Vascular tissue fragility assessed by a new double stain method

Although matrix metalloproteinases (MMPs) are known to be involved in the development of atherosclerosis and the instability of atheromatous plaques, much remains to be learned about their roles at the tissue level. To help clarify this area, we established a new double staining method using film in situ zymography and immunohistochemistry. Using this technique, a comprehensive analysis of the gelatinolytic activity in human vessel tissue demonstrated that gelatinolytic activity is enhanced in the shoulder region and fibrous cap at superficial areas of the atheromatous plaque in the presence of thrombolysis. Enzyme assay clarified high activity in the superficial area (7.50 ± 5.04 U/mg weight; P < 0.001). Gelatin zymography also indicated that addition of the antiplatelet agent, trapidil, alters the amount of secretion of MMP-2 and MMP-9 and their activation ratio. This novel approach to detect the activity of gelatinases in resected tissues may aid in the selection of optimal treatment of individual patients.