Induction of HHcy enhances vascular inflammation and expression and activity of MMP-9 and increases elastolysis in aortic tissue from apoE-null mice. ApoE-null mice were fed the indicated diet for 8 weeks. (a–d) EN-RAGE. In a, aortae were removed, and lysates were prepared. Protein (10 μg) was subjected to immunoblotting with rabbit anti–EN-RAGE IgG (2 μg/ml). ^AP < 0.01 vs. diets A and C. In b–d, immunohistochemistry was performed on atherosclerotic lesions from mice fed diet A, B, or C. Scale bar, 50 μm. (e) TNF-α. Plasma was retrieved from mice, and ELISA was performed for detection of murine TNF-α. ^AP < 0.01 vs. diets A and C. (f and g) MMP-9. In f, lysates from aorta were subjected to immunoblotting using anti–MMP-9 IgG (1 μg/ml). In g, zymography was performed using gelatin-laden gels. In f and g, ^BP < 0.05 vs. diets A and C. In a, f, and g, molecular weight markers are indicated. Densitometric analysis was performed; pixel units from aortic tissue derived from mice receiving diet A were arbitrarily assigned a relative value of 1. In a, e, f, and g, immunoblots or zymograms were performed on n = 7 mice/diet; representative experiments are shown. In b–d, immunohistochemistry was performed on n = 5 mice/diet; representative experiments are shown. (h). Determination of extent of elastolysis. Lesions were assessed for extent of elastolysis as described above. All lesions in n = 10 mice/diet were examined. ^AP < 0.01 vs. diets A and C.