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Abstract
Purpose
We investigated the extent of adenosine A1 agonist-induced expression and regulation of matrix metalloproteinase 2 (MMP-2) synthesis in human trabecular meshwork cells (HTMC).
Methods
Primary HTMC cultures were exposed to 0.1 or 1.0 µM N6-cyclohexyladenosine (CHA) for 2 h in the presence or absence of an inhibitor thereof, 8-cyclopentyl-1,3-dimethylxanthine (CPT). The expression level of mRNA encoding MMP-2 was assessed via reverse transcription-polymerase chain reaction, and the levels of tissue inhibitor of metalloproteinase 2 (TIMP2) and membrane-type-1 MMP (MT1-MMP) measured by Western blotting. The permeability of the HTMC monolayer was assessed with the aid of carboxyfluorescein.
Results
CHA at 1.0 µM increased the permeability of the HTMC monolayer (p = 0.003) and CHA at both 0.1 and 1.0 µM significantly increased MMP-2 mRNA expression, which was inhibited by co-exposure to CPT (all p < 0.05). CHA increased MMP-2 activity, decreased that of TIMP2, and increased that of MT1-MMP (all p < 0.05).
Figures and Tables
Figure 1
Exposure to 1.0 µM N6-cyclohexyladenosine (CHA) increased the permeability of carboxyfluorescin significantly compared to control. Exposure to the 10 µM 8-cyclopentyl-1,3-dimethylxanthine (CPT) decreased permeability significantly when co-exposed to 0.1 or 1.0 µM CHA (*). When co-exposed to CPT, 0.1 or 1.0 µM CHA decreased permeability significantly compared with exposure to CHA alone (**). Carboxyfluorescin intensity of outer chamber normalized to the mean value obtained using non-exposed control (permeability 100%). *,**p < 0.05.
Figure 2
Exposure to 0.1, 1.0 µM N6-cyclohexyladenosine (CHA) increased significantly the expression of matrix metalloproteinase 2 (MMP2) mRNA with or without co-exposure to 10 µM 8-cyclopentyl-1,3-dimethylxanthine (CPT) compared to control. Co-exposure to the 10 µM CPT with 0.1 or 1.0 µM CHA decreased the expression of MMP2 mRNA compared to exposure to CHA alone, respectively. β-actin used as internal standard. *p < 0.05.
Figure 3
Exposure to 0.1, 1.0 µM N6-cyclohexyladenosine (CHA) increased significantly the activity of matrix metalloproteinase 2 with or without co-exposure to 10 µM 8-cyclopentyl-1,3-dimethylxanthine (CPT) compared to control. GAPDH used as internal standard. *p < 0.05.
Figure 4
Exposure to 1.0 µM N6-cyclohexyladenosine (CHA) decreased significantly the activity of tissue inhibitor of metalloproteinase 2 (TIMP2) with or without co-exposure to 10 µM 8-cyclopentyl-1,3-dimethylxanthine (CPT) compared to control. Exposure to the 10 µM CPT alone did not affect the activity of TIMP2. GAPDH used as internal standard. *p < 0.05.
Figure 5
Exposure to 1.0 µM N6-cyclohexyladenosine (CHA) increased significantly the activity of membrane-type-1 matrix metalloproteinase (MT1-MMP). Co-exposure to the 10 µM 8-cyclopentyl-1,3-dimethylxanthine (CPT) with 0.1 or 1.0 µM CHA decreased the activity of MT1-MMP. GAPDH used as internal standard. *p < 0.05.
Notes
References
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