Korean J Ophthalmol.  2016 Feb;30(1):66-75. 10.3341/kjo.2016.30.1.66.

Effect of Nitric Oxide on the Expression of Matrix Metalloproteinase and Its Association with Migration of Cultured Trabecular Meshwork Cells

• Kim JW

• KMID: 2363893
• DOI: http://doi.org/10.3341/kjo.2016.30.1.66

Abstract

PURPOSE
To determine the effect of exogenous nitric oxide (NO) on the migration of trabecular meshwork (TM) cells and its association with expression of matrix metalloproteinases (MMPs).
METHODS
Primary human TM cells treated with 1 or 10 microM S-nitroso-N-acetyl-penicillamine (SNAP) and examined for changes in adherence. TM cells were seeded onto transwell culture inserts, and changes in their migratory activity were quantified. Reverse transcription polymerase chain reaction was performed to determine the relative changes in mRNA expression of MMPs and tissue inhibitor of metalloproteinases (TIMPs).
RESULTS
Treatment with SNAP did not significantly suppress TM cell adhesion or migration (p > 0.05). Treatment of TM cells with 10 microM SNAP decreased expression of MMP-2 and increased expression of membrane type MMP-1 and TIMP-2. Treatment with interleukin-1alpha triggered MMP-3 expression but did not exert significant effects on MMP-3 activation in response to SNAP.
CONCLUSIONS
These data suggest that NO revealed no significant effect on the migration of TM cells because NO decreased MMP-2 and increased TIMP-2 expression. Although expression of certain MMPs and TIMPs change in response to NO donors, NO may modulate trabecular outflow by changing the cellular production of extracellular matrix without having a significant effect on the migration of TM cells.

Keyword

Matrix metalloproteinase; Migration; Nitric oxide; Trabecular meshwork cells

MeSH Terms

Cell Movement/*drug effects
Cell Survival/drug effects
Cells, Cultured
DNA Primers/chemistry
Gene Expression Regulation, Enzymologic/*physiology
Humans
Matrix Metalloproteinases/*genetics
Nitric Oxide Donors/*pharmacology
RNA, Messenger/genetics
Real-Time Polymerase Chain Reaction
S-Nitroso-N-Acetylpenicillamine/*pharmacology
Tissue Inhibitor of Metalloproteinase-2/*genetics
Trabecular Meshwork/cytology/*drug effects/enzymology
DNA Primers
Matrix Metalloproteinases
Nitric Oxide Donors
RNA, Messenger
S-Nitroso-N-Acetylpenicillamine
Tissue Inhibitor of Metalloproteinase-2

Figure

• 

  Fig. 1 Effect of S-nitroso-N-acetyl-penicillamine (SNAP) with interleukin-1α (IL-1α) on the production of nitric oxide (NO) in trabecular meshwork (TM) cells. SNAP significantly increased NO production in a dose-dependent manner (*p < 0.05). Production of NO in TM cells incubated with IL-1α was not different compared with the control. Addition of Nω-nitro-L-arginine methyl ester (L) to 10 µM SNAP significantly inhibited NO production (**p < 0.05). Values are presented as means ± standard error of the mean.

• 

  Fig. 2 Effect of Nω-nitro-L-arginine methyl ester (L-NAME), interleukin-1α (IL-1α), and S-nitroso-N-acetyl-penicillamine (SNAP) on the survival of trabecular meshwork cells. L-NAME, IL-1α, and SNAP did not have different effects on survival compared with the control (p > 0.05). Values are presented as means ± standard error of the mean.

• 

  Fig. 3 Effect of Nω-nitro-L-arginine methyl ester (L-NAME) and S-nitroso-N-acetyl-penicillamine (SNAP) on the adhesion of trabecular meshwork cells. L-NAME and SNAP did not have a significant effect on cell adhesion compared with the control (p > 0.05). Values are presented as means ± standard error of the mean.

• 

  Fig. 4 Effect of Nω-nitro-L-arginine methyl ester (L-NAME) and S-nitroso-N-acetyl-penicillamine (SNAP) on the migration of trabecular meshwork cells. L-NAME and SNAP did not have a significant effect on cell migration significantly compared with the control (p > 0.05). Values are presented as means ± standard error of the mean.

• 

  Fig. 5 Effect of S-nitroso-N-acetyl-penicillamine (SNAP) on matrix metalloproteinase (MMP)-2 (A) and MT1-MMP (B) mRNA activity. Trabecular meshwork cells showed a significant decrease in MMP-2 levels and increased MT1-MMP in response to 10 µM SNAP compared to the non-exposed control (*p < 0.05).

• 

  Fig. 6 Effect of S-nitroso-N-acetyl-penicillamine (SNAP) on matrix metalloproteinase (MMP)-3 mRNA activity stimulated by interkeukin-1α. Trabecular meshwork cells did not show a significant difference in MMP-3 levels in response to SNAP compared to the non-exposed control (p > 0.05). MMP-9 was not expressed in trabecular meshwork cells after stimulation with interkeukin-1α.

• 

  Fig. 7 Effect of S-nitroso-N-acetyl-penicillamine (SNAP) on tissue inhibitor of metalloproteinase (TIMP)-1 (A) and TIMP-2 (B) mRNA activity. Trabecular meshwork cells did not show a significant difference in TIMP-1 levels (p > 0.05) but showed a significant increase in TIMP-2 levels in response to 10 µM SNAP compared to the non-exposed control (*p < 0.05).

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