Supplementary Material for: Increased Shedding of Microvesicles from Intimal Smooth Muscle Cells in Athero-Prone Areas of the Human Aorta: Implications for Understanding of the Predisease Stage

<b><i>Objective:</i></b> This study evaluated whether a change in the content of matrix microvesicles might occur at the preatherosclerotic stage. <b><i>Methods:</i></b> Applying quantitative electron microscopic and immunohistochemical analyses, two areas of grossly normal segments of the thoracic aorta were compared: atherosclerosis-prone (AP) areas, situated at the dorsal aspect of the aorta along the rows of intercostal branch origins, and atherosclerosis-resistant (AR) areas, situated at the corresponding sites of the ventral aspect of the aorta. <b><i>Results:</i></b> The electron microscopic analysis showed that there were 1.4 times more microvesicles in AP areas than AR areas (p = 0.019). It was found that matrix microvesicles originated as a result of blebbing and shedding of surface membranes of smooth muscle cells. A quantitative analysis of the expression of ADP-ribosylation factor 6 (ARF6), which is known to be involved in membrane trafficking and microvesicle formation, showed that ARF6 expression was 1.3 times higher in AP areas than that in AR areas (p = 0.006). There was a positive correlation between the content of matrix microparticles and the expression of ARF6 by intimal smooth muscle cells (r = 0.61; p < 0.0001). <b><i>Conclusion:</i></b> The present study supports the concept that alterations of the arterial intima occur at the predisease stage.