The no donor DETA-NONOate decreases MMP-9 expression and activity in abdominal aortic explants

Abstract

Objective: The goal of this project is to examine the role of an exogenous NO donor, DETA-NONOate (DETA), on MMP-9, MMP-2, and TIMP-1 expression and activity in interleukin-1β (IL-1β) induced rat aortic explants (RAE). Methods: RAEs were incubated with IL-1β (2 ng/ml) and increasing concentrations of DETA (0, 5.0, 50, 100, and 500 μM) (n = 3 per group). Messenger RNA (mRNA) was extracted from cells after 24 hours and analyzed for MMP-9, MMP-2, and TIMP-1 expression levels by real time RT-PCR. Media at 48 hours was collected and assayed for NO2 and NO3 (NOx) by the Saville Assay, MMP-9 and MMP-2 activity by gelatin zymography, and TIMP-1 activity by reverse zymography. All statistical analyses were performed by ANOVA and Pearson correlation. Results: DETA administration resulted in a dose-dependent increase in media NOx concentration (0.001 +/- 0.0003 ng NOx / mg protein to 0.062 +/- 0.004 ng NOx / mg protein, p < 0.01). In RAE, MMP-9 expression and activity decreased significantly in a dose dependent fashion with increasing DETA concentrations (p < 0.01). At the maximal dose of 500 μM DETA, a 78% decrease in MMP-9 expression (p < 0.05) and a 72% decrease in pro-MMP-9 activity (p < 0.05) was demonstrated compared to RAE treated with IL-1β alone (0 μM DETA). There were no significant differences seen in MMP-2 and TIMP-1 expression or activity in response to DETA exposure. Conclusion: The NO donor DETA-NONOate decreased IL-1β induced MMP-9 expression and activity in RAE in a dose dependent fashion. These data suggest that NO donors may be beneficial in decreasing MMP-9 levels, an enzyme believed to be critical in vessel wall remodeling, and therefore may serve to inhibit MMP-9 dependent vessel wall degradation seen during AAA formation.