Calmodulin Expression Distinguishes the Smooth Muscle Cell Population of Human Carotid Plaque

Abstract

Several observations suggest the expansion of a distinct medial smooth muscle cell (SMC) subset in atherosclerosis and restenosis. We characterized the phenotypic features of SMC subsets in cultures derived from human carotid endarterectomy specimens. Specimens comprised an undiseased portion (thin intimal thickening with the underlying media) and a diseased portion (atherosclerotic plaque with the underlying media). From plaque tissues of the diseased portion, only macrophage-derived foam cells were retrieved. From medial tissues, two SMC phenotypes were isolated: large SMCs (flat with a monolayered growth pattern, from the undiseased portion) and small SMCs (fusiform and growing in multilayers, from the undiseased and diseased portions after co-culture with macrophage-derived foam cells). Small SMCs displayed higher proliferative and migratory activities and were less differentiated than large SMCs. Proteomic analysis showed that calmodulin was predominant in small SMCs. Co-culture of large SMCs with macrophage-derived foam cells induced a transition to the small phenotype with increased calmodulin expression. The calmodulin inhibitor W-7 decreased the proliferation of small SMCs and prevented the large to small phenotypic transition. In vivo, calmodulin was markedly expressed in SMCs of atherosclerotic plaques and was barely detectable in the media. Macrophage-derived foam cells promote selective migration from the media of atheroma-prone SMCs characterized by calmodulin overexpression. Further studies of small SMCs could be instrumental in understanding atherosclerosis pathogenesis and in planning therapeutic strategies.

Eukaryotic elongation factor 2 kinase controls proliferation and migration of vascular smooth muscle cells

“…inhibits proliferation (Coen et al 2013) and migration (Tada et al 2008) of SMCs also support the present results. Several reports showed that PDGF-BB increased intracellular Ca 2+ concentration through induction of diacylglycerol and inositol 1,4,5-trisphosphate via acti-vation of phospholipase C, which leads to the activation of CaM-dependent signals including Akt and ERK (Rhee 2001, Egan et al 2005, Farrer et al 2013.…”

“…Previous reports demonstrating that W7, a specific CaM inhibitor, inhibits proliferation (Coen et al . ) and migration (Tada et al . ) of SMCs also support the present results.…”

Cell‐specific diversity in the expression and organization of cytoplasmic plaque proteins of apical junctions

“…Immunohistochemical staining was performed on 4‐μm‐thick paraffin sections of porcine coronary artery (collected from 8‐month‐old pigs obtained from a nearby slaughterhouse) and on samples from human carotid endarterectomy (approved by the ethics committee of S. Anna University Hospital, Ferrara, Italy). Immunoreactivity was intensified in citrate buffer for 5 min using a pressure cooker.…”

“…Slides were counterstained with hemalum and mounted in Eukitt (Kindler, Freiburg, Germany). Images were taken on an Axioskop microscope (Carl Zeiss, Jena, Germany) equipped with Plan‐Neofluar 20/0.3× and 40/0.75× objectives and a high‐sensitivity, high‐resolution digital color camera (Axiocam, Carl Zeiss) …”

“…Images were taken on an Axioskop microscope (Carl Zeiss, Jena, Germany) equipped with Plan-Neofluar 20/0.3× and 40/0.75× objectives and a high-sensitivity, high-resolution digital color camera (Axiocam, Carl Zeiss). 61,62 Proximity ligation assay The following primary antibodies were used for the proximity ligation assay (PLA): rabbit anti-PLEKHA7 R30388 (1:10000), mouse anti-Ecadherin (1:2000, BD610181), mouse antiparacingulin (E-6) (1:100, Santa Cruz, sc-377525), mouse anti-ZO-1 (1:1000, Invitrogen, 33-9100), mouse anti-occludin (1:200, Invitrogen, 33-1500), mouse anti-cingulin 22BD5A1 (1:2000), rabbit anti-ZO-1 (1:200, Invitrogen, 61-7300), rabbit antiparacingulin n.821 (1:1000), rabbit anti-cingulin C532 (1:10000), and rabbit anti-phospho-histone H3 (1:200, Merck Millipore, 06-570). The assay was performed according to the manufacturer's instructions (Duolink R In Situ Red Starter Kit Mouse/ Rabbit, Sigma).…”