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Characterization of Carotid Smooth Muscle Cells during Phenotypic Transition.

ABSTRACT: Vascular smooth muscle cells (VSMCs) are central players in carotid atherosclerosis plaque development. Although the precise mechanisms involved in plaque destabilization are not completely understood, it is known that VSMC proliferation and migration participate in plaque stabilization. In this study, we analyzed expression patterns of genes involved in carotid atherosclerosis development (e.g., transcription factors of regulation of SMC genes) of VSMCs located inside or outside the plaque lesion that may give clues about changes in phenotypic plasticity during atherosclerosis. VSMCs were isolated from 39 carotid plaques extracted from symptomatic and asymptomatic patients by endarterectomy. Specific biomarker expression, related with VSMC phenotype, was analyzed by qPCR, western immunoblot, and confocal microscopy. MYH11, CNN1, SRF, MKL2, and CALD1 were significantly underexpressed in VSMCs from plaques compared with VSMCs from a macroscopically intact (MIT) region, while SPP1, KLF4, MAPLC3B, CD68, and LGALS3 were found significantly upregulated in plaque VSMCs versus MIT VSMCs. The gene expression pattern of arterial VSMCs from a healthy donor treated with 7-ketocholesterol showed high similarity with the expression pattern of carotid plaque VSMCs. Our results indicate that VSMCs isolated from plaque show a typical SMC dedifferentiated phenotype with macrophage-like features compared with VSMCs isolated from a MIT region of the carotid artery. Additionally, MYH11, KLF5, and SPP1 expression patterns were found to be associated with symptomatology of human carotid atherosclerosis.

SUBMITTER: Goikuria H

PROVIDER: S-EPMC5870355 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Characterization of Carotid Smooth Muscle Cells during Phenotypic Transition.

Goikuria Haize H Freijo Maria Del Mar MDM Vega Manrique Reyes R Sastre María M Elizagaray Elena E Lorenzo Ana A Vandenbroeck Koen K Alloza Iraide I

Cells 20180318 3

Vascular smooth muscle cells (VSMCs) are central players in carotid atherosclerosis plaque development. Although the precise mechanisms involved in plaque destabilization are not completely understood, it is known that VSMC proliferation and migration participate in plaque stabilization. In this study, we analyzed expression patterns of genes involved in carotid atherosclerosis development (e.g., transcription factors of regulation of SMC genes) of VSMCs located inside or outside the plaque lesi ...[more]

PMID: 29562638

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Project description:Atherosclerosis, the leading cause of death, is a vascular disease of chronic inflammation. We recently showed that angiopoietin-like 4 (ANGPTL4) promotes cardiac repair by suppressing pathological inflammation. Given the fundamental contribution of inflammation to atherosclerosis, we assessed the role of ANGPTL4 in the development of atherosclerosis and determined whether ANGPTL4 regulates atherosclerotic plaque stability. We injected ANGPTL4 protein twice a week into atherosclerotic Apoe-/- mice and analyzed the atherosclerotic lesion size, inflammation, and plaque stability. In atherosclerotic mice, ANGPTL4 reduced atherosclerotic plaque size and vascular inflammation. In the atherosclerotic lesions and fibrous caps, the number of α-SMA(+), SM22α(+), and SM-MHC(+) cells was higher, while the number of CD68(+) and Mac2(+) cells was lower in the ANGPTL4 group. Most importantly, the fibrous cap was significantly thicker in the ANGPTL4 group than in the control group. Smooth muscle cells (SMCs) isolated from atherosclerotic aortas showed significantly increased expression of CD68 and Krüppel-like factor 4 (KLF4), a modulator of the vascular SMC phenotype, along with downregulation of α-SMA, and these changes were attenuated by ANGPTL4 treatment. Furthermore, ANGPTL4 reduced TNFα-induced NADPH oxidase 1 (NOX1), a major source of reactive oxygen species, resulting in the attenuation of KLF4-mediated SMC phenotypic changes. We showed that acute myocardial infarction (AMI) patients with higher levels of ANGPTL4 had fewer vascular events than AMI patients with lower levels of ANGPTL4 (p < 0.05). Our results reveal that ANGPTL4 treatment inhibits atherogenesis and suggest that targeting vascular stability and inflammation may serve as a novel therapeutic strategy to prevent and treat atherosclerosis. Even more importantly, ANGPTL4 treatment inhibited the phenotypic changes of SMCs into macrophage-like cells by downregulating NOX1 activation of KLF4, leading to the formation of more stable plaques.

Project description:Background3,3'Diindolylmethane (DIM), a natural phytochemical, has shown inhibitory effects on the growth and migration of a variety of cancer cells; however, whether DIM has similar effects on vascular smooth muscle cells (VSMCs) remains unknown. The purpose of this study was to assess the effects of DIM on the proliferation and migration of cultured VSMCs and neointima formation in a carotid injury model, as well as the related cell signaling mechanisms.Methodology/principal findingsDIM dose-dependently inhibited the platelet-derived growth factor (PDGF)-BB-induced proliferation of VSMCs without cell cytotoxicity. This inhibition was caused by a G0/G1 phase cell cycle arrest demonstrated by fluorescence-activated cell-sorting analysis. We also showed that DIM-induced growth inhibition was associated with the inhibition of the expression of cyclin D1 and cyclin-dependent kinase (CDK) 4/6 as well as an increase in p27(Kip1) levels in PDGF-stimulated VSMCs. Moreover, DIM was also found to modulate migration of VSMCs and smooth muscle-specific contractile marker expression. Mechanistically, DIM negatively modulated PDGF-BB-induced phosphorylation of PDGF-recptor? (PDGF-R?) and the activities of downstream signaling molecules including Akt/glycogen synthase kinase(GSK)3?, extracellular signal-regulated kinase1/2 (ERK1/2), and signal transducers and activators of transcription 3 (STAT3). Our in vivo studies using a mouse carotid arterial injury model revealed that treatment with 150 mg/kg DIM resulted in significant reduction of the neointima/media ratio and proliferating cell nuclear antigen (PCNA)-positive cells, without affecting apoptosis of vascular cells and reendothelialization. Infiltration of inflammatory cells was also inhibited by DIM administration.ConclusionThese results demonstrate that DIM can suppress the phenotypic modulation of VSMCs and neointima hyperplasia after vascular injury. These beneficial effects on VSMCs were at least partly mediated by the inhibition of PDGF-R? and the activities of downstream signaling pathways. The results suggest that DIM has the potential to be a candidate for the prevention of restenosis.

Project description:Key pointsSmooth muscle cell (SMC) phenotypic conversion from a contractile to a migratory phenotype is proposed to underlie cardiovascular disease but its contribution to vascular remodelling and even its existence have recently been questioned. Tracking the fate of individual SMCs is difficult as no specific markers of migratory SMCs exist. This study used a novel, prolonged time-lapse imaging approach to continuously track the behaviour of unambiguously identified, fully differentiated SMCs. In response to serum, highly-elongated, contractile SMCs initially rounded up, before spreading and migrating and these migratory cells displayed clear phagocytic activity. This study provides a direct demonstration of the transition of fully contractile SMCs to a non-contractile, migratory phenotype with phagocytic capacity that may act as a macrophage-like cell.AbstractAtherosclerotic plaques are populated with smooth muscle cells (SMCs) and macrophages. SMCs are thought to accumulate in plaques because fully differentiated, contractile SMCs reprogramme into a 'synthetic' migratory phenotype, so-called phenotypic modulation, whilst plaque macrophages are thought to derive from blood-borne myeloid cells. Recently, these views have been challenged, with reports that SMC phenotypic modulation may not occur during vascular remodelling and that plaque macrophages may not be of haematopoietic origin. Following the fate of SMCs is complicated by the lack of specific markers for the migratory phenotype and direct demonstrations of phenotypic modulation are lacking. Therefore, we employed long-term, high-resolution, time-lapse microscopy to track the fate of unambiguously identified, fully-differentiated, contractile SMCs in response to the growth factors present in serum. Phenotypic modulation was clearly observed. The highly elongated, contractile SMCs initially rounded up, for 1-3 days, before spreading outwards. Once spread, the SMCs became motile and displayed dynamic cell-cell communication behaviours. Significantly, they also displayed clear evidence of phagocytic activity. This macrophage-like behaviour was confirmed by their internalisation of 1 ?m fluorescent latex beads. However, migratory SMCs did not uptake acetylated low-density lipoprotein or express the classic macrophage marker CD68. These results directly demonstrate that SMCs may rapidly undergo phenotypic modulation and develop phagocytic capabilities. Resident SMCs may provide a potential source of macrophages in vascular remodelling.

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Characterization of Carotid Smooth Muscle Cells during Phenotypic Transition.

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Characterization of Carotid Smooth Muscle Cells during Phenotypic Transition.

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