In Situ Localization and Quantification of mRNA for 92-kD Type IV Collagenase and Its Inhibitor in Aneurysmal, Occlusive, and Normal Aorta

Abstract

Abstract Ninety-two-kilodalton type IV collagenase (MMP-9) is present in aortic aneurysms and may be important to the pathogenesis of this disease. Alteration in expression of MMP-9 or its inhibitor, the tissue inhibitor of metalloproteinase type 1 (TIMP-1), could increase degradation of extracellular matrix and lead to aneurysm formation. The purpose of this study was (1) to measure tissue levels of MMP-9 and TIMP-1 mRNA in aneurysmal (AAA), atherosclerotic occlusive (AOD), and normal (NL) human infrarenal aorta; (2) to test for their expression by cultured AAA and NL vascular smooth muscle cells (VSMCs); and (3) to locate in situ the cells responsible for mRNA production within AAA, AOD, and NL aortic wall. Total RNA extracted from AAA (n=8), AOD (n=8), and NL (n=7) tissue was subjected to Northern analysis. Signals for MMP-9 and TIMP-1 were normalized to α-tubulin. Mean values±SEM were compared by ANOVA. NL and AAA VSMCs were cultured, passaged, and grown to confluence before RNA extraction and Northern analysis. In situ hybridization with digoxigenin-labeled RNA probes localized cells responsible for MMP-9 and TIMP-1 mRNA expression within sections of AAA (n=5), AOD (n=2), and NL (n=2) aorta. MMP-9 mRNA levels were significantly greater in AAA (0.855±0.180) than NL (0.046±0.23) ( P <.02), but differences between AOD (0.406±0.196) and AAA or AOD and NL were not significant. Differences in TIMP-1 mRNA levels between tissue types were not significant (AAA, 1.17±0.123; AOD, 1.79±0.351; NL, 0.652±0.378). Cultured AAA and NL aortic VSMCs constitutively expressed mRNA for TIMP-1 but not MMP-9. In situ hybridization of AAA and AOD tissue localized MMP-9 mRNA to adventitial macrophages in areas of neovascularization and TIMP-1 mRNA to adventitial VSMCs. MMP-9 mRNA levels are significantly greater in aneurysmal than normal aorta. Cultured VSMCs constitutively express TIMP-1 but not MMP-9. In the diseased aortic wall, MMP-9 mRNA is found in adventitial macrophages and TIMP-1 mRNA in adventitial VSMCs. Localization of MMP-9 mRNA expression to discrete areas surrounding vasa vasorum suggests that the enzyme is responsible for localized matrix alterations associated with neovascularization.