Visualization of endothelial barrier damage prior to formation of atherosclerotic plaques.

En-face fat staining is frequently used to visualize atherosclerotic lesions. This method, however, is not suitable to visualize endothelial barrier damage prior to microscopically detectable morphological alterations of the arterial wall such as sub-endothelial lipid deposition. To enable the investigation of early endothelial barrier damage and in particular the initial steps of atherosclerosis, a new method has to fulfill three requirements: (i) easy and fast to perform, (ii) low cost of applicability without requirement for highly sophisticated technical equipment, and (iii) reliable reproducibility of valid results. To this end, we used intracardial Evans blue dye injection after washout of blood and measured dye deposition within the aortic wall as a parameter of endothelial barrier leakiness, which is recognized as one of the earliest signs of atherosclerotic plaque formation. These analyses were performed in ApoE , LDL receptor and Cc1 mouse models which have been reported to develop aortic plaques with or without high cholesterol diet. Our data show that sub-endothelial dye deposition is a reliable and reproducible readout parameter to assess endothelial barrier damage. Along these lines, measurements of aortic intima areas with Evans blue deposition in relation to total intima circumference enabled quantitative assessments of the results. Our technique enables the imaging of endothelial barrier damage prior to detectable aortic lipid deposition and plaque development. Thus, it will facilitate the detection of the initial vascular pathogenetic processes that lead to cardiovascular diseases. It will also enable the testing of new drugs and therapeutic procedures to prevent these disorders.