with an A260/A280 optical density ratio of 1.8 to 2.0.

Gene Chip Microarray
From each control or stretch sample, 9 µg of total purified RNA was provided to the Oregon Health and Science University Spotted Microarray Core (OHSU SMC) for processing (http://www.ohsu.edu/gmsr/smc/index.shtml). The stretch or the nonstretch control RNA was labeled by reverse transcription using either fluorescein-modified or biotinylated nucleotides and an oligo-dT primer. These labeled probes were mixed and hybridized to the spotted chips at 65°C for 16 hours (M-series LifterSlips; Erie Scientific, Portsmouth, NH) in a deep well hybridization chamber (TeleChem, Sunnyvale, CA). Hybridized arrays were then probed with Tyramide signal amplification (TSA) kits (PerkinElmer, Boston, MA), with horseradish peroxidase-conjugated anti-fluorescein antibody or streptavidin. Slides were developed with Cy3- and Cy5-tyramine and scanned (ScanArray 4000 XL with ScanArray Express software; Perkin-Elmer).

The two types of OHSU SMC microarray chips that were used had duplicate separated spottings of approximately 5700 or 8400 human cDNA clones per slide. The data presented herein are from the 8400 chips, but very similar results were obtained earlier with the 5700 chips. The libraries used were prepared by the IMAGE consortium32 and distributed in sequence-verified form by Invitrogen (Carlsbad, CA). Amplified PCR products were printed onto microarray slides (UltraGAPS; Corning Costar, Corning, NY) using microarray printing pins (TeleChem 16 CMP-3) and an array printer (Cartesian PixSys 5500 XL; Genomic Solutions, Irvine, CA). Twenty-three plant genes (Arabidopsis thaliana) spotted twice per chip, were used for standard reference providing both a positive and a negative control. In addition, 176 spots were left blank on each chip to provide another type of negative control. Scanned images were analyzed by computer (ImaGene; BioDiscovery, Marina del Rey, CA) to determine relative fluorescence intensity for each label; to identify spot locations, shapes, sizes, and anomalies; and to determine spot-specific background intensity for each label. At each of the three time points (12, 24, and 48 hours), three totally independent experiments were conducted. Each sample from each experiment was analyzed on two separate identical microchips. On each chip, each of the 8400 genes was spotted twice in separate regions. Consequently, at each of the three time points, there were 12 separate stretch and control data pairs for each gene.

Microarray spot fluorescence intensities for control and stretch samples with associated background values were then analyzed separately at each of the three time points. To determine statistical significance, the data from each chip with the duplicate spot values were separated into half chips, with one stretch and one control value and their respective specific backgrounds for each of the approximately 8400 cDNAs. The stretch and control data from each half-chip were then corrected for background and normalized via Lowess33 (GeneSpring software; Silicon Genetics, Redwood City, CA). These normalized fluorescence intensities were then combined into a single parallel data set for each time point, to identify those genes that achieved statistical significance using significance analysis of microarrays (SAM).34 Twelve normalized stretch and 12 normalized control fluorescence intensities for each gene at each time point were thus used to determine which changes were statistically significant. The number of permutations was set at 1000; the analysis was in a paired format; and the nearest number imputer was set to 10 in the SAM software (http://www.stat.stanford.edu/~tibs/SAM/ provided in the public domain by Stanford University, Stanford, CA). The false-discovery rate {Delta} parameter was then adjusted to produce a median value of less than 0.5 falsely significant genes from the complete set. A separate evaluation was conducted by using two criteria to determine which gene changes achieved biological significance. To do this, all 12 of the stretch and control fluorescence spot intensity pairs with their associated backgrounds were combined into a single long data set. They were corrected for background and then normalized via Lowess (GeneSpring; Silicon Genetics), as though they were replicates on one large microchip. These data were then filtered to select genes that exhibited at least a 1.5-fold average increase or at least a 50% average decrease. These were then further filtered to eliminate genes expressed at low levels both before and after mechanical stretching. Spots with average normalized fluorescence intensities below 300 for both control and stretch, after the background was subtracted, were eliminated. This lower expression threshold was selected because it was more than 2 standard deviations above the average specific background on these chips. The subset of genes that passed all three of these selection criteria (i.e., exhibited both statistical and biological significance for at least one time point) was retained.

Quantitative RT-PCR
Quantitative RT-PCR was performed (LightCycler and one-step Lightcycler-RNA amplification Kit, SYBR Green I; Roche Diagnostics, Indianapolis, IN).35 Primer pairs were designed in adjacent exons so that amplification of potential traces of contaminating genomic DNA would be easily identified. After the RT cycle (55°C, 10 minutes), 40 PCR cycles (94°C, 30 seconds; 50°C [primer dependent], 10 seconds; and 72°C, 14 seconds) were used. Fluorescence was acquired at the end of each extension step, and a melting curve was obtained at the end of each run. In a few cases in which multiple peaks were observed in the melting curve, the fluorescence was acquired in a separate step after each cycle at a temperature above the melting temperature of the additional nonspecific peak. After the PCR, products were analyzed on gels to verify band sizes and purity. In some cases, the product was also sequenced. Data were analyzed by the system software (LightCycler; Roche Diagnostics), by using a 10-point dilution standard curve and the crossing-point method to determine the relative template concentration of samples. Because fibronectin exhibits alternative splicing, several additional primers were designed to determine which splice forms were expressed. Twenty genes were evaluated by qRT-PCR, including several that increased, several that decreased, and several that did not show any change on the microarrays at any time after stretching.

Western Immunoblots
Western immunoblots, transferred electrophoretically from standard SDS-PAGE gels to nitrocellulose or PVDF membranes, were probed with the indicated primary antibodies. Detection used the appropriate secondary antibodies with conjugated horseradish peroxidase and chemiluminescence according to the manufacturer’s instructions (SuperSignal; Pierce)5 14 31 36 Autoradiographs were scanned and relative band density analyzed37 with a densitometry program (UVP, Upland, CA). For both the qRT-PCR and the Western immunoblot data, Student’s t-test or Mann-Whitney ranked sum analysis was used to determine significance, when comparing treatment results. All experiments presented were repeated at least three times, and typical gels were selected for presentation.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Normalization and Significance
When the microarray data sets were normalized by Lowess after the backgrounds were subtracted, flat and well-centered ratio-intensity (R-I) curves33 were obtained. Figure 1 shows a typical R-I curve for the SMC8400 chip and 24-hour data. At each time point, a few hundred genes achieved statistically significant changes, when analyzed by SAM34 in each 12 x 12 stretch–control data set. As the {Delta} parameter decreased incrementally, a lower plateau was reached at approximately a median number of 0.5 falsely significant genes per data set. Additional increments reduced the number of genes without reducing the false-discovery rate. Therefore, the highest number of genes that achieved this false-discovery rate was accepted. Further filtering of this group for biological significance, in terms of expression level in the TM and magnitude of changes with mechanical stretch, gave 126 genes that achieved a 1.5-fold or larger increase and 29 genes that achieved a 50% or greater decrease at one or more of the three time points. Analysis of the results from the SMC5700 chips showed good agreement for the genes common to both arrays (data not shown). The OHSU SMC microarray facility also conducted a detailed data analysis in which somewhat different specific normalization and significance testing approaches were used. Their lists of changed genes were very similar to those we obtained, as detailed earlier.



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  		FIGURE 1. Typical R-I plot after Lowess normalization. All the 24-hour stretch and control data from the SMC8400 chip, which included three completely separate experiments with two chips for each experiment and with two spots per chip for each gene (approximately 192,000 data points after individual local background subtractions), were subjected to Lowess normalization in the software (GeneSpring; Silicon Genetics, Redwood City, CA). The means of these normalized stretch and control data were then plotted, as indicated on the axes.

 
Filtering the data from all three time points, after subtraction of the local background and normalization via Lowess but with no other restrictions, simply requiring the mean expression levels to reach a lower threshold level of greater than 300 relative fluorescence units, produced a group of well over 3000 genes that are clearly expressed at significant levels by TM cells.

Effects of Mechanical Stretch on Specific TM Gene Expression
Twenty-seven transcripts that code for ECM molecules or for molecules integrally involved in ECM regulation or in cell adhesion were upregulated, and two were downregulated, according to the stringent statistical and biological criteria described earlier (Table 1) . Several were elevated at all three time points, but most showed significant increases at only one or two time points. Several of those that did not reach the significance criteria were nonetheless clearly affected. As an example, fibronectin 1 was elevated at 24 hours based on our statistical significance criteria, but only 1.33-fold, and thus is labeled NS (not significant) in Table 1 at this time point.


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  		TABLE 1. Genes Involved in ECM, ECM Regulation, or Cell Adhesion

 
Transcript levels for 42 genes involved in cytoskeletal function or in a variety of other forms of cellular regulation were increased, and 9 were decreased at one or more of the three time points (Table 2) . Transcript levels of 20 genes that are involved in transcription or translation were increased, and levels of 13 genes were decreased at one or more time points (Table 3) . Thirty-seven genes involved in cellular stress responses, cellular metabolism, or other cellular processes were upregulated, and five were downregulated at one or more time points (Table 4) .


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  		TABLE 2. Genes Involved in the Cytoskeleton or in Cytoskeletal, and Other Cellular Regulation

 

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  		TABLE 3. Genes Involved in Transcription or Translation Regulation Affected by Mechanical Stretch

 

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  		TABLE 4. Genes Involved in Stress, Metabolism and Other Processes That Are Affected by Mechanical Stretching

 
Effects of Mechanical Stretching on TM Genes Associated with Primary Open-Angle Glaucoma
We also looked specifically in the microarray data for changes in genes that have been identified as active in primary open-angle glaucoma, (i.e., myocilin/TIGR, optineurin and WDR36)38 39 40 and for genes that reside in some of the chromosomal regions to which glaucoma loci have been mapped. Myocilin/TIGR (GLC1A) was not represented on this microarray, although it has been shown to be elevated in the TM early after mechanical stretch or elevated IOP.18 21 23 Optineurin (GLC1E) and WDR36 (GLC1G) were both represented on our microarray. However, neither showed significant changes in expression with mechanical stretch at any of the three time points. Although positive relative fluorescence was observed for both transcripts, neither was expressed at sufficiently high levels in the TM under our culture conditions, with or without stretch to reach the very stringent expression level cutoff that we used as part of our biological significance criteria.

The chromosomal location of each gene that reached both statistical and biological significance in our studies is included in the last columns in Tables 1 2 3 and 4 . Analysis of the actual chromosomal location of each possibly relevant gene relative to the markers used to map these glaucoma loci as localized on the most recent human chromosomal maps, showed that most of the changed genes in Tables 1 2 3 and 4 are not actually within any of the mapped glaucoma loci. A summary of changed genes that are located within several mapped loci is shown in Table 5 . None were within the GLC1B, GLC1C, GLC1D, GLC1F, or GLC1G loci.40 41 42 43 44 Several changed genes were identified in some of the other loci that have been mapped but not named, as indicated in Table 5 .45 46 47


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  		TABLE 5. Stretch-Modified Genes and Mapped Primary Open-Angle Glaucoma Loci

 
Alternative Analyses of Genes Affected by Mechanical Stretch in Microarrays
Quantitative RT-PCR was used to evaluate mRNA levels at all three time points of 20 of the genes that were elevated, decreased, or unaffected by stretch in the microarray analysis. Several examples (i.e., metallothionein 1R, tenascin C, MMP-15, SPARC and mimecan transcript levels measured by microarray and qRT-PCR) are compared at select times after stretching in Figure 2 . Some of the other genes for which mRNA levels were compared by qRT-PCR, included MMP-2, MMP-14, and TIMP-2, which were not changed (data not shown). These also showed good agreement between the two methods. Although the exact changes (x-fold) were seldom identical when the microarray and qRT-PCR were compared, all showed similar responses to stretching by both methods.



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  		FIGURE 2. Comparison of typical microarray and qRT-PCR analysis. Log base 2 plot of the ratio of stretch to control responses as analyzed by microarray compared with qRT-PCR is shown for several genes at the indicated time points. The positions of the cutoff points used for the microarray studies (a decrease to 50% or a 1.5-fold increase) are shown by the vertical lines and are labeled above the lines for reference.

 
The fibronectin microarray results were also compared with results from qRT-PCR and from Western immunoblots at 24 hours after stretching (Fig. 3) . The microarray values for fibronectin at 24 hours showed a 1.33-fold increase (Fig. 3C) . Although this reached the level of high statistical significance by SAM, it did not achieve the cutoff filter level of a >1.5-fold increase and is listed as NS in Table 1 . The results of the comparison of stretch and control cells evaluated by qRT-PCR (Fig. 3D) and by Western immunoblot (Figs. 3A 3B) show similar or larger increases in mRNA and protein levels.



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  		FIGURE 3. Effects of mechanical stretching on fibronectin mRNA and protein levels. Cells were stretched for 24 hours and cellular protein or total RNA extracted and analyzed. (A) Relative band density of scans of Western immunoblots from three separate experiments. The mean ± SD with paired t-test significance is shown. (B) A typical image of the Western immunoblot bands. (C) All 36 stretch and control data pairs from seven separate experiments at the 24-hour time point from all the SMC5700 and SMC8400 microarray chips were compared. The mean relative spot fluorescence intensities with standard deviations and significance from paired t-test are shown. The microarray stretch/control ratio at this time point was ~1.3. (D) The results from 18 qRT-PCRs from two separate experiments with six different primer sets are shown with mean, standard deviations, and significance from paired t-test, as indicated.

 
Several other sets of fibronectin PCR primers were used to look for possible changes in mRNA splicing with stretch (Fig. 4) . One pair of primers, one in the 12th and one in the 14th fibronectin type III domains, were used to look for the presence of the 13th type III domain, EIIIA (Fig. 4) . Only the smaller (337-bp) PCR product in which this domain was spliced out was detectable in stretched (S) and in nonstretched control (C) TM cells. A primer pair was made in the seventh and ninth fibronectin type III domains to look at splicing of the eighth type III domain, EIIIB. Although at least 90% of the transcripts had EIIIB spliced out and gave the 406-bp PCR product, approximately 10% of the transcripts gave the 678-bp PCR product with the EIIIB domain spliced in. This ratio did not change significantly with stretching. In the fibronectin variable region within the 17th type III domain, often referred to as the connecting segment (IIICS) domain, both variant (V)1 and V3 are often alternatively spliced. We made a pair of primers in the 16th type III domain matched with one in the V2 domain to look for the V1 domain, and a pair of primers in the V2 domain matched to one in the 18th type III domain to look for the V3 domain. The V2 domain is present in all the known splice variants containing either of the other variable domains. Stretched cells expressed exclusively a splice variant that included V1 and V3, giving, respectively, the 294- and the 264-bp PCR products. Nonstretched control TM cells, however, expressed approximately 70% of transcripts containing V1, V2, and V3 and 30% containing V2 but without V1 and V3—that is, they gave PCR products of only 219 and 171 bp, respectively. When splice variants were evaluated at the other two time points (i.e., at 12 and 48 hours; data not shown), the same patterns noted at 24 hours were observed.



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  		FIGURE 4. Effects of mechanical stretching on fibronectin mRNA splice variants. Cells were stretched for 24 hours and total RNA extracted and analyzed by RT-PCR, with several PCR primer sets. A diagram is shown with the domain structure of fibronectin 1 including the alternatively spliced exons EIIIB, EIIIA, and III CS containing variant regions V1, V2, and V3 as indicated. The position of PCR primers and full-length product sizes are indicated above the respective splice variants. The size of the exon variably spliced is shown below each exon. Agarose gel with ethidium bromide-stained PCR products from cells that were stretched (S) or controls (C) for 24 hours are shown, with markers as indicated. The position of the predicted bands, with (+) and without (–) the indicated spliced domains, are shown next to each gel.

 
Western immunoblots at 24 and 48 hours showed tenascin C increases (Fig. 5) that are compatible with the microarray data (Table 1) and with the qRT-PCR data (Fig. 2) . Western immunoblots on several other proteins (data not shown), also showed expression patterns compatible with the microarray data.



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  		FIGURE 5. Comparison of tenascin C protein levels after 24 and 48 hours of stretching. Extracted protein was subjected to Western immunoblot analysis to determine tenascin C levels. The relative density of the tenascin C band on Western immunoblots is compared. Replicate numbers (n) from three separate experiments and significance (P) levels from paired t-tests are shown.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Only 155 of approximately 8000 genes met our very stringent biological and statistical significance criteria for change in mRNA levels with stretching at one or more time points (Tables 1 2 3 4) . This number is likely to be an underestimate of the total number of affected genes, but the confidence that these genes changed expression levels in a biologically significant manner is high. Our selection criteria, requiring an increase of at least 1.5-fold or a decrease of 50%, seems to provide a reasonable compromise between including changes that are not really of biological significance and excluding changes that are. Requiring either the control or the stretched cellular expression levels to be several standard deviations above the subtracted background also seems reasonable, but is somewhat arbitrary. The biological relevance of the absolute abundance of a transcript, as roughly approximated by the relative fluorescence levels on the microarray and of a given x-fold change in mRNA levels is most certainly very gene specific and highly dependent on the specific cellular context. Additional variables like translational efficiency, protein half-life, posttranslational modifications, and the functional and biological efficiency of each protein molecule further complicate selection of these criteria.

The 8000 genes on this microarray represent approximately 25% of the human genome. Although it seems possible that a few of the porcine probe sequences do not hybridize with high affinity to the human cDNAs spotted on the microarrays, this number is probably small. Because human and porcine sequences are generally similar, microarray hybridization conditions are of only moderate stringency, and both the labeled porcine probes and the spotted human cDNAs are several hundred base pairs in length, lack of cross-species hybridization should be minimal in these studies. This selected subset of 126 increased and 29 decreased mRNAs should provide useful insights into this homeostatic process.

The genes that were changed by stretching displayed varied temporal patterns, with some changing at only one time point and others at two or all three points. One gene, IGF-binding protein 4 proteinase, was elevated 1.5-fold at 12 hours, not significantly changed at 24 hours, and significantly decreased (to 0.54-fold) at 48 hours. This temporal variation in gene expression is not surprising and suggests that the TM cell response to stretching is both complex and intricately coordinated. The distribution of the changes suggests a bias favoring only a few gene functional groups. Genes involved in ECM, cytoskeleton, or stress responses or in their regulation comprised a disproportionately large fraction of those we identified. The genes in this microarray are biased toward genes with known functions and include a wide representation of genes involved in most major biological processes. A fairly predictable and significant number of genes involved in transcription, transcriptional regulation, translation, or overall cellular regulation were significantly affected by stretching. Perhaps not too surprisingly, few genes involved in apoptosis, cellular proliferation, most aspects of cellular metabolism, or most other major cellular functions were affected. Stretching is presumably a normal homeostatic adjustment signal requiring only a moderate and focused response from these cells.

In the current literature, microarray and cDNA library studies of human TM cells subjected to elevated pressures in perfused organ culture are somewhat comparable to our results.22 24 48 The TM cells in these studies, which mostly involved experimental conditions different from ours, were probably responding to mechanical stretching that is functionally somewhat similar to the process we used. Matrix Gla protein, several metallothioneins, alpha B crystallin, alpha tubulin, transgelin, chaperonin-containing TCP1, and periostin were among the genes showing similar changes. Because of the very complex structural organization of the cells and the ECM within the TM in vivo, the relative degree to which different TM cells are exposed to stretching and distortion with increasing IOP is difficult to predict. It seems probable that each TM cell will experience different degrees of stretching and distortion with elevated IOP. In our cell culture stretch model, more uniformity is likely to be attained. However, different regions of the membranes in our model may also experience different degrees of stretch/distortion. We estimate that many of the cells in our model are experiencing degrees and types of stretching and distortion that are similar to that of many of the cells in the TM in vivo. However, it is very clear that additional detailed biophysical analysis of the TM is needed to clarify this contention.

In terms of our working hypothesis of IOP homeostasis, the changes in ECM or ECM-related genes listed in Table 1 are of particular interest. In addition to the initiation of ECM turnover by the concerted action of MMP-2, TIMP2, and MMP-14,14 28 subsequent biosynthetic replacement of the degraded ECM components is likely to be an integral aspect of the homeostatic IOP adjustment. This probably entails modifying the composition or organization of the ECM to change the outflow resistance and thus modulate IOP. One group of ECM genes that are affected by stretching includes: NELL2, tenascin C, SPARC, fibronectin, laminin {gamma}1 chain, and collagen XIV. A common feature of this group is that they are all modular matricellular ECM proteins composed of numerous repeat domains containing motifs, such as EGF-like, calcium-binding EGF-like, thrombospondin 1, fibronectin type III, von Willebrand factor C, heparin-binding or integrin-binding RGD motif.49 50 51 52 53 54 55 56 57 58 59 60 Domains of these types commonly serve as specific binding cassettes for protein–protein, protein–glycosaminoglycan, or protein–cell interactions and function in ECM and tissue structure and organization. Their elevation during stretching is likely to be involved in maintaining and facilitating the tissue and ECM reorganization associated with the ECM turnover process initiated by the MMPs. Because they bind glycosaminoglycans (GAGs) and proteoglycans, which are thought to provide at least a portion of the outflow resistance,3 4 61 they could also directly affect outflow by changing resistance component orientation.

The increase in fibronectin levels and the splicing changes, which increase the fraction of the protein that contains V1 and V3 domains, are intriguing. Perfusion of anterior segment organ cultures with the second heparin binding (Hep II) fragment of fibronectin—the 14th to 16th type III domains as indicated in Figure 4 —reversibly increases outflow facility.57 In another tissue, it has been shown that alternative splicing of the V1 and V3 domains affect several specific biological activities of the adjacent Hep II fragment without affecting other activities.56 Fibronectin has an RGDS cell-binding site in the 11th type III domain that binds to {alpha}5ß1 integrins (Fig. 4) . An {alpha}4ß1 integrin-binding site is found in the distal portion of the Hep II region. In the alternatively spliced variable regions of IIICS, V1 contains an LDV and V3 contains an REDV, both of which are also {alpha}4ß1 integrin-binding motifs.62 63 Thus, TM cells switch from a mix of splice variants, with and without these two sites, to exclusively expressing the form that contains these two additional cell-binding motifs. In addition, the V2 domain of fibronectin contains a GAG-binding site, and these splice variants may impact binding to CD44 and syndecan 2 (both discussed later). Although we did not see changes in fibronectin EIIIA or EIIIB exon splicing with stretching, we detected very low levels of EIIIB in both stretched and control cells. A previous report did not detect any of either of these exons in serum-free control TM cells, although both were detected after TGF-ß2 or serum treatment.64

Of interest, laminin {gamma}1 was increased 1.5-fold at 12 hours, but {alpha}2, {alpha}4, {alpha}5, ß1, and the laminin receptor 1 were totally unaffected at all three time points. The laminins are a central component of cellular basement membranes, acting as scaffolds for the recruitment of other ECM components.65 Because laminins 1 to 15 are different heterotrimeric combinations of the {alpha}1 to 5, ß1 to 4, and {gamma}1 to 3 subunits, an increase in {gamma}1 may reflect a partial shift from one laminin form to another,58 at a time when the ECM is undergoing remodeling and change. The {gamma}1-subunit of laminin is also involved in binding to nidogen and in polymer formation.66 Although the primary cell-binding sites on laminin are in the laminin {alpha}-subunit, cell-binding sites have been identified on the {gamma}1 subunit of laminin.67 68 69 Based on relative fluorescence intensities in our microarrays, the {alpha}2 subunit was expressed at low to negligible levels, ß1 only at moderately low levels; but {alpha}4 and {alpha}5, {gamma}1, and laminin receptor 1 were all expressed at relatively high levels by the TM.

A second group of ECM genes that changed expression with stretching includes several proteoglycans, some of which may serve a direct role in outflow resistance.3 4 6 61 Chondroitin sulfate proteoglycan 4, fibromodulin, biglycan, syndecan 2, and the part-time proteoglycan and hyaluronan GAG receptor, CD44, increased with stretching, whereas mimecan decreased. Presumably, one likely way to adjust the outflow resistance in response to mechanical stretching would be to adjust the proteoglycan/GAG levels or composition in the TM’s ECM. Although it is tempting to speculate on the molecular mode by which increasing one or more of these proteoglycans or of decreasing mimecan, could decrease the outflow resistance, more information is needed to understand the process. Changes in the GAG side chains are also probable modifiers of the outflow resistance.

The increase in CD44, the hyaluronan receptor, is very likely to be important in this process. A relationship between hyaluronan levels and the trabecular outflow resistance is generally accepted.70 Several recent studies have demonstrated the involvement of CD44 in TM cellular function and have provided hypotheses explaining relationships with the outflow resistance.71 72 CD44 is also integrally involved and interactive with MMP-2 and -14,73 74 75 both of which are upregulated at the translational but not the transcriptional level in TM cells by mechanical stretching.14 28 CD44 exists in forms, with and without GAG side chains, and the form with chondroitin–dermatan sulfates binds to collagen XIV, which is also increased with stretching.

Syndecan 2, a transmembrane heparan sulfate proteoglycan,76 77 was upregulated at 24 hours (Table 1) . It interacts with {alpha}5ß1 integrin in cellular signaling and with the Hep II, V1, and V2 regions of fibronectin. This upregulation also affects cytoskeletal organization in an ezrin-dependent step via Rho A GTPase activation.78 79 Syndecan 2 is phosphorylated on two C-terminal tyrosines by the kinase domain of EphB2 receptors as a step in signaling to the cytoskeleton.77 Although EphB2 levels were not upregulated, its ligand, ephrin-B2, was upregulated by stretching. Thus, several proteins that have important interactions with each other are changed, further increasing the relevance of this process. CD44 also contains a cytoplasmic ezrin-binding domain, linking it to this same signaling system and to the Rho A-Rho kinase–mediated cytoskeletal regulation.80 81 82 83 Therefore, the syndecan 2, ephrin-B2, and CD44 upregulation by stretching may be closely linked in maintaining the TM’s ECM and may be involved in, among other events, the Rho A–cytoskeletal modulatory changes, which also occurred with mechanical stretching of TM cells (Table 2) .

The cytoskeletal changes observed (Table 2) involve components of all three filament systems—microfilaments, microtubules, and intermediate filaments—which suggests significant cross-talk between systems.84 Early cytoskeletal changes after mechanical stretching of TM cells have been reported,15 and sustained responses and long-term differences can be expected. Agents that manipulate the TM cell cytoskeleton have been shown to affect outflow facility.85 86 87 88 89 Whether the cytoskeletal changes are a direct cause or an effect of the ECM changes and outflow resistance adjustments remains unclear.

Several other intriguing gene expression changes are notable, such as increases in the chemokines, metallothionines, and metastasis-associated protein 1. The magnitude of some of these changes suggests important, if only partially understood, regulatory processes. Several metallothionein mRNAs increase from low or negligible levels to quite high levels, increasing between 20- to more than 100-fold at one time point. Functions of these genes in other tissues include heavy metal, particularly zinc, detoxification90 and homeostasis.91 This suggests that TM cells are responding to elevated or released zinc or to oxidative stress in association with the stretching.92 A zinc transporter and several other stress genes are also induced. The several chemokine ligand increases and the large metastasis-associated 1 increases are similarly intriguing and similarly enigmatic.

Although the number of genes with expression levels that are significantly changed by mechanical stretch is not large, the potential involvement of many of them in maintaining and adjusting the TM’s ECM and putatively the outflow resistance is likely. Understanding this complex process that appears to be responsible for maintaining IOP homeostasis clearly requires additional studies focused on the function of these proteins in outflow pathway biology.


   Footnotes
 
Supported by National Institutes of Health Grants EY003279, EY008247, and EY010572 and by grants from the Glaucoma Research Foundation (San Francisco, CA), Research to Prevent Blindness, and Alcon Labs (Fort Worth, TX).

Submitted for publication January 21, 2005; revised April 11, 2005; accepted April 22, 2005.

Disclosure: V. Vittal, None; A. Rose, None; K.E. Gregory, None; M.J. Kelley, None; T.S. Acott, Alcon Labs (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: Ted S. Acott, Casey Eye Institute (CERES), Oregon Health and Science University, 3375 SW Terwilliger, Portland, OR 97239-4197; acott{at}ohsu.edu.


   References
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 Abstract
 Materials and Methods
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 References
 

  1. Quigley HA. Open-angle glaucoma. N Engl J Med. 1993;328:1097–1106.[Free Full Text]
  2. Quigley HA. Number of people with glaucoma worldwide. Br J Ophthalmol. 1996;80:389–393.[Abstract/Free Full Text]
  3. Acott TS, Wirtz MK. Biochemistry of aqueous outflow. Ritch R Shields MB Krupin T eds. The Glaucomas. 1996;1:281–305. Mosby St. Louis.
  4. Acott TS. Biochemistry of aqueous humor outflow. Kaufman PL Mittag TW eds. Textbook of Ophthalmology. 1994;7:1.47–41.78. Mosby London.
  5. Alexander JP, Samples JR, Van Buskirk EM, Acott TS. Expression of matrix metalloproteinases and inhibitor by human trabecular meshwork. Invest Ophthalmol Vis Sci. 1991;32:172–180.[Abstract/Free Full Text]
  6. Acott TS. Trabecular extracellular matrix regulation. Drance SM Van Buskirk EM Neufeld AH eds. Pharmacology of Glaucoma. 1992;125–157. Williams & Wilkins Baltimore.
  7. Bradley JMB, Vranka JA, Colvis CM, et al. Effects of matrix metalloproteinase activity on outflow in perfused human organ culture. Invest Ophthalmol Vis Sci. 1998;39:2649–2658.[Abstract/Free Full Text]
  8. Parshley DE, Bradley JMB, Fisk A, et al. Laser trabeculoplasty induces stromelysin expression by trabecular juxtacanalicular cells. Invest Ophthalmol Vis Sci. 1996;37:795–804.[Abstract/Free Full Text]
  9. Parshley DE, Bradley JMB, Samples JR, Van Buskirk EM, Acott TS. Early changes in matrix metalloproteinases and inhibitors after in vivo laser treatment to the trabecular meshwork. Cur Eye Res. 1995;14:537–544.
  10. Bradley JMB, Anderssohn AM, Colvis CM, et al. Mediation of laser trabeculoplasty-induced matrix metalloproteinase expression by IL-1ß and TNF{alpha}. Invest Ophthalmol Vis Sci. 2000;41:422–430.[Abstract/Free Full Text]
  11. Bill A, Maepea O. Mechanisms and routes of aqueous humor drainage. Albert DM Jakotiec FA eds. Principles and Practices of Ophthalmology. 1994;206–226. WB Saunders Philadelphia.
  12. Acott TS. Receptor biology and glaucoma: integrins in the eye. Anderson DR Drance SM eds. Encounters in Glaucoma Research 1: Receptor Biology and Glaucoma. 1994;97–135. Foglizza Editore Milan, Italy.
  13. Zhou LL, Zhang S-R, Yue BYJT. Adhesion of human trabecular meshwork cells to extracellular matrix proteins: roles and distribution of integrin receptors. Invest Ophthalmol Vis Sci. 1996;37:104–113.[Abstract/Free Full Text]
  14. Bradley JMB, Kelley MJ, Zhu XH, Anderssohn AM, Alexander JP, Acott TS. Effects of mechanical stretching on trabecular matrix metalloproteinases. Invest Ophthalmol Vis Sci. 2001;42:1505–1513.[Abstract/Free Full Text]
  15. Tumminia SJ, Mitton KP, Arora J, Zelenka P, Epstein PL, Russell P. Mechanical stretch alters the actin cytoskeletal network and signal transduction in human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 1998;39:1361–1371.[Abstract/Free Full Text]
  16. WuDunn D. The effect of mechanical strain on matrix metalloproteinase production by bovine trabecular meshwork cells. Curr Eye Res. 2001;22:394–397.[CrossRef][ISI][Medline][Order article via Infotrieve]
  17. Okada Y, Matsuo T, Ohtsuki H. Bovine trabecular cells produce TIMP-1 and MMP-2 in response to mechanical stretching. Jpn J Ophthalmol. 1998;42:90–94.[CrossRef][Medline][Order article via Infotrieve]
  18. Sato Y, Matouo T, Ohtsuki H. A novel gene (oculomedin) induced by mechanical stretching of human trabecular cells of the eye. Biochem Biophys Res Commun. 1999;259:349–351.[CrossRef][ISI][Medline][Order article via Infotrieve]
  19. Stamer WD, Roberts BC, Epstein DL. Hydraulic pressure stimulates adenosine 3', 5'-cyclic monophosphate accumulation in endothelial cells from Schlemm’s canal. Invest Ophthalmol Vis Sci. 1999;40:1983–1988.[Abstract/Free Full Text]
  20. Mitton KP, Tumminia SJ, Arora J, Zelenka P, Epstein DL, Russell P. Transient loss of {alpha}B-crystallin: an early cellular response to mechanical stretch. Biochem Biophys Res Commun. 1997;235:69–73.[CrossRef][ISI][Medline][Order article via Infotrieve]
  21. Tamm ER, Russell P, Epstein DL, Johnson DH, Piatigorsky J. Modulation of myocillin: TIGR expression in human trabecular meshwork. Invest Ophthalmol Vis Sci. 1999;40:2577–2582.[Abstract/Free Full Text]
  22. Gonzalez P, Epstein DL, Borras T. Genes upregulated in the human trabecular meshwork in response to elevated intraocular pressure. Invest Ophthalmol Vis Sci. 2000;41:352–361.[Abstract/Free Full Text]
  23. Borras T, Rowlette LL, Tamm ER, Gottanka J, Epstein DL. Effects of elevated intraocular pressure on outflow facility and TIGR/MYOC expression in perfused human anterior segments. Invest Ophthalmol Vis Sci. 2002;43:33–40.[Abstract/Free Full Text]
  24. Borras T. Gene expression in the trabecular meshwork and the influence of intraocular pressure. Prog Retin Eye Res. 2003;22:435–463.[CrossRef][ISI][Medline][Order article via Infotrieve]
  25. Polansky JR, Wood IS, Maglio MT, Alvarado JA. Trabecular meshwork cell culture in glaucoma research: evaluation of biological activity and structural properties of human trabecular cells in vitro. Ophthalmology. 1984;91:580–595.[ISI][Medline][Order article via Infotrieve]
  26. Polansky JR, Weinreb R, Alvarado JA. Studies on human trabecular cells propagated in vitro. Vision Res. 1981;21:155–160.[CrossRef][ISI][Medline][Order article via Infotrieve]
  27. Stamer WD, Seftor RE, Williams SK, Samaha HA, Snyder RW. Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion. Curr Eye Res. 1995;14:611–617.[ISI][Medline][Order article via Infotrieve]
  28. Bradley JM, Kelley MJ, Rose A, Acott TS. Signaling pathways used in trabecular matrix metalloproteinase response to mechanical stretch. Invest Ophthalmol Vis Sci. 2003;44:5174–5181.[Abstract/Free Full Text]
  29. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 1989; 2nd ed. Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY.
  30. Harlow E, Lane D. Antibodies: A Laboratory Manual. 1988; Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY.
  31. Alexander JP, Acott TS. Involvement of protein kinase C in TNF{alpha} regulation of trabecular matrix metalloproteinases and TIMPs. Invest Ophthalmol Vis Sci. 2001;42:2831–2838.[Abstract/Free Full Text]
  32. Lennon G, Auffray C, Polymeropoulos M, Soares MB. The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression. Genomics. 1996;33:151–152.[CrossRef][ISI][Medline][Order article via Infotrieve]
  33. Quackenbush J. Microarray data normalization and transformation. Nat Genet. 2002;32(suppl 2)496–501.
  34. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA. 2001;98:5116–5121.[Abstract/Free Full Text]
  35. Rasmussen R. Quantification on the LightCycler. Meuer S Wittwer C Nakagawara K-I eds. Rapid Cycle Real-Time PCR: Methods and Applications. 2001;21–34. Springer New York.
  36. Alexander JP, Bradley JMB, Gabourel JD, Acott TS. Expression of matrix metalloproteinases and inhibitor by human retinal pigment epithelium. Invest Ophthalmol Vis Sci. 1990;31:2520–2528.[Abstract/Free Full Text]
  37. Samples JR, Alexander JP, Acott TS. Regulation of the levels of human trabecular matrix metalloproteinases and inhibitor by interleukin-1 and dexamethasone. Invest Ophthalmol Vis Sci. 1993;34:3386–3395.[Abstract/Free Full Text]
  38. Stone EM, Fingert JH, Alward WLM, et al. Identification of a gene that causes primary open angle glaucoma. Science. 1997;275:668–670.[Abstract/Free Full Text]
  39. Rezaie T, Child A, Hitchings R, et al. Adult-onset primary open-angle glaucoma caused by mutations in optineurin. Science. 2002;295:1077–1079.[Abstract/Free Full Text]
  40. Monemi S, Spaeth G, Dasilva A, et al. Identification of a novel adult-onset primary open-angle glaucoma (POAG) gene on 5q22.1. Hum Mol Genet. 2005;14:725–733.[Abstract/Free Full Text]
  41. Stoilova D, Child A, Trifan OC, Crick RP, Coakes RL, Sarfarazi M. Localization of a locus (GLC1B) for adult-onset primary open angle glaucoma to the 2cen-q13 region. Genomics. 1996;36:142–150.[CrossRef][ISI][Medline][Order article via Infotrieve]
  42. Wirtz MK, Samples JR, Kramer PL, et al. Mapping a gene for adult-onset primary open-angle glaucoma to chromosome 3q. Am J Hum Genet. 1997;60:296–304.[ISI][Medline][Order article via Infotrieve]
  43. Trifan OC, Traboulsi EI, Stoilova D, et al. A third locus (GLC1D) for adult-onset primary open-angle glaucoma maps to the 8q23 region. Am J Ophthalmol. 1998;126:17–28.[CrossRef][ISI][Medline][Order article via Infotrieve]
  44. Wirtz MK, Samples JR, Rust K, et al. GLC1F, a new primary open-angle glaucoma locus, maps to 7q35–q36. Arch Ophthalmol. 1999;117:237–241.[Abstract/Free Full Text]
  45. Wiggs J, Allingham R, Hossain A, et al. Genome-wide scan for adult onset primary open angle glaucoma. Hum Mol Genet. 2000;9:1109–1117.[Abstract/Free Full Text]
  46. Nemesure B, Jiao X, He Q, et al. A genome-wide scan for primary open-angle glaucoma (POAG): the Barbados Family Study of Open-Angle Glaucoma. Hum Genet. 2003;112:600–609.[ISI][Medline][Order article via Infotrieve]
  47. Wiggs JL, Lynch S, Ynagi G, et al. A genomewide scan identifies novel early-onset primary open-angle glaucoma loci on 9q22 and 20p12. Am J Hum Genet. 2004;74:1314–1320.[CrossRef][ISI][Medline][Order article via Infotrieve]
  48. Gonzalez P, Zigler JS, Jr, Epstein DL, Borras T. Identification and isolation of differentially expressed genes from very small tissue samples. BioTechniques. 1999;26:884–886, 888–892.
  49. Kuroda S, Oyasu M, Kawakami M, et al. Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2. Biochem Biophys Res Commun. 1999;265:79–86.[CrossRef][ISI][Medline][Order article via Infotrieve]
  50. Ghert MA, Qi WN, Erickson HP, Block JA, Scully SP. Tenascin-C splice variant adhesive/anti-adhesive effects on chondrosarcoma cell attachment to fibronectin. Cell Struct Funct. 2001;26:179–187.[CrossRef][ISI][Medline][Order article via Infotrieve]
  51. Joester A, Faissner A. The structure and function of tenascins in the nervous system. Matrix Biol. 2001;20:13–22.[CrossRef][ISI][Medline][Order article via Infotrieve]
  52. Yan Q, Sage EH. SPARC, a matricellular glycoprotein with important biological functions. J Histochem Cytochem. 1999;47:1495–1506.[Free Full Text]
  53. Rhee DJ, Fariss RN, Brekken R, Sage EH, Russell P. The matricellular protein SPARC is expressed in human trabecular meshwork. Exp Eye Res. 2003;77:601–607.[CrossRef][ISI][Medline][Order article via Infotrieve]
  54. Bornstein P, Sage EH. Matricellular proteins: extracellular modulators of cell function. Curr Opin Cell Biol. 2002;14:608–616.[CrossRef][ISI][Medline][Order article via Infotrieve]
  55. Hohenester E, Engel J. Domain structure and organisation in extracellular matrix proteins. Matrix Biol. 2002;21:115–128.[CrossRef][ISI][Medline][Order article via Infotrieve]
  56. Santas AJ, Peterson JA, Halbleib JL, Craig SE, Humphries MJ, Peters DM. Alternative splicing of the IIICS domain in fibronectin governs the role of the heparin II domain in fibrillogenesis and cell spreading. J Biol Chem. 2002;277:13650–13658.[Abstract/Free Full Text]
  57. Santas AJ, Bahler C, Peterson JA, et al. Effect of heparin II domain of fibronectin on aqueous outflow in cultured anterior segments of human eyes. Invest Ophthalmol Vis Sci. 2003;44:4796–4804.[Abstract/Free Full Text]
  58. 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]
  59. Ehnis T, Dieterich W, Bauer M, Lampe B, Schuppan D. A chondroitin/dermatan sulfate form of CD44 is a receptor for collagen XIV (undulin). Exp Cell Res. 1996;229:388–397.[CrossRef][ISI][Medline][Order article via Infotrieve]
  60. Gerecke DR, Foley JW, Castagnola P, et al. Type XIV collagen is encoded by alternative transcripts with distinct 5' regions and is a multidomain protein with homologies to von Willebrand’s factor, fibronectin, and other matrix proteins. J Biol Chem. 1993;268:12177–12184.[Abstract/Free Full Text]
  61. Yue BYJT. The extracellular matrix and its modulation in the trabecular meshwork. Surv Ophthalmol. 1996;40:379–390.[ISI][Medline][Order article via Infotrieve]
  62. Mostafavi-Pour Z, Askari JA, Whittard JD, Humphries MJ. Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion to fibronectin splice variants. Matrix Biol. 2001;20:63–73.[CrossRef][ISI][Medline][Order article via Infotrieve]
  63. Yamada KM. Fibronectin and other cell interactive glycoproteins. Hay ED eds. Cell Biology of Extracellular Matrix. 1991;111–146. Plenum New York.
  64. Li J, Tripathi BJ, Tripathi RC. Modulation of pre-mRNA splicing and protein production of fibronectin by TGF-beta2 in porcine trabecular cells. Invest Ophthalmol Vis Sci. 2000;41:3437–3443.[Abstract/Free Full Text]
  65. Sasaki T, Fassler R, Hohenester E. Laminin: the crux of basement membrane assembly. J Cell Biol. 2004;164:959–963.[Abstract/Free Full Text]
  66. Kadoya Y, Salmivirta K, Talts JF, et al. Importance of nidogen binding to laminin gamma1 for branching epithelial morphogenesis of the submandibular gland. Development. 1997;124:683–691.[Abstract]
  67. Kuratomi Y, Nomizu M, Tanaka K, et al. Laminin gamma 1 chain peptide, C-16 (KAFDITYVRLKF), promotes migration, MMP-9 secretion, and pulmonary metastasis of B16–F10 mouse melanoma cells. Br J Cancer. 2002;86:1169–1173.[CrossRef][ISI][Medline][Order article via Infotrieve]
  68. Nomizu M, Kuratomi Y, Song SY, et al. Identification of cell binding sequences in mouse laminin gamma1 chain by systematic peptide screening. J Biol Chem. 1997;272:32198–32205.[Abstract/Free Full Text]
  69. Ponce ML, Nomizu M, Delgado MC, et al. Identification of endothelial cell binding sites on the laminin gamma 1 chain. Circ Res. 1999;84:688–694.[Abstract/Free Full Text]
  70. Knepper PA, Farbman AI, Telser AG. Exogenous hyaluronidases and degradation of hyaluronic acid in the rabbit eye. Invest Ophthalmol Vis Sci. 1984;25:286–293.[Abstract/Free Full Text]
  71. Knepper PA, Mayanil CS, Goossens W, et al. Aqueous humor in primary open-angle glaucoma contains an increased level of CD44S. Invest Ophthalmol Vis Sci. 2002;43:133–139.[Abstract/Free Full Text]
  72. Knepper PA, Goossens W, Mayanil CS. CD44H localization in primary open-angle glaucoma. Invest Ophthalmol Vis Sci. 1998;39:673–680.[Abstract/Free Full Text]
  73. Isacke CM, Yarwood H. The hyaluronan receptor, CD44. Int J Biochem Cell Biol. 2002;34:718–721.[CrossRef][ISI][Medline][Order article via Infotrieve]
  74. Harigaya K. Role of CD44 and MT1-MMP in colorectal cancer invasion and metastasis [in Japanese]. Nippon Rinsho. 2003;61(suppl 7)111–115.
  75. Seiki M, Mori H, Kajita M, Uekita T, Itoh Y. Membrane-type 1 matrix metalloproteinase and cell migration. Biochem Soc Symp. 2003;70:253–262.
  76. Couchman JR, Chen L, Woods A. Syndecans and cell adhesion. Int Rev Cytol. 2001;207:113–150.[ISI][Medline][Order article via Infotrieve]
  77. Couchman JR. Syndecans: proteoglycan regulators of cell-surface microdomains?. Nat Rev Mol Cell Biol. 2003;4:926–937.