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(Investigative Ophthalmology and Visual Science. 2003;44:3485-3493.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0756
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Expression of Matrix Metalloproteinases and Their Inhibitors in Human Trabecular Meshwork Cells

Iok-Hou Pang, Peggy E. Hellberg, Debra L. Fleenor, Nasreen Jacobson, and Abbot F. Clark

From Alcon Research, Ltd., Fort Worth, Texas.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Matrix metalloproteinases (MMPs) are involved in trabecular meshwork (TM) extracellular matrix metabolism and have been shown to increase aqueous outflow facility. The purpose of this study was to characterize effects of cytokines, a phorbol ester, and prostanoids on the expression of MMP-1, -2, -3, and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 in cultured human TM cells.

METHODS. Five human TM cell strains were treated with selected compounds. Levels of proMMPs and TIMPs in cell media were quantified by ELISA. MMP-3 activity was assayed by casein zymography.

RESULTS. All human TM cell strains produced detectable basal amounts of proMMPs and TIMPs. 12-O-tetradecanoyl-phorbol-13-acetate was effective in increasing the levels of proMMP-1, -3, and -9 and TIMP-1. Its effect on proMMP-1 was concentration-dependent with an EC50 of 2 to 3 nM. Interleukin (IL)-1{alpha} did not affect levels of proMMP-1 and -2 or the TIMPs, but was most efficacious in increasing proMMP-3 production with an EC50 of 0.5 ng/mL. The IL-1{alpha}–induced upregulation of proMMP-3 correlated with an increase in MMP-3 activity. Tumor necrosis factor-{alpha} activated proMMP-3 production in some but not all cell strains. Platelet-derived growth factor-BB was generally ineffective in modulating MMP and TIMP levels. Prostaglandins E2 and F2{alpha} at 10 µM did not affect levels of proMMP-1 or -3.

CONCLUSIONS. The expression of the different MMPs and TIMPs in human TM cells was independently regulated. Production of MMP-3 was maximally activated by IL-1{alpha}. The IL-1{alpha}–stimulated expression of MMP-3 provides a probable mechanism for IL-1{alpha}–enhanced aqueous outflow.

Glaucoma is one of the leading causes of blindness in the world.1 Although the etiology of primary open-angle glaucoma, one of the most prominent form of glaucoma, is not fully elucidated, ocular hypertension due to compromised aqueous outflow facility is a major risk factor. Histologic and morphologic studies2 3 4 5 6 7 have demonstrated that there is an excessive accumulation of extracellular matrix in the trabecular meshwork (TM) of glaucomatous eyes, which probably contributes to impeded aqueous outflow. Therapeutic manipulations that eliminate the excessive extracellular matrix should theoretically improve outflow facility and consequently lower intraocular pressure (IOP).

Recently, matrix metalloproteinases (MMPs) have been proposed as important enzymes regulating the turnover of extracellular matrix in the TM.8 9 10 MMPs are a family of zinc-containing neutral proteinases involved in the regulated degradation of extracellular matrix.11 12 13 14 There are more than 20 members in this gene family. They share many common structural and functional features but differ in substrate specificity. The MMPs are secreted from cells as proenzymes and must be activated by proteolytic cleavage. Their enzymatic activities are inhibited by specific inhibitors, such as tissue inhibitors of metalloproteinases (TIMPs).

Activation of these enzymes should reduce the excessive accumulation of extracellular matrix molecules, such as proteoglycans, collagens, fibronectins, and laminin, in the glaucomatous eye and in turn may decrease hydrodynamic resistance of the outflow pathway. Indeed, perfusion with 20 µg of purified MMPs, containing equal concentrations of MMP-2 (gelatinase A), MMP-3 (stromelysin-1), and MMP-9 (gelatinase B), in anterior segments of the human eye increased outflow facility by more than 50%, lasting for at least 5 days.15 Similarly, interleukin (IL)-1{alpha}, a cytokine known to increase the expression of MMPs in the TM,10 also produced a long-lasting augmentation of outflow facility when perfused in the anterior segment.15 Consistent with these findings, inhibitors of MMPs, such as the TIMPs, minocycline, or L-tryptophan hydroxamate, suppressed aqueous outflow.15 Taken together, these data strongly suggest that MMPs play a significant role in the regulation of aqueous humor outflow facility by controlling extracellular matrix turnover in the TM. In fact, TM expression of MMP-3 and -9 is enhanced after clinical laser treatment for glaucoma, and this enhancement may be responsible for mediating the ocular hypotensive effect of trabeculoplasty.16 17

Most studies regarding TM expression of MMPs were performed on cultured animal TM cells18 or human donor TM tissue,10 which contains more than one cell type. Thus, there is limited detailed information about the regulation of expression of MMPs in human TM cells. The present study was designed to demonstrate the presence of the proenzymes of MMP-1 (interstitial collagenase), -2, -3, and -9 and TIMP-1 and -2 in cultured human TM cells and to demonstrate the involvement of selected trophic factors, cytokines, and neurotransmitters in the regulation of MMP expression in these cells.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Culture of Human TM Cells
Five human TM cell lines were used in the study: TM10A, TM16A, TM30A, TM35D, and TM332/344. Cells were isolated, characterized, and cultured as described elsewhere.19 20 21 They were maintained at 5% CO2 and 37°C in a medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) with stabilized L-glutamine (Glutamax I; Gibco/Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and 50 µg/mL gentamicin (Gibco/Invitrogen). The cells were allowed to reach approximately 90% confluence. Before treatment, cells were serum deprived for 24 hours. Cell medium was then replaced with serum-free medium containing the appropriate test compound. The plate was returned to the culture incubator and allowed to incubate for the indicated amount of time. Cell media were then removed and used for the quantification of proMMPs and TIMPs by ELISA and active MMP-3 by zymography.

ProMMP and TIMP Assays
Twenty-four-well TM cell cultures were serum deprived for 24 hours, followed by 24 hours of treatment with test agents in serum-free medium. Final volume per well was 300 µL, 250 µL of which was collected, with 100 µL used for proMMP and TIMP quantification by commercially available ELISA kits. For proMMP-1 and -9 and TIMP-1 and -2, specific assay kits (Biotrak; Oncogene Research Products, San Diego, CA) were used. For proMMP-2 and -3, other specific assay kits (Bindazyme; The Binding Site, Birmingham, UK) were used. Assays were performed according to the manufacturer’s instructions. Briefly, for the proMMP-1, proMMP-9, and TIMP assay kits (Biotrak; Oncogene Research Products), 96-well microtiter plates coated with the specific primary antibody were incubated with known amounts of standards or TM cell medium samples. The plate was incubated for 2 hours at room temperature and then washed with wash buffer (6.7 mM sodium phosphate buffer [pH 7.5] containing 0.033% Tween 20). Then, 100 µL of specific secondary antibody (polyclonal rabbit anti-proMMP or anti-TIMP) was added to each well and incubated at room temperature for 2 hours. The wells were again washed and incubated with 100 µL of donkey anti-rabbit antibody conjugated with horseradish peroxidase. After a 1-hour incubation at room temperature, the wells were washed again. The 3,3',5,5''-tetramethylbenzimide (TMB) substrate was then added to each well and incubated for 20 to 30 minutes. Reactions were stopped by the addition of 100 µL 1 M sulfuric acid and the resultant yellow color was read at 450 nm with a microplate reader (MR5000; Dynatech, Cambridge, MA).

The proMMP-2 and -3 assay kits (Bindazyme; The Binding Site) used microtiter plates precoated with affinity purified antibody to proMMP-2 or proMMP-3. After the addition of standard samples or TM cell medium, the plate was incubated for 1 hour at room temperature and washed with wash buffer. Biotinylated antibody (100 µL) was added to each well, and the plate was incubated for another hour. After another wash, 100 µL of streptavidin peroxidase was added to each well and incubated for 30 minutes. The washing step was repeated, and 100 µL of TMB was added to the wells. The assay was stopped after 10 minutes by adding 3 M phosphoric acid to each well, and the resultant yellow color was read at 450 nm.

The concentration of proMMPs or TIMPs in TM cell medium was calculated by comparison with their respective standard curves. In these assays, the detectable limits (as defined by the minimal amount that produced a statistically significant change in the ELISA signal) for proMMP-1, 0.6 ng/mL; proMMP-2, 2.0 ng/mL; proMMP-3, 0.3 ng/mL; proMMP-9, 0.1 ng/mL; TIMP-1, 0.4 ng/mL; and TIMP-2, 1 ng/mL.

Zymography
Twelve-well TM cell cultures were serum deprived for 24 hours, followed by a 24-hour treatment with test agents. Final volume per well was 600 µL, 500 µL of which was collected and concentrated approximately sevenfold using microconcentrator units (Nanosep; Pall Filtron, Northborough, MA; molecular mass cutoff, 10 kDa). Final volume of each concentrated sample was adjusted to 75 µL by adding serum-free DMEM. Concentrated samples were then mixed with equal volumes of 2x Tris-glycine-SDS zymography sample buffer (Novex; Invitrogen, Carlsbad, CA) and allowed to stand at room temperature for 10 minutes. Fifty microliters of each sample was loaded onto precast 12% casein minigels (Bio-Rad, Hercules, CA) and electrophoresed at constant voltage (100 V) in a Tris-glycine-SDS (25 mM-192 mM-0.1%; pH 8.3) buffer, with cooling. The resultant gels were incubated for 1 hour at room temperature in renaturation buffer (Bio-Rad), then transferred to development buffer (Bio-Rad) for 48 hours at 37°C. Developed gels were stained for at least 1 hour in 0.5% Coomassie blue solution, then destained until clear bands were visible against the blue background. The gels were then scanned (Precision ScanPro; Hewlitt-Packard, Palo Alto, CA, or ScanWizard Pro, Microtek Lab, Redondo Beach, CA) and analyzed for relative densities on computer (Gellab II+; Scanalytics, Fairfax, VA).

Test Compounds
IL-1{alpha}, tumor necrosis factor-{alpha} (TNF{alpha}), platelet-derived growth factor-BB (PDGF-BB), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and prostaglandin E2 and F2{alpha} were all purchased from Sigma-Aldrich (St. Louis, MO). Stock solutions of the prostanoids were prepared in ethanol and diluted 1:1000 in cell culture medium for treatment. Stock solutions of all other compounds were prepared in DMEM. Control wells contained the same amount of respective vehicle.

Statistical Analysis
Data are presented as the mean ± SEM. Statistical comparisons among groups were performed by one-way ANOVA followed by Dunnett test versus the vehicle control group. P < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
The five TM cell lines used in this study had been well characterized previously.19 20 21 They were established from TM tissues of six individuals. The donors’ ages ranged from 48 days to 57 years. All cells were derived from explant cultures of TM tissues,22 23 24 except TM332/344, which was obtained by matrix digestion of combined TM tissues from two donors.25 None of the donors was known to have glaucoma (Table 1) .


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  		TABLE 1. Basic Information of Human Trabecular Meshwork Cell Strains

 
All the cultured human TM cells produced the examined MMPs and TIMPs, but seemingly with different rates. For example, the basal levels of proMMP-1 among the various cell lines ranged from 1.41 ng/24 h per well in the TM35D cells to 22.6 ng/24 h per well in TM30A cells, a 16-fold difference in basal levels. Similarly, levels of proMMP-3 and TIMP-1 also varied by 10- to 15-fold between the highest and lowest producers. In contrast, basal proMMP-2 and -9 and TIMP-2 levels were rather similar among the cell lines (Table 2) . It is important to point out that because the basal levels of proMMP-3 in some cell lines and proMMP-9 in all three cell lines tested were close to the detection limit of the respective assay, we may not have been able to detect small changes in the enzyme levels, although large increases were clearly observable. Furthermore, because of this limitation, the quantitation of changes of the two enzymes in these cells is not expected to be as accurate as that in the cells with higher basal levels.


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  		TABLE 2. Basal Levels of ProMMPs and TIMPs in Various Human TM Cells

 
Initially, four compounds were tested for their effects on MMP and TIMP expression. They were chosen because these compounds were shown to upregulate the expression of MMPs and TIMPs in various tissues and cells, including porcine TM cells and human TM tissues. In our study, these stimuli also upregulated the expression of specific MMPs and TIMPs in human TM cells. Figure 1 demonstrates the effects of these compounds on the production of proMMPs and TIMPs in TM35D cells. A 24-hour exposure to TPA (25 ng/mL; 40 nM) significantly (P < 0.05) enhanced the accumulation of proMMP-1 (200% ± 8%, mean ± SEM, n = 10), proMMP-9 (4918% ± 460, n = 8), and TIMP-1 (253% ± 31%, n = 8). ProMMP-1 expression was dependent on, but not linearly correlated with, TPA incubation time; the rate of proMMP-1 accumulation decreased substantially after the first 24 hours (Fig. 2A) . The stimulatory effect of TPA was concentration-dependent with a calculated EC50 of 2.62 nM (Fig. 2B) . The maximal effect was produced by a TPA concentration of 10 nM or higher and ranged from 200% to 500% of concentration in the control. Among the compounds tested, IL-1{alpha} (25 ng/mL; 1.4 nM) was the most efficacious in stimulating the expression of proMMP-3 (516% ± 39%, n = 12; after a 24-hour incubation) in TM35D cells (Fig. 1) . Its effect was also time- and concentration-dependent with a calculated EC50 of 0.42 ng/mL (23 pM; Fig. 3 ). The IL-1{alpha}-upregulated MMP-3 expression correlated well with an increase in MMP-3 activity. As shown by zymography (Fig. 4) , IL-1{alpha} produced a concentration-dependent increase in casein hydrolytic activity at a molecular mass of approximately 50 kDa, which is identical with the molecular mass of active MMP-3. IL-1{alpha} also stimulated the expression of MMP-9, but did not seem to affect the expression of other MMPs or TIMPs. Similarly, TNF{alpha} (25 ng/mL; 1.4 nM) also increased the production of proMMP-3 (300% ± 44%, n = 12), although its efficacy was lower than that of IL-1{alpha} (Fig. 1) . PDGF-BB (25 ng/mL; 1 nM) upregulated the expression of TIMP-1 only (196% ± 24%, n = 8; Fig. 1 ). None of these tested compounds affected the expression of MMP-2 or TIMP-2 in TM35D cells.



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  		FIGURE 1. ProMMP and TIMP levels in TM35D cell supernatants after treatment with various test agents. Data reflect mean ± SEM. *Significant at P < 0.05 versus the respective vehicle group.

 


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  		FIGURE 2. Time- and concentration-dependence of TPA-stimulated proMMP-1 expression in TM35D cells. (A) Effect of incubation time on proMMP-1 production (n = 8–10). (B) Concentration–response curve of the effect of a 24-hour exposure to TPA on proMMP-1 levels in TM35D cells. Data are the mean ± SEM (n = 3).

 


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  		FIGURE 3. Time- and concentration-dependence of IL-1{alpha}–stimulated proMMP-3 expression in TM35D cells. (A) Effect of incubation time on proMMP-3 production. Data are the mean ± SEM (n = 8–10). (B) Concentration–response curve of the effect of a 24-hour exposure to IL-1{alpha} on proMMP-3 levels in TM35D cells (n = 4).

 


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  		FIGURE 4. Zymographic analysis of IL-1{alpha}–induced increase in MMP-3 activity. (A) A representative casein zymograph of concentrated supernatants of TM35D cells treated with various amounts of IL-1{alpha} for 24 hours. Numbers on the left side of gel image represents masses (in kilodaltons) of molecular standards. (B) Concentration-dependent increases in casein hydrolytic activity of supernatants of TM35D cells after a 24-hour exposure to IL-1{alpha}. Data are the mean ± SEM of results in three to nine independent studies. *Significant increase (P < 0.05) when compared with vehicle control by ANOVA followed by the Dunnett test.

 
All five human TM cell lines showed similar stimulation profiles. The phorbol ester TPA was the most consistent and efficacious stimulator for proMMP-1 (Table 3) . TNF{alpha} also increased the production of proMMP-1, but only in the TM10A cells was the increase statistically significant (P < 0.05). PDGF-BB increased proMMP-1 level in TM30A cells but not in any other cell lines. ProMMP-2 did not respond to any of the stimuli in the three cell lines tested (Table 4) . IL-1{alpha} was the most efficacious and consistent stimulator for proMMP-3 in all TM cell lines, though this stimulatory effect ranged from a modest 146% to a dramatic 7882% above control among different cell lines. TPA and TNF{alpha} also were effective in upregulating the expression of MMP-3 in all five cells tested, and their action was statistically significant in three of the five cell lines (Table 5) . However, the efficacies of TPA and TNF{alpha} were always less than that of IL-1{alpha}. TPA also was very effective in increasing the accumulation of proMMP-9 in the three TM cell lines tested, whereas IL-1{alpha} was significantly less efficacious and was only effective in two of the three cell strains (Table 6) .


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  		TABLE 3. Effects of Various Compounds on ProMMP-1 Expression in Various TM Cell Strains

 

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  		TABLE 4. Effects of Various Compounds on ProMMP-2 Expression in Various TM Cell Strains

 

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  		TABLE 5. Effects of Various Compounds on ProMMP-3 Expression in Various TM Cell Strains

 

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  		TABLE 6. Effects of Various Compounds on ProMMP-9 Expression in Various TM Cell Strains

 
In addition to the stimulation of MMP expression, TPA also enhanced the production of TIMP-1 in all cells tested. PDGF-BB significantly increased TIMP-1 production in the TM35D cells, and a trend toward increase was observed in all tested cells (Table 7) . Similar to proMMP-2 expression, TPA, IL-1{alpha}, TNF, or PDGF-BB did not affect TIMP-2 expression in the cultured human TM cells (Table 8) .


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  		TABLE 7. Effects of Various Compounds on TIMP-1 Expression in Various TM Cell Strains

 

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  		TABLE 8. Effects of Various Compounds on TIMP-2 Expression in Various TM Cell Strains

 
Recently, Lindsey et al.27 28 and Weinreb et al.29 have demonstrated that FP prostaglandin receptor agonists upregulate the expression of MMP-1 and -3 in cultured human ciliary muscle cells. They have further proposed that this mechanism may contribute to the IOP-lowering effect of FP agonists. Hence, we also examined the effects of prostaglandins on MMP expression in the cultured human TM cells. We found that incubation of the TM16A or TM35D cells with PGE2 (10 µM) and PGF2{alpha} (10 µM) did not affect the proMMP-1 and -3 levels (Table 9) , suggesting that, in contrast to the ciliary muscle, EP and FP prostaglandin receptor agonists do not regulate MMP-1 and -3 production in the TM.


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  		TABLE 9. Effects of Prostaglandins E2 and F2{alpha} on MMP-1 and -3 Expression in TM Cells

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
We have demonstrated that cultured human TM cells express and secrete MMPs and TIMPs. MMP expression was measured by ELISA for the respective proMMPs, which are inactive zymogens requiring enzymatic hydrolysis for activation.30 Okada et al.26 demonstrated that changes in secreted proMMP levels correlate with changes in total MMP levels, and upregulation of proMMP correlates with increased MMP activity in various cell types.31 32 33 Similarly, the change in TM proMMP-3 levels induced by IL-1{alpha} in this study correlated with the change of MMP-3 zymographic activities (Figs. 3 4) . Therefore, it is thought that the changes in proMMP levels in the TM cells also reflect changes in MMP activities.

All five human TM cell strains expressed a quantifiable level of each of the proMMPs and TIMPs evaluated, although the basal levels among the different strains were not always similar. The cause of these differences is unclear, although they were independent of donor age, isolation methods, growth rates, or passage numbers of the cells used in the study. Furthermore, cell strains that produced high levels of a particular MMP or TIMP did not always produce high levels of other MMPs or TIMPs.

Despite these interstrain differences in basal levels, the various cell strains responded to the stimulants in a similar and consistent manner. For example, TPA was always the most efficacious stimulator in the production of MMP-1, and likewise, IL-1{alpha} was the most efficacious inducer of MMP-3 expression in all five TM cell strains. This suggests that the regulatory functions represented by these compounds are shared by most human TM cells and are not peculiarities of a particular cell strain.

It is interesting to note that the expression of the different MMPs and TIMPs in the TM cells was obviously independently regulated. Even though TPA upregulated the production of MMP-1, -3, and -9, and TIMP-1, it did not have any significant effect on the expression of MMP-2 or TIMP-2. Similarly, IL-1{alpha} stimulation was selective for MMP-3, and did not affect the expression of MMP-1 and -2 and TIMP-1 or -2. The selective regulation of individual MMPs and TIMPs in the human TM cells correlates with findings in cultured porcine TM cells.18 It also indicates that the stimulant-induced upregulation was not due to a broad trophic effect on the cells, suggesting that specific signaling pathways are involved.

The TPA and IL-1{alpha} stimulation profiles on MMP and TIMP synthesis in the cultured human TM cells were generally comparable to those reported in human TM explant organ cultures and TM cells derived from other species. We found that TPA increased the production of MMP-1, -3, and -9 and TIMP-1, but not of MMP-2 or TIMP-2. Alexander et al.,18 reported that TPA enhances gelatinase activities corresponding to gelatinases A (MMP-2) and B (MMP-9), as well as protein levels of stromelysin (MMP-3) and TIMP-1 in cultured porcine TM cells. The same researchers also demonstrated similar increases in gelatinases and stromelysin activities effected by TPA in human TM tissue explants and cells.8 We showed that IL-1{alpha} selectively induced the expression of MMP-3 from all five human TM cell strains. By using zymography and Western immunoblots, Samples et al.,10 reported that stromelysin production in TM tissue explants is augmented by IL-1{alpha}. Similar results in porcine TM cells were presented by Alexander et al.,18 who demonstrated that IL-1{alpha} stimulates, to a lesser degree, MMP-1 and -9 and TIMP-1 expression and does not affect MMP-2 or TIMP-2 expression.

In our study, the response of human TM cells to TNF{alpha} did not seem to correlate with that previously reported in porcine TM cells. In human TM cells, TNF{alpha} upregulated the production of MMP-3 and -1 in some, but not all, cell strains tested. It had no statistically significant effect on the immunoreactivities of proMMP-2 and -9 and TIMP-1 or -2. Yet in porcine TM cells, TNF{alpha} clearly increased MMP-1, -3, and -9, and TIMP-1 activities and/or protein levels in the culture media. It did not affect MMP-2 activity and significantly decreased the protein content of TIMP-2.18 The difference between the human and porcine TM cells is even more striking in their responses to PDGF-BB. In the porcine cells, PDGF-BB stimulated the production of MMP-1, -3, and -9, and TIMP-1,18 but in the human TM cells, PDGF-BB in general did not affect levels of MMPs and TIMPs. In only one of all the cell strains tested, did the growth factor slightly increase the amount of proMMP-1 or TIMP-1. Currently, the significance of these differences between the porcine and human TM cells is not clear. It may represent species differences in the regulation of MMPs and TIMPs expression.

Our finding that IL-1{alpha} maximally activated MMP-3 production in a potent and concentration-dependent fashion is highly interesting. MMPs are categorized into collagenases, gelatinases, and stromelysins according to their substrate specificity. Stromelysins, such as MMP-3, distinguish themselves from the collagenases by their activity against numerous structural extracellular matrix glycoproteins, including proteoglycans, fibronectin, laminin, gelatins, and collagens types III, IV, V, and IX.34 These glycoproteins are present in the TM and have been theorized to be involved in the regulation of aqueous humor outflow thereby modulating IOP. Hence, MMP-3 can directly modify aqueous outflow by catalyzing the degradation of these molecules. Moreover, MMP-3 is able to "superactivate" the 92-kDa type IV collagenase to participate in a proteolytic cascade with other MMPs.34 35 36 A similar cascade, if it exists in the TM, can cause additional hydrolysis of other matrix molecules. These characteristics of MMP-3 predict that it can be an important modulator of aqueous humor hydrodynamics in the eye. Indeed, when perfused in organ culture of human ocular anterior segments, MMP-3 alone is sufficient to decrease the aqueous outflow resistance and raise outflow facility.15

The probable involvement of MMP-3 in IOP regulation implies that modulating MMP-3 expression may be important in modulating IOP. Expression of MMP-3 is tightly controlled by a variety of physiologic and pharmacologic agents. Thus far, nearly all regulatory factors have been shown to function by transcriptional mechanisms.37 The IL-1{alpha}–mediated MMP-3 increase demonstrated in this study also involved signaling pathways related to transcriptional events.38 The biological significance of the IL-1{alpha} effect is quite obvious. Intracameral injection of IL-1{alpha} lowers IOP in the rat,39 and also increases aqueous outflow facility in human ocular perfusion organ culture.15 This cytokine was also shown to be one of the mediators of the clinical IOP-lowering effect induced by laser trabeculoplasty.40 Its stimulatory action on MMP-3 production demonstrated in the human TM cells provides a probable mechanism for these aqueous outflow effects of IL-1{alpha}.

In this study, we also found that EP and FP prostaglandin receptor agonists did not affect MMP-1 or -3 expression in cultured human TM cells. Lindsey et al.27 28 41 and Weinreb et al.29 showed that FP agonists upregulated MMPs in cultured human ciliary muscle cells and suggested that this action may be responsible for the uveoscleral outflow effect of FP agonists. Their hypothesis was supported by morphologic changes observed in the ciliary muscle of monkeys treated with topical application of prostaglandin F2{alpha}, an FP agonist. In these animals, the intercellular space of the ciliary muscle was enlarged and depleted of extracellular matrix, a finding consistent with the activation of MMP.42 43 44 Our finding demonstrates that prostaglandins did not affect TM cell MMP-1 or -3 levels, which agrees with findings that FP compounds do not seem to affect conventional aqueous outflow through the TM.45

In conclusion, we have shown that cultured human TM cells expressed various MMPs and TIMPs. Their expression was modulated independently by different regulatory molecules. We also demonstrated that IL-1{alpha} was the most efficacious in the activation of MMP-3 expression, which may mediate the ocular hypotensive effect of IL-1{alpha}. These results suggest that manipulation of TM production of MMP may provide a new and effective therapy for lowering IOP in glaucoma.


   Acknowledgements
 
The authors thank Mari Engler and Sherry English-Wright for providing initial cultures of the human TM cells used in the study; H. Thomas Steely for assistance in the analysis of zymograms; Ted Acott for technical advice in zymography; Paula Billman and Central Florida Lions Eye and Tissue Bank for procurement of donor tissues.


   Footnotes
 
Supported by Alcon Research, Ltd.

Submitted for publication July 26, 2002; revised January 9, February 27, and March 27, 2003; accepted April 7, 2003.

Disclosure: I.-H. Pang, Alcon Research, Ltd. (E, P); P.E. Hellberg, Alcon Research, Ltd. (E); D.L. Fleenor, Alcon Research, Ltd. (E, P); N. Jacobson, Alcon Research, Ltd. (E); A.F. Clark, Alcon Research, Ltd. (E, P)

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: Iok-Hou Pang, Alcon Research, Ltd., R3-24, 6201 South Freeway, Fort Worth, TX 76134; iok-hou.pang{at}alconlabs.com.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Quigley, HA. (1996) Number of people with glaucoma worldwide Br J Ophthalmol 80,389-393[Abstract/Free Full Text]
  2. Segawa, K. (1979) Electron microscopic changes in the trabecular tissue in primary open angle glaucoma Bellows, JG eds. Glaucoma: Contemporary International Concepts ,17-23 Masson Publishing New York.
  3. Rohen, JW, Linner, E, Witmer, R. (1973) Electron microscopic studies on the trabecular meshwork in two cases of corticosteroid-glaucoma Exp Eye Res 17,19-31[CrossRef][Medline][Order article via Infotrieve]
  4. Lütjen-Drecoll, E, Shimizu, T, Rohrbach, M, Rohen, JW. (1986) Quantitative analysis of "plaque material" between ciliary muscle tips in normal- and glaucomatous eyes Exp Eye Res 42,457-465[CrossRef][Medline][Order article via Infotrieve]
  5. Rohen, JW, Lütjen-Drecoll, E, Flügel, C, Meyer, M, Grierson, I. (1993) Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma (POAG) Exp Eye Res 56,683-692[CrossRef][Medline][Order article via Infotrieve]
  6. Knepper, PA, Goossens, W, Hvizd, M, Palmberg, PF. (1996) Glycosaminoglycans of the human trabecular meshwork in primary open-angle glaucoma Invest Ophthalmol Vis Sci 37,1360-1367[Abstract/Free Full Text]
  7. Acott, TS, Kingsley, PD, Samples, JR, Van Buskirk, EM. (1988) Human trabecular meshwork organ culture: morphology and glycosaminoglycan synthesis Invest Ophthalmol Vis Sci 29,90-100[Abstract/Free Full Text]
  8. 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]
  9. Acott, TS. (1992) Trabecular extracellular matrix regulation Drance, SM Van Buskirk, EM Neufeld, AH eds. Pharmacology of Glaucoma ,125-127 Williams & Wilkins Baltimore.
  10. Samples, JR, Alexander, JP, Acott, TS. (1993) Regulation of the levels of human trabecular matrix metalloproteinases and inhibitor by interleukin-1 and dexamethasone Invest Ophthalmol Vis Sci 34,3386-3395[Abstract/Free Full Text]
  11. Baramova, E, Foidart, J-M. (1995) Matrix metalloproteinase family Cell Biol Int 19,239-242[Medline][Order article via Infotrieve]
  12. Yong, VW, Krekoski, CA, Forsyth, PA, Bell, R, Edwards, DR. (1998) Matrix metalloproteinases and diseases of the CNS Trends Neurosci 21,75-80[CrossRef][Medline][Order article via Infotrieve]
  13. Clark, AF. (1998) New discoveries on the roles of matrix metalloproteinases in ocular cell biology and pathology Invest Ophthalmol Vis Sci 39,2514-2516[Free Full Text]
  14. Lukes, A, Mun Bryce, S, Lukes, M, Rosenberg, GA. (1999) Extracellular matrix degradation by metalloproteinases and central nervous system diseases Mol Neurobiol 19,267-284[Medline][Order article via Infotrieve]
  15. Bradley, JM, Vranka, J, Colvis, CM, et al (1998) Effect of matrix metalloproteinases activity on outflow in perfused human organ culture Invest Ophthalmol Vis Sci 39,2649-2658[Abstract/Free Full Text]
  16. Parshley, DE, Bradley, JM, Samples, JR, Van Buskirk, EM, Acott, TS. (1995) Early changes in matrix metalloproteinases and inhibitors after in vitro laser treatment to the trabecular meshwork Curr Eye Res 14,537-544[Medline][Order article via Infotrieve]
  17. Parshley, DE, Bradley, JM, Fisk, A, et al (1996) Laser trabeculoplasty induces stromelysin expression by trabecular juxtacanalicular cells Invest Ophthalmol Vis Sci 37,795-804[Abstract/Free Full Text]
  18. Alexander, JP, Samples, JR, Acott, TS. (1998) Growth factor and cytokine modulation of trabecular meshwork matrix metalloproteinase and TIMP expression Curr Eye Res 17,276-285[CrossRef][Medline][Order article via Infotrieve]
  19. Jacobson, N, Andrews, M, Shepard, AR, et al (2001) Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor Hum Mol Genet 10,117-125[Abstract/Free Full Text]
  20. Wordinger, RJ, Lambert, W, Agarwal, R, Talati, M, Clark, AF. (2000) Human trabecular meshwork cells secrete neurotrophins and express neurotrophin receptors (Trk) Invest Ophthalmol Vis Sci 41,3833-3841[Abstract/Free Full Text]
  21. Steely, HT, Jr, English Wright, SL, Clark, AF. (2000) The similarity of protein expression in trabecular meshwork and lamina cribrosa: implications for glaucoma Exp Eye Res 70,17-30[CrossRef][Medline][Order article via Infotrieve]
  22. Steely, HT, Browder, SL, Julian, MB, Miggans, ST, Wilson, KL, Clark, AF. (1992) The effects of dexamethasone on fibronectin expression in cultured human trabecular meshwork cells Invest Ophthalmol Vis Sci 33,2242-2250[Abstract/Free Full Text]
  23. Wilson, K, McCartney, MD, Miggans, ST, Clark, AF. (1993) Dexamethasone induced ultrastructural changes in cultured human trabecular meshwork cells Curr Eye Res 12,783-793[Medline][Order article via Infotrieve]
  24. Clark, AF, Wilson, K, McCartney, MD, Miggans, ST, Kunkle, M, Howe, W. (1994) Glucocorticoid-induced formation of cross-linked actin networks in cultured human trabecular meshwork cells Invest Ophthalmol Vis Sci 35,281-294[Abstract/Free Full Text]
  25. Stamer, WD, Seftor, RE, Williams, SK, Samaha, HA, Snyder, RW. (1995) Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion Curr Eye Res 14,611-617[Medline][Order article via Infotrieve]
  26. Okada, Y, Harris, ED, Nagase, H. (1988) The precursor of a metalloendopeptidase from human rheumatoid synovial fibroblasts: purification and mechanisms of activation by endopeptidases and 4-aminophenylmercuric acetate Biochem J 254,731-741[Medline][Order article via Infotrieve]
  27. Lindsey, JD, Kashiwagi, K, Boyle, D, Kashiwagi, F, Firestein, GS, Weinreb, RN. (1996) Prostaglandins increase proMMP-1 and proMMP-3 secretion by human ciliary smooth muscle cells Curr Eye Res 15,869-875[Medline][Order article via Infotrieve]
  28. Lindsey, JD, Kashiwagi, K, Kashiwagi, F, Weinreb, RN. (1997) Prostaglandins alter extracellular matrix adjacent to human ciliary muscle cells in vitro Invest Ophthalmol Vis Sci 38,2214-2223[Abstract/Free Full Text]
  29. Weinreb, RN, Kashiwagi, K, Kashiwagi, F, Tsukahara, S, Lindsey, JD. (1997) Prostaglandins increase matrix metalloproteinase release from human ciliary smooth muscle cells Invest Ophthalmol Vis Sci 38,2772-2780[Abstract/Free Full Text]
  30. Murphy, G, Cockett, MI, Stephens, PE, Smith, BJ, Docherty, AJ. (1987) Stromelysin is an activator of procollagenase; a study with natural and recombinant enzymes Biochem J 248,265-268[Medline][Order article via Infotrieve]
  31. Salamonsen, LA, Nagase, H, Woolley, DE. (1991) Production of matrix metalloproteinase 3 (stromelysin) by cultured ovine endometrial cells J Cell Sci 100,381-385[Abstract/Free Full Text]
  32. Di Girolamo, N, Lloyd, A, McCluskey, P, Filipic, M, Wakefield, D. (1997) Increased expression of matrix metalloproteinases in vivo and scleritis tissue and in vitro in cultured human scleral fibroblasts Am J Pathol 150,653-666[Abstract]
  33. Yee, J, Kuncio, GS, Bhandari, B, Shihab, FS, Neilson, EG. (1997) Identification of promoter activity and differential expression of transcripts encoding the murine stromelysin-1 gene in renal cells Kidney Int 52,120-129[Medline][Order article via Infotrieve]
  34. Matrisian, L. (1992) The matrix-degrading metalloproteinases Bioessays 14,455-463[CrossRef][Medline][Order article via Infotrieve]
  35. Alexander, CM, Werb, Z. (1989) Proteinases and extracellular matrix remodeling Curr Opin Cell Biol 1,974-982[CrossRef][Medline][Order article via Infotrieve]
  36. Murphy, G, Nagase, H, Brinckerhoff, CE. (1988) Relationship of procollagenase activator, stromelysin and matrix metalloproteinase 3 Coll Relat Res 8,389-391[Medline][Order article via Infotrieve]
  37. Welgus, HG. (1991) Stromelysin: structure and function Agents Actions Suppl 35,61-67[Medline][Order article via Infotrieve]
  38. Fleenor, DL, Pang, I-H, Clark, AF. (2003) Involvement of AP-1 in interleukin-1{alpha}–stimulated MMP-3 expression in human trabecular meshwork cells Invest Ophthalmol Vis Sci 44,3494-3501[Abstract/Free Full Text]
  39. Kee, C, Seo, K. (1997) The effect of interleukin-1alpha on outflow facility in rat eyes J Glaucoma 6,246-249[Medline][Order article via Infotrieve]
  40. Bradley, JM, Anderssohn, AM, Colvis, CM, et al (2000) Mediation of laser trabeculoplasty-induced matrix metalloproteinase expression by IL-1beta and TNFalpha Invest Ophthalmol Vis Sci 41,422-430[Abstract/Free Full Text]
  41. Lindsey, JD, Kashiwagi, K, Kashiwagi, F, Weinreb, RN. (1997) Prostaglandin action on ciliary smooth muscle extracellular matrix metabolism: implications for uveoscleral outflow Surv Ophthalmol 41(suppl 2),S53-S59
  42. Lütjen-Drecoll, E, Tamm, E. (1988) Morphological study of the anterior segment of cynomolgus monkey eyes following treatment with prostaglandin F2 alpha Exp Eye Res 47,761-769[CrossRef][Medline][Order article via Infotrieve]
  43. Lütjen-Drecoll, E, Tamm, E. (1989) The effects of ocular hypotensive doses of PGF2 alpha-isopropylester on anterior segment morphology Prog Clin Biol Res 312,437-446[Medline][Order article via Infotrieve]
  44. Tamm, E, Lütjen-Drecoll, E, Rohen, JW. (1990) Age-related changes of the ciliary muscle in comparison with changes induced by treatment with prostaglandin F2 alpha: an ultrastructural study in rhesus and cynomolgus monkeys Mech Ageing Dev 51,101-120[CrossRef][Medline][Order article via Infotrieve]
  45. Gabelt, BT, Kaufman, PL. (1989) Prostaglandin F2 alpha increases uveoscleral outflow in the cynomolgus monkey Exp Eye Res 49,389-402[CrossRef][Medline][Order article via Infotrieve]



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