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Keywords:

  • matrix metalloproteases;
  • elevated hydrostatic pressure;
  • interleukin-1

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Purpose: We investigated the effects of interleukin-1β (Il-1β) and dexamethasone (Dex) on the expression of matrix metalloprotease-1, -2, -3 and -14 (membrane type-1 MMP−MT1-MMP) as well as tissue inhibitors of matrix metalloproteases (TIMP-1 and -2) mRNA by trabecular cells exposed not only to normal, but also to elevated levels of hydrostatic pressure.

Methods: Confluent primary cultures of porcine trabecular cells were incubated in a serum-free medium (SFM) as controls, or in SFM containing either 10 ng/ml Il-1β or 10 nm Dex and exposed to pressures of 15 mmHg or 50 mmHg (corresponding to normal and high intraocular pressure, respectively) in specially designed pressure chambers. After 72 hours, total RNA was extracted from the harvested cells, reverse transcribed and amplified using primers specific to MMP-1, -2, -3 and -14, and TIMP-1 and -2.

Results: The most significant changes were detected in the levels of MMP-3 mRNA in control cells (2.4-fold increase), of TIMP-1 and -2 mRNA in cells treated with Il-1β (2.6-fold increase) and of MMP-3 mRNA in cells treated with Dex (3.5-fold increase) exposed to 50 mmHg pressure.

Conclusion: Because MMP-3 (stromelysin) mRNA showed the highest upregulation, our findings suggest that trabecular cells preferentially degrade and turn over the proteoglycan components of the extracellular matrix in response to short-term exposure to increased hydrostatic pressure with and without Dex as a homeostatic mechanism.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Matrix metalloproteases (MMPs) and tissue inhibitors of matrix metalloproteases (TIMPs) have a crucial role in the turnover of the extracellular matrix (ECM) in the trabecular meshwork and hence in the regulation of intraocular pressure (Alexander et al. 1991; Acott 1992; Nagase 1998; Schlötzer-Schrehardt et al. 2003). Particularly in the case of primary open-angle glaucoma (POAG), an increase in a few ECM components and subsequent elevated outflow resistance has been found (Rodriques et al. 1976; Alvarado et al. 1984; Abare et al. 1985; Babizhayev & Brodskaya 1989; Luetjen-Drecoll et al. 1989; Rohen & Luetjen-Drecoll 1989; Gong & Fredo 1994; Gonzales-Avila et al. 1995; Knepper et al. 1996).

Interleukin-1 (IL-1) is a proinflammatory cytokine that induces upregulation of MMP and TIMP expression (Samples et al. 1993; Alexander et al. 1998; Bradley et al. 2000; Pang et al. 2000).

Dexamethasone (Dex) is a widely used anti-inflammatory immune modulator, which can increase intraocular pressure (IOP) in a distinctive manner, and is able to downregulate MMP expression (Jonat et al. 1990; Tripathi et al. 1999). Our previous studies revealed upregulated expression of MMP-2, -3 and MMP-14 (MT1-MMP) mRNA and of TIMP-1 and -2 mRNA by trabecular cells exposed to increased hydrostatic pressure (Ehrich et al. 2001). The goal of this study was to determine whether, under the influence of increased hydrostatic pressure, IL-1α and Dex treatment modulates the MMP and TIMP expression of trabecular meshwork (TM) cells at the transcription level.

Material and Methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Trabecular meshwork preparation

Eyes of domestic pigs were obtained within 1 hour of death and immediately bisected equatorially. Dissection of the trabecular meshwork was carried out according to our previously documented technique (Tripathi et al. 1989). The excised trabecular meshwork tissue samples were transferred into 25 ml modified culture flasks.

Trabecular cell cultures

The explants were incubated in Dulbecco's modified Eagle's medium (low glucose, l-glutamine) supplemented with 1000 units penicillin G, 200 µg streptomycin and 15% newborn calf serum (Gibco, Santa Clara, California, USA) at 37 ° in humidified 95% air with 5% carbon dioxide. After 7 days the tissue pieces were removed and the cells were incubated for another week. As a result of our earlier investigations on changes in protein expression patterns (Tripathi et al. 1996), only primary cell cultures were used in all experiments.

Pressure chamber

The modified culture flasks were filled completely with serum-free medium (SFM), SFM containing 10 ng/Ml porcine IL-1α (R&D Systems, Minneapolis, Minnesota, USA), or SFM containing 10 nm Dex (SIGMA, St. Louis, Missouri, USA) and sealed. A hydrostatic pressure of 15 mmHg or 50 mmHg was generated (Fig. 1).

image

Figure 1. A modified flask allowing medium flow and generation.

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In order to maintain a gas exchange, a flow of medium at a rate of 2.5 µl per minute from reservoir A through the flask into reservoir B was maintained. After 72 hours exposure to hydrostatic pressure, the trabecular cells were harvested.

RNA extraction and purification

Total cellular RNA was extracted and purified using an SV Total RNA Isolation System (Promega, Madison, Wisconsin, USA). The 100 µl obtained were stored at − 80 °. The amount of total RNA and its purity were verified using a spectrophotometer (DU-65, Beckman, Fullerton, California, USA) at 260 nm and 280 nm absorption, respectively.

Primer design

Specific primers were designed using a computer program (omiga 2.0; Oxford Molecular Ltd., San Diego, CA, USA) and porcine GenBank sequences.

For semiquantative analysis of mRNA expression, cDNA samples adjusted to equal GAPDH inputs were used.

RT-PCR, agarose gel electrophoresis and laser densitometry

For the one-step, two-enzyme protocol an Access reverse transcription polymerase chain reaction (RT-PCR) system kit (Promega, Madison, Wisconsin, USA) was used. In each case 500 ng total RNA was imported into the cDNA synthesis for semiquantitative comparison. The number of PCR cycles was chosen so as to remain within the linear amplification phase.

Ten microlitres of each sample were run in a 2% agarose gel (Agarose, LMP, preparative grade for small fragments; Promega, Madison, Wisconsin, USA) in a horizontal electrophoresis unit (BIORAD; DNA Sub Cell, Richmond, California, USA) at 36 V and 20 mA overnight. The gels were analysed by laser densitometry using an Ultrascan XL (LKB, Paramus, NJ, USA) and the amounts of DNA were semiquantitatively evaluated with the Gelscan XL software.

For the statistical evaluations paired t-test and Kruskal–Wallis one-way anova were used.

The amplification products were confirmed by Southern blot and hybridization using specific biotin labelled probes and visualized with a BlueGene-Kit (Gibco BRL; Life Technologies, Gaithersburg, Maryland, USA).

Il-1α elisa

Earlier experiments in our laboratory had indicated some similarities in MMP regulation between highly increased hydrostatic pressure and IL-1α treatment. Therefore, we investigated the cell culture supernatant for autocrine IL-1α with a solid phase sandwich ELISA (BioSource, Camarillo, California, USA) specific for porcine IL-1α. We concentrated the supernatant by using a centrifugal filter device (Centriplus® YM-3; Millipore, Bedford, Massachusetts, USA) 15-fold. The sensitivity of the ELISA was 1 pg/ml.

Results

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

There were no morphological differences between trabecular cells exposed to either 15 mmHg or 50 mmHg. Cell viability (93% ± 2 for SFM-treated cells, 91% ± 3.5 for IL-1 and 94% ± 2.5 for Dex) was counted by 0.4% trypan blue staining in a standard haemacytometer chamber. Increased hydrostatic pressure for 72 hours did not result in significantly more dead cells. We were able to extract 6.5–7.6 µg total RNA from each flask and no significant differences were observed between the 15 mmHg and the 50 mmHg groups.

Statistics

No significant changes were found for MMP-1 (SFM15 versus SFM50, Dex15 versus Dex50 and SFM15 versus IL15), MMP-2 (IL15 versus IL50 and Dex15 versus Dex50), MMP-14 (Dex15 versus Dex50 and SFM50 versus IL50), TIMP-1 (SFM15 versus SFM50, SFM15 versus IL15 and SFM50 versus Dex50), or TIMP-2 (Dex15 versus Dex50 and SFM15 versus IL-15). Fig. 2 shows comparisons of MMP and TIMP mRNA expression in SFM, medium containing IL or Dex, exposed to 15 mmHg or 50 mmHg.

image

Figure 2. Box plots comparing MMP and TIMP mRNA expression in SFM, medium containing IL or Dex, exposed to 15 mmHg or 50 mmHg (measured by laser densitimetry, in arbitrary units, difference from background).

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The differences between all other pairs were statistically significant (p < 0.001).

Effect of increased hydrostatic pressure

The most significant changes were detected for MMP-3 in control cells (+ 2.4-fold) and cells treated with Dex (+ 2.6-fold), and for TIMP-1 and -2 in cells treated with interleukin (+ 2.3-fold and + 2.6-fold, respectively).

Effect of interleukin-1α

The largest changes at 15 mmHg were detected for MMP-2 (+ 1.6-fold) and MMP-3 (+ 1.6-fold) and at 50 mmHg hydrostatic pressure for MMP-1 (+ 1.8-fold), TIMP-1 (+ 2.3-fold) and TIMP-2 (+ 2.7-fold).

Effect of dexamethasone

The downregulating effect of Dex on mRNA expression was greatest in the 15 mmHg group for MMP-14 (− 2.6-fold) and in the 50 mmHg group for MMP-2 (− 3.2-fold), MMP-3 (− 2.6-fold), and MMP-14 (− 3.6-fold).

Interleukin-1α ELISA

No autocrine IL-1α was detectable by ELISA in either the SFM or Dex group. The amount of IL-1 in the culture medium that was treated with 10 ng/ml IL-1α decreased to 2 ng/ml detectable IL-1α after 72 hours.

Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Trabecular meshwork cells produce various MMPs and TIMPs in order to regulate the ECM turnover (Alexander et al. 1991). Various cytokines and growth factors, such as interleukin, transforming growth factor, or tumour necrosis factor-α, influence MMP and TIMP mRNA expression (Samples et al. 1993, 1998; Snyder et al. 1993; Kee & Seo 1997; Alexander et al. 1998; Bradley et al. 2000; Pang et al. 2000; Alexander & Acott 2003).

MMP-1 is thought to be involved in the regulation of the uveoscleral outflow (Gaton et al. 1999), and is increased in the aqueous humour of patients with uveitis (Di Girolamo et al. 1996). In our earlier investigations this collagenase did not show any significant change in mRNA expression in response to highly increased hydrostatic pressure. However, the mRNA expression of TM cells was elevated by a factor of 1.6 under treatment with IL-1β and the influence of increased pressure, whereas IL-1β or elevated pressure alone did not induce noticeable changes.

Interleukin-1β increases the activation of transcription factor AP-1 (activator protein-1), which promotes and subsequently upregulates the transcription of collagenases and stromelysin (Frisch & Ruley 1987; Hungness et al. 2000). Presumably one response to increased hydrostatic pressure is upregulation of c-Fos (subunit of AP-1), initiated by the opening of Ca+/Na ± channels in the cell membrane, as has been shown for other mechanical stimuli (Gasull et al. 2003).

Glucocorticoid hormones such as Dex downmodulate AP-1 (c-Fos/c-Jun) activity with the glucocorticoid receptor (GP) protein by transcriptional cross-talk and subsequently interfere with promoter activation (Jonat et al. 1990; Goettlicher et al. 1998). This leads to downregulation of MMP mRNA expression. Consequently, in our experiments Dex decreased MMP-1 mRNA expression according to the surrounding pressure.

MMP-2 and -14 showed similar reactions. In this context it is remarkable that MMP-14 (MT1-MMP) is a membrane-type protease, whereas all other investigated proteins circulate in the intercellular spaces. Therefore, MMP-14 is able to cleave only ECM material that is in direct contact with the trabecular meshwork cells, such as fibronectin (Ohuchi et al. 1997). With a 2.4-fold elevation MMP-3 showed the strongest reaction in untreated cells to highly increased hydrostatic pressure. Because MMP-3 is able to cleave glycosaminoglycans (GAGs) and their proteoglycans, which fill the intercellular spaces in the trabecular meshwork and are thought to be the putative outflow source (Murphy et al. 1991; Knepper et al. 1996; Wirtz et al. 1997), MMP-3 perhaps acts as a short-term response to highly increased hydrostatic pressure, considering that the half-life period for these ECM materials in the TM is about 1.5 days (Bradley et al. 1998). It has been shown by means of immunohistochemistry that MMP-3 induces early changes in the trabecular meshwork in the juxtacanalicular region after laser trabeculoplasty (Parshley et al. 1995, 1996). Tissue inhibitors of matrix metalloproteases are quite unspecific for MMPs, except for TIMP-1, which is unable to bind to any membrane-type matrix metalloproteases (Nagase et al. 1999), and TIMP-2, which is mandatory for MMP-14 controlled MMP-2 activation (Yu et al. 1998). If we consider all measured elevations in MMP mRNA, there is, despite significant upregulation of TIMP-2, a clear shift in balance toward MMP action.

In summary, the effects of IL-1β and Dex on MMP and TIMP mRNA expression in TM cells exposed to highly increased hydrostatic pressure differed. Under Dex treatment all investigated proteins except TIMP-1 showed a similar pattern of mRNA expression in the 15 mmHg and 50 mmHg groups, compared with untreated cells, but at a lower level. These findings suggest one reason why glucocorticoids are able to increase IOP. Because this interference presumably takes place in the MMP promoter regions by the GR protein, it could be one explanation why there are distinctive differences in IOP in humans responding to glucocorticoid treatment.

Acknowledgements

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This research was supported, in part, by NIH grant EY-08707, the Vision Research Foundation and Gertrud-Krusen-Stiftung.

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  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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