|Molecular Vision 2004;
Received 21 April 2004 | Accepted 6 October 2004 | Published 7 October 2004
Fibronectin overexpression inhibits trabecular meshwork cell monolayer permeability
An-Fei Li,1,2,3 Nobuhiro Tane,1
Departments of 1Ophthalmology and 4Medicine, Boston University School of Medicine, Boston, MA; 2Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan; 3Department of Ophthalmology, National Yang-Ming University, Taipei, Taiwan
Correspondence to: Sayon Roy, Department of Ophthalmology, Boston University School of Medicine; 715 Albany Street, Boston, MA, 02118; Phone: (617) 638-4110; FAX: (617) 638-4177; email: email@example.com
Purpose: To study whether excess synthesis of an extracellular matrix (ECM) component, fibronectin (FN), underlying the monolayer of human trabecular meshwork (HTM) cells, influences permeability.
Methods: To upregulate FN expression, HTM cells were grown in high glucose (30 mM) medium for 10 days. In parallel, cells were grown in normal (5 mM) medium as control, and two separate groups of HTM cells were grown in high glucose medium for transfection with FN antisense phosphorothioate oligonucleotides (AS-FN oligos) to modulate high glucose induced FN overexpression, or random phosphorothioate oligonucleotides (Ran oligos) as control. FN protein expression and distribution was assessed by western blot analysis and immunofluorescence microscopy. In parallel, HTM cells were grown in transwell plates in normal or high glucose medium to perform in vitro permeability (IVP) assays and to assess transelectrical resistance (TER).
Results: Western blot analysis showed FN expression was upregulated by 27% (p=0.018) in HTM cells grown in high glucose medium compared to cells grown in normal medium. Immunofluorescence microscopy showed intense FN immunostaining, and IVP results showed a consistent reduction in monolayer permeability (13% reduction, p=0.004) in cells grown in high glucose medium compared to cells grown in normal medium. When cells grown in high glucose medium were transfected with AS-FN oligos FN expression was reduced by 33% (p=0.009) and resulted in increased permeability to near normal levels (98±7% of control, p=0.01), whereas random oligos had no effect on either FN expression or IVP. TER was significantly increased across TM cell monolayers grown in high glucose compared to those grown in normal medium (143±11% of control, p=0.001), which was reduced when cells were transfected with AS-FN oligos (109±7% of control, p=0.02) whereas cells transfected with random oligos showed no change.
Conclusions: Excess FN synthesis by trabecular meshwork cells may contribute to blockage in aqueous outflow associated with the development of primary angle open glaucoma (POAG).
The trabecular meshwork (TM) represents the main pathway for aqueous outflow and plays an important role in the maintenance of intraocular pressure. The cells lining the trabecular meshwork synthesize various extracellular matrix (ECM) components  that are believed to influence patency of the aqueous channels. An altered composition of ECM in trabecular meshwork may contribute to an increase in outflow resistance . An increase in matrix metalloproteinase activity has been shown to mediate rapid ECM turnover and increase aqueous outflow facility in organ cultures of the anterior segment . Also, an increase in ECM content has been reported to be associated with the development of increased intraocular pressure (IOP) in primary angle open glaucoma (POAG) [4-7]. However, a direct link between accumulation of ECM in the aqueous channels and blockage of aqueous outflow still remains to be established.
Studies have shown that excess fibronectin (FN) deposition in the trabecular meshwork occurs in corticosteroid induced glaucoma, POAG, and in aging eyes associated with increased resistance in outflow facility [8-12]. These reports suggest that reduced outflow facility in POAG may be caused, at least in part, by excess synthesis and accumulation of ECM components such as FN.
Several studies have indicated a higher incidence or prevalence of glaucoma in diabetic patients [13-18] and an association between higher IOP and diabetes [19-26]. While there is general agreement regarding the association between the prevalence of high IOP and diabetes, there is some controversy regarding the association between diabetes and glaucoma [22,27-29]. Recently, we have found that a high glucose condition increases FN synthesis in TM cells , which raises the possibility that high glucose levels in the aqueous humor of diabetics  may trigger excess ECM synthesis, including FN, and contribute to development of higher IOP in diabetics. Because a direct link between increased FN synthesis by TM cells and its influence on aqueous outflow has not been established, we have investigated the effects of increased FN synthesis on paracellular permeability in TM cell monolayers.
In this study, we hypothesized that aqueous outflow through the paracellular pathway in the trabecular meshwork may be hindered by excess deposition of FN. To test this hypothesis we have used antisense oligos against FN transcript to reduce excess FN synthesis in HTM cells grown in high glucose medium and assessed its effect on permeability in HTM cell monolayers.
TM cells from human eyes were isolated by methods described previously . Briefly, the anterior segments were removed from human donor eyes under a dissecting microscope and thin strips of TM were carefully placed in 35 mm petri dishes and allowed to attach to the dish in a humidified incubator at 37 °C and 5% CO2. Immediately after attachment, the explants were fed with complete Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS. Cells from the explants appeared within 3-5 days with ruffled edges, numerous cell extensions, and overlapping processes typical of trabecular meshwork cells. Frozen stocks of third to fifth passage human trabecular meshwork (HTM) cells were plated onto dishes and grown to confluency. Upon reaching confluency the cells were trypsinized and plated in DMEM with 10% FBS (Sigma, St. Louis, MO), antibiotics, and antimycotics. HTM cells were cultured in normal glucose (5 mM) medium or high glucose (30 mM) medium for 10 days. To induce FN overexpression, trabecular meshwork cells were grown to semi-confluency and exposed to high (30 mM) D-glucose medium for 10 days. As a control, cells were grown in normal (5 mM) medium. In each experiment, trabecular meshwork cell isolates from at least four different eyes were examined.
Transfection with antisense oligonucleotide
Cell transfection was performed on subconfluent HTM cell cultures with 0.04 μM FN antisense phosphorothioate oligonucleotides (AS-FN oligos) or 0.04 μM random oligos synthesized by Oligos, Etc (Wilsonville, OR). AS-FN oligos were targeted against the translation initiation site of the FN transcript to modulate FN overexpression. The 17-mer AS-FN oligo was designed from a published sequence (NM_054034, base 259-276). The sequence for AS-FN oligo was 5'-CCT AAG CAT GTT GAG AC-3'and the sequence for the random oligo was: 5'-TAT GGT ACG TGT CGT CCT-3'. Transfection was performed in OptiMem, a reduced serum medium, with 8 μM Lipofectin (GYBCO, Grand Island, NY) and 0.04 μM either antisense or random oligos for 4 h at 37 °C and then allowed to grow in complete medium for three days before cells were harvested for analysis.
Western blot analysis
Cells were washed with phosphate buffered saline (PBS) and lysed in buffer containing 10 mM Tris pH 7.4 (Sigma), 1 mM EDTA, and 0.1% Triton-X100 (Sigma). Protein content in the cell lysates was determined by the BCA protein assay method (Pierce, Rockfors, IL). In each lane, 15 μg of total protein was loaded and electrophoresed in 6% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). After electrophoresis, the samples were transferred onto nitrocellulose membrane according to the procedure of Towbin . The membranes were blocked with 5% nonfat dry milk in Tris buffered saline (TBS) for 2 h and incubated with rabbit anti-rat FN polyclonal antibody (Chemicon, Temecula, CA) solution (1:1000) in 0.2% nonfat dry milk for 1 h at room temperature. After 3 washes in TBS containing 0.1% Tween-20, the membranes were incubated with goat anti-rabbit IgG conjugated with alkaline phosphatase (Sigma) for 1 h. The membranes were then washed as above, applied to the Immun-Star chemiluminescent substrate (BioRad) and exposed to x-ray film (Fuji, Tokyo, Japan). Quantitative analysis of densitometric signals from Western blots was performed using National Institutes of Health (NIH) image analysis software. To determine actin protein levels, the membranes exposed previously to FN antibody were washed with TTBS for 5 min at room temperature and then at 70 °C for 30 min in buffer containing 62.5 mM Tris, pH 6.8 (Sigma), 2% SDS, and 100 mM 2-Mercaptoethanol (Sigma). Finally, after a 5 min wash at room temperature, the membranes were blocked and incubated with mouse anti β-actin antibody solution (1:10,000; Abcam, Cambridge UK) in 0.2% nonfat dry milk for 16 h, incubated with antibody solution containing anti mouse IgG antibody conjugated with alkaline phosphatase (1:15000; Sigma) for 1 h at room temperature and applied to the Immun-Star chemiluminescent substrate and exposed to X-ray film as described previously. Densitometric analyses of the luminescent signals were performed at nonsaturating exposures with NIH image analysis software.
To study the localization and distribution of FN protein in HTM cells, immunofluorescence staining for FN was performed in cells plated onto glass coverslips. Briefly, the cells were fixed with 4% paraformaldehyde in PBS, blocked with 0.2% bovine serum albumin in PBS for 15 min, and incubated with rabbit anti-rat FN polyclonal antibody (Chemicon CA) solution (1:100) for 1 h in a moist chamber. After three washes with 0.2% BSA-PBS, they were incubated with goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC; Jackson immunoresearch labs, PA) for 1 h at 37 °C. After PBS wash, the cells on coverslips were mounted in Slow-Fade (Molecular Probes, OR) and examined. Negative control samples were processed in the same manner except that the primary antibody was omitted. The cells were viewed and photographed with a Nikon Diaphot fluorescence microscope and a Kodak MDS290 digital camera. The fluorescence intensity of the FN signal was analyzed using NIH image analysis software.
In vitro permeability assay
The in vitro permeability (IVP) assay was performed as reported earlier [34-36]. Briefly, cells were plated onto cell culture inserts of transwell plates (Falcon, Paramus, NJ) and allowed to grow in normal or high glucose medium. After one week in culture when cells reached subconfluency, cells grown in high glucose medium were transfected with AS-FN oligos or random oligos as described earlier. Three days after transfection, the cells were subjected to IVP assay. Briefly, on the day of assay, medium in both the upper and lower chamber was replaced with fresh DMEM. Medium in the upper chamber contained 0.5 mg/ml fluorescein (Molecular Probes, Eugene, OR). After incubation for 1 h at 37 °C, aliquots from the lower chamber were analyzed for fluorescein concentration at 490 nm using a spectrophotometric microplate reader (SpectraMax Gemini Vmax; Molecular Devices, Sunnyvale, CA).
Transelectrical resistance (TER) across monolayer of HTM cells was determined by measuring electrical resistance using a voltohmmeter according to the manufacturer's instructions (EVOM; World Precision Instruments, Inc, Sarasota, FL). These cells were grown in high glucose medium for 10 days and transfected with AS-FN oligos or random oligos for TER measurements. Prior to TER assessments, medium in both chambers of each well was replaced with normal medium. The TER (ohms per square centimeter) of the insert alone in the normal medium was measured as a baseline value and subtracted from each TER data obtained with cells in the inserts. The measurements were repeated at least three times for each well, and each experiment was repeated four times.
Cell proliferation assay
The effect of antisense oligo transfection on cell proliferation was studied in HTM cells grown in normal or high glucose medium. Cells grown in high glucose medium were transfected with FN-AS oligos or random oligos. In each experiment, an equal number of cells (50,000) were seeded at the start of the experiment. Cell counts were monitored for 10 days. Cells were washed twice with Calcium-free PBS, trypsinized, and counted in duplicate in a cell counter (Coulter Electronics, Inc., Hialeah, FL) for each experiment. Hemocytometer counts were randomly performed to confirm the Coulter counter counts.
Data were expressed as mean±standard deviation. Comparison between groups was performed using one-way ANOVA and Student's t-test. To determine statistical significance, an α level of 0.05 was chosen.
Effect of high glucose on FN protein expression and distribution in HTM cells
HTM cells grown in high glucose medium for 10 days exhibited increased FN protein expression compared to cells grown in normal medium (127±14% of control, p=0.018, n=5). When HTM cells grown in high glucose medium were transfected with FN antisense oligos, the cells showed a significant reduction in FN protein level (94±15% of control verse 127±14% of control, p=0.009, n=5) compared to cells transfected with random oligos or untransfected cells. Cells transfected with random oligos had no effect on FN protein expression. The actin protein level, used as an internal control, was similar in all groups (Figure 1). The distribution and localization of FN protein in HTM cells was assessed by immunofluorescence microscopy. A modest but significant increase in FN immunostaining was observed in TM cells grown in high glucose medium, compared to cells grown in normal medium (18% increase, p=0.009, n=6). This increase was corrected to near normal levels when the high glucose cells were transfected with AS-FN oligos. Both the untransfected cells and the cells grown in high glucose medium transfected with random oligos, did not exhibit any change in the distribution or localization of FN protein (Figure 2).
In vitro permeability assay and TER measurement performed on monolayers of HTM cells
IVP assay performed in monolayers of HTM cells revealed that cells grown in high glucose medium had less fluorescein permeance compared to cells grown in normal medium (87±9% of control, p=0.004, n=9). When FN expression was downregulated with antisense FN oligo permeance was facilitated (98±8% of control, p=0.01, n=9) compared to cells grown in high glucose medium (Figure 3). Cells transfected with random oligos showed no effect on IVP. In addition to the IVP assay, we measured TER across the TM cell monolayer, which is independent of fluorescein movement. The TER was significantly increased across the TM cell monolayer in cells grown in high glucose compared to those grown in normal medium (143±11% of control, p=0.001), and was reduced when cells grown in high glucose were transfected with AS-FN oligos (109±7% of control, p=0.02) whereas cells transfected with random oligos showed no change.
Effect of AS-FN oligo transfection on cell proliferation
The effect of high glucose on TM cell proliferation was examined in HTM cells grown in high glucose medium for 10 days. The results showed reduced cell number (76.9±5% of control, p=0.0004, n=6) compared to cells grown in normal medium (Figure 4). HTM cells grown in high glucose medium and transfected with AS-FN or random oligos showed no difference in cell numbers compared to untransfected cells. This observation indicates that the transfection process itself had no effect on cell proliferation and that the cells tolerated the oligos well (Figure 4).
To identify mechanisms leading to resistance of aqueous outflow associated with POAG, we explored the relationship between excess FN deposition and trabecular meshwork cell monolayer permeability. In this study, our findings indicate that overexpression of FN by trabecular meshwork cells may contribute to blockage of aqueous outflow. We also determined that antisense oligos targeted to the translation initiation site of the FN transcript may be useful in reducing such blockage, and that inhibition of FN overexpression could facilitate paracellular permeability in TM cell monolayers.
Although FN deposition in trabecular meshwork is known to occur with progression of glaucoma , the exact mechanism(s) leading to elevated IOP with respect to quantitative changes in ECM proteins is still unclear. Our finding that five or more HTM cell isolates tested in our laboratory responded to high glucose conditions with increased resistance to monolayer cell permeability suggests that high glucose induced excess FN synthesis in TM cells may contribute to blockage of aqueous outflow. Since glucose concentration in aqueous humor is elevated in diabetic patients  it is possible that increased FN deposition by TM cells may occur in diabetic patients and result in reduced aqueous outflow, which, in turn, may partly contribute to the development of increased IOP. These findings provide a cellular basis for clinical observations from epidemiological studies reporting diabetic individuals to exhibit significantly increased IOP [13-26]. Overall, these findings underscore the importance of excess FN deposition by TM cells, which may play a role in obstructing the passage of aqueous outflow.
Several studies have indicated that excess matrix deposition in the TM could lead to reduced aqueous outflow. These reports were largely based on indirect studies in which tissues derived from glaucomatous samples showed increased deposition of FN or other ECM components compared to nonglaucomatous samples [4,6,7]. This study provides for the first time direct evidence that excess FN synthesis could contribute to reduced trabecular meshwork cell monolayer permeability and therefore influence aqueous outflow. By applying AS-FN oligos and reducing FN overexpression an increase in TM cell monolayer permeability was achieved, indicating that FN overexpression played a direct role in the blockage of TM cell monolayer permeability. However, overexpression of other ECM components such as laminin and collagen type IV may also contribute to aqueous outflow resistance. In line with our current result, studies have reported that inhibition of ECM deposition can result in increased uveoscleral outflow [37,38]. Overall, these findings suggest that FN deposition in the aqueous outflow path may contribute to outflow resistance.
Other studies indicate that altered FN expression may directly or indirectly influence aqueous outflow. For example, in the juxtacanalicular tissue (JCT), where FN is extensively co-localized with myocilin [39,40], a protein believed to regulate aqueous outflow, it is possible FN may influence myocillin function and indirectly influence aqueous outflow. In another study, FN in aqueous humor was reported to exhibit powerful chemoattractant characteristics toward trabecular meshwork cells, which may partly be responsible for TM cell loss in POAG . Taken together these findings suggest that FN mediated cellular interactions may influence aqueous outflow.
Under high glucose conditions, we have observed reduced proliferation of HTM cells similar to our observation in vascular endothelial cells [42,43] and bovine TM cells  grown in high glucose. Studies on microvascular endothelial cell proliferation have indicated that with increasing duration of exposure to high glucose the antiproliferative effect becomes more pronounced . Currently it is not well understood how high glucose affects cell proliferation. Studies indicate that high glucose induces apoptosis in vascular endothelial cells and pericytes, and in turn, might influence cell proliferation and growth [44,45]. In diabetic individuals, the anti-proliferative effect of high glucose on TM cells may play a role in TM cell loss known to be associated with POAG .
The advantages of various in vitro model systems are often accompanied by certain drawbacks, and therefore it is important to note that the present data are initial steps in understanding the overall process of the role of excess matrix production in aqueous outflow associated with POAG. The cell monolayer culture system used in this study provides an opportunity to assess whether overproduction of ECM by the TM cells may contribute to reduced aqueous outflow but does not take into account other biological processes as well as the anatomical structures involved in the outflow passage in an in vivo system. For example, matrix accumulation is not only the result of excess production of ECM components but is also a dynamic process where proteases such as matrix metalloproteinases and tissue inhibitor of metalloproteinases [3,46] play a role in regulating the degradation of the matrix. Overall, our findings are indicative of a promising first step towards understanding the role of excess matrix accumulation in the outflow passage.
In this study we have observed that antisense oligos can specifically reduce FN overexpression in HTM cells. To the best of our knowledge this is the second report in which specific gene expression was downregulated using antisense oligos in HTM cells. The other report had shown that antisense oligos reduced expression of the tight junction protein occludin, in HTM cells . Several strategies have been used for modulating gene expression in human trabecular meshwork cells including the antisense mRNA approach that requires virus mediated plasmid transfer . This study demonstrates that antisense oligos offer a powerful, non-virally mediated means to downregulate specific gene expression in HTM cells. In addition, the finding that cell transfection with antisense FN was well-tolerated by the human trabecular meshwork cells indicate that antisense strategy may be useful for facilitating aqueous outflow. Further studies are necessary to understand the role of excess FN accumulation in aqueous outflow resistance and its consequence in pathogenesis of primary open angle glaucoma.
Research work was supported by grants from the American Diabetes Association, and the National Eye Institute, NIH (EY11990-01A1), and in part by departmental grants from MA Lions Eye Research Foundation, Inc., and Research to Prevent Blindness, Inc.
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