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Dexamethasone decreases tissue plasminogen activator activity in trabecular meshwork organ and cell cultures
R E Seftor1, W D Stamer, E A Seftor
1Pediatric Research Institute, Cardinal Glennon Children's Hospital, St. Louis University Health Sciences Center, St. Louis, Missouri, and *Department of Ophthalmology, University of Arizona, Tucson, Arizona, U.S.A.
Abstract:
Previously, we observed that dexamethasone decreased the extracellular activity of some matrix metalloproteinases and tissue plasminogen activator in trabecular meshwork containing organ explants and cell cultures. We have extended these studies to determine whether the decrease in extracellular tissue plasminogen activator activity is the result of a decrease in the expression of tissue plasminogen activator, a change in the extracellular level of plasminogen activator inhibitor-1, or a combination of both. We demonstrate that dexamethasone decreases the steady-state levels of tissue plasminogen activator mRNA by 75% in trabecular meshwork cells using Northern blot analysis, and show that this occurs concurrently with a 31% increase in the levels of extracellular plasminogen activator inhibitor-1 as determined by enzyme-linked immunosorbent assay analysis. A 38% increase was also measured in the extracellular plasminogen activator inhibitor-1 levels in trabecular meshwork containing organ explants after treatment. The amount of active, extracellular tissue plasminogen activator decreased by 72% in trabecular meshwork organ explant cultures, and 96% in trabecular meshwork cell cultures after treatment as determined using a parabolic rate assay for tissue plasminogen activator activity. These data suggest that dexamethasone decreases the extracellular activity of tissue plasminogen activator in trabecular meshwork containing organ and cell cultures by decreasing the expression of tissue plasminogen activator and increasing the extracellular levels of plasminogen activator inhibitor-1.
Insights
Dexamethasone reduces extracellular tissue plasminogen activator activity in trabecular meshwork by lowering its mRNA expression and increasing plasminogen activator inhibitor-1 levels.
Area of Science:
- Ocular Biology
- Biochemistry
- Pharmacology
Background:
- Dexamethasone previously shown to decrease extracellular matrix metalloproteinases and tissue plasminogen activator activity.
- Trabecular meshwork (TM) plays a crucial role in regulating intraocular pressure.
Purpose of the Study:
- To investigate the mechanisms by which dexamethasone decreases extracellular tissue plasminogen activator (tPA) activity in TM.
- To determine if the decrease is due to reduced tPA expression or altered levels of plasminogen activator inhibitor-1 (PAI-1).
Main Methods:
- Northern blot analysis to measure tPA mRNA levels in TM cells.
- Enzyme-linked immunosorbent assay (ELISA) to quantify extracellular PAI-1 levels in TM cells and organ explants.
- Parabolic rate assay to determine active extracellular tPA activity in TM cultures.
Main Results:
- Dexamethasone decreased steady-state tPA mRNA levels by 75% in TM cells.
- Extracellular PAI-1 levels increased by 31% in TM cells and 38% in TM organ explants.
- Active extracellular tPA activity decreased by 72% in TM organ explants and 96% in TM cell cultures.
Conclusions:
- Dexamethasone reduces extracellular tPA activity in the trabecular meshwork.
- This reduction is mediated by decreased tPA gene expression and increased extracellular PAI-1 levels.
