Modulated smooth muscle cells accumulate late in human coronary atherosclerosis and are temporally and spatially linked to necrotic core formation

Background and AimsProliferation of arterial smooth muscle cells (SMCs) and their modulation to alternative mesenchymal phenotypes is a central mechanism in the growth of atherosclerotic lesions. The underlying processes have been studied extensively in mouse models, but a detailed analysis of when and where modulated SMCs accumulate in human atherosclerosis is lacking. The present study mapped modulated SMC subtypes during the progression of human coronary atherosclerosis and explored their associations with disease processes in human carotid plaques. MethodsMultiplex immunostaining protocols based on single-cell RNA sequencing-validated markers were established to detect SMCs, modulated SMCs, and macrophages in sections of left anterior descending arteries from forensic autopsies. The material comprised 44 arterial segments from 38 individuals, encompassing samples with normal intima, eccentric intimal thickening, pathological intimal thickening, and fibroatheroma. A similar analysis of carotid endarterectomy samples allowed examination of the involvement of modulated SMCs in fibrosis, calcification, and apoptosis. Coronary and carotid sections were analyzed by machine learning-assisted cell classification, enabling phenotyping of entire plaques at high microscopic resolution. ResultsCells co-expressing contractile and modulated SMC markers were present in normal human coronary arteries, but fully modulated SMCs, with complete loss of detectable contractile protein expression, did not accumulate substantially until the fibroatheroma stage, where they were located preferentially around the necrotic core. SMC subtypes showed no preferential co-localization with areas of fibrosis or calcification; however, osteoprotegerin secreted by modulated SMCs was found bound to calcium deposits. Modulated SMCs accounted for 35-53% of all apoptotic remnants for which a cell origin could be determined. ConclusionsFully modulated SMCs expand at the fibroatheroma stage, localize around the necrotic core region, and account for many apoptotic remnants in plaques.