S2, online supplementary file]. In recent atherotrombotic occlusion, vascularization, expression of the highly active remodeling process, was also observed [Fig. S3, online supplementary file]. Vascularization was not detected in the hyperechoic with acoustic shadow calcific tissue, nor in the hypoechoic necrotic and hemorrhagic areas. Moreover, plaque vascularization is present in almost every plaque, regardless the degree of stenosis. In acute symptomatic patients a completely different pattern of vascularization was detected with ultrasound and validated by post-operative histology in a first paper published from our group

AZD6244 [41]. In the first seconds after contrast agent administration, no vascularization seemed to be identified in the hypoechoic areas. Few seconds later, vascularization presented as a major diffuse area of contrast enhancement at the base of the plaques, due to an agglomerate of many small microvessels, difficult to differentiate from each other, while the residual hypoechoic part of the plaque, corresponding to the necrotic or hemorrhagic contents, remained avascularized. In operated patients, carotid

endoarterectomies were carefully performed in order to obtain selleck kinase inhibitor the whole plaque with minimal trauma. The pathologist evaluated the removed plaques after formalin fixation: the pathologist and the sonographers discussed the regions of interest previously observed at ultrasound imaging. The intra-operative macroscopic findings confirmed the presence of the these unstable plaques observed at contrast ultrasound. The microscopic

findings confirmed the presence of plaque vascularization in the ultrasound contrast-enhanced areas. Symptomatic carotid plaques showed a relevant increased number of small (diameter 20–30 μm), immature microvessels in respect to asymptomatic ones, consisting with a strong neoangiogenetic activity. Angiogenesis was less represented in asymptomatic plaques that underwent surgery, with microvessels of a higher caliber (80–100 μm). Immunostaining with VEGF, MMP3, CD 31 and CD 34 depicted a different distribution pattern between asymptomatic and symptomatic lesions: while in the former antigenic activity was of a lesser degree and localized mainly along the microvessels course, in symptomatic plaques a high antigenic fixation was observed also in the external part of the plaque, closer to the adventitial layers. In the same areas, an inflammatory infiltrate constituted by macrophagic foam cells and T lymphocytes, indicative of high plaque activity was detected, with small areas of hemorrhage expression of microvessels rupture.