Abstract 512: The Long Non-coding Rna MIAT Regulates Smooth Muscle Cell Proliferation and Macrophage Activity in Advanced Atherosclerotic Lesions

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Yuhuang Li ◽  
Hong Jin ◽  
Ljubica Perisic ◽  
Ekaterina Chernogubova ◽  
Alexandra Bäcklund ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Oxidized Ldl ◽  
In Silico Analysis ◽  
Human Monocyte ◽  
Carotid Plaques ◽  
Knock Down ◽  
Plaque Stability ◽  
Atherosclerotic Lesions ◽  
Markers Of Inflammation

Background: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators in various biological processes and diseases. Here we sought to identify and functionally characterize the lncRNA MIAT as a novel regulator in atherosclerotic plaque stability. Methods and results: We profiled RNA transcript expression in patients with advanced atherosclerotic lesions from the Biobank of Karolinska Endarterectomies (BiKE). By microarray analysis, lncRNA MIAT was identified as one of the most highly up-regulated non-coding RNAs in carotid plaques compared to iliac artery controls, which was confirmed by qRT-PCR and in situ hybridization. Additional in silico analysis indicated a substantial positive correlation of MIAT with markers of inflammation, apoptosis and matrix degradation in carotid plaques. Experimental knock-down of MIAT, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) not only markedly decreased proliferation and migration rates of cultured human carotid artery smooth muscle cells (hCASMCs), but also increased their levels of apoptosis. In addition, MIAT inhibition significantly impaired oxidized LDL (oxLDL) uptake of murine peritoneal as well as human monocyte-differentiated macrophages in vitro. In contrast, induction of MIAT expression by lipoprotein-a (LPa) treatment, displayed the opposite effect. Conditioned medium from macrophage cultures after MIAT knock-down substantially decreased hCASMC proliferation, indicating a potential involvement of MIAT in macrophage-SMC interactions during advanced stages of atherosclerosis. Conclusion: The lncRNA MIAT is a novel regulator of cellular processes in atherosclerosis and plaque stability, which influences SMC proliferation and apoptosis and interacts with disease-triggering macrophages.

Human tribbles homologue 2 is expressed in unstable regions of carotid plaques and regulates macrophage IL-10 in vitro

2009 ◽  
Vol 116 (3) ◽  
pp. 241-248 ◽  
Author(s):  
Jingti Deng ◽  
Christian H. James ◽  
Lisa Patel ◽  
Alberto Smith ◽  
Kevin G. Burnand ◽  
...  
Keyword(s):  
Oxidized Ldl ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Human Monocyte ◽  
Interleukin 10 ◽  
Plaque Instability ◽  
Signalling Molecules ◽  
Atherosclerotic Lesions ◽  
Protein Kinase Signalling

Mammalian orthologues of the Drosophila tribbles protein (Trb1, Trb2 and Trb3) are a recently described family of signalling molecules that regulate gene expression by modulation of protein kinase signalling pathways. In the present study, a screen for mRNA species specifically regulated in vulnerable regions of human atherosclerotic plaque demonstrated the up-regulation of both Trb1 and Trb2, the latter by more than 8-fold. In vitro experiments in primary human monocyte-derived macrophages showed that Trb2 expression was up-regulated by treatment with oxidized LDL (low-density lipoprotein), and that expression of recombinant Trb2 specifically reduced macrophage levels of IL-10 (interleukin-10) mRNA. Our results thus identify Trb2 as a highly regulated gene in vulnerable atherosclerotic lesions, and demonstrate inhibition of macrophage IL-10 biosynthesis as a potential pro-inflammatory consequence of high Trb2 expression, which may contribute to plaque instability.

Abstract 149: Pluripotency Factors Krüppel-Like Factor 4 and Octamer-binding Transcription Factor 4 Alter Indices of Plaque Stability in Long Term Western Diet Fed Mice by Regulating Smooth Muscle Cell Phenotypes

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Laura S Shankman ◽  
Olga A Cherepanova ◽  
Delphine Gomez ◽  
Gary K Owens
Keyword(s):  
Smooth Muscle ◽  
Foam Cell ◽  
Lineage Tracing ◽  
Phenotypic Switching ◽  
Marker Genes ◽  
Plaque Stability ◽  
Pluripotency Factors ◽  
Fibrous Cap ◽  
Atherosclerotic Lesions ◽  
Life Threatening

The bulk of life threatening thrombotic events have been associated with disruption of the fibrous cap, an atheroprotective layer of smooth muscle α-actin positive (ACTA2+) cells that form around the plaque, and the presence of a large foam cell-laden necrotic core within the plaque. Despite the overwhelming research demonstrating that ACTA2+ cells are beneficial for plaque stability, and cells positive for macrophage-markers are detrimental, there are major ambiguities regarding the origins of these cells, and their role in lesion stability. To clearly define the contribution of smooth muscle cells (SMCs) within atherosclerotic lesions, we generated SMC specific lineage tracing Apoe-/- mice containing a SM myosin heavy chain ( Myh11 ) tamoxifen-inducible cre-recombinase gene and a floxed STOP ROSA eYFP gene ( Myh11 YFP ApoE-/- mice) thus allowing activation of eYFP exclusively in fully differentiated SMCs before the onset of atherosclerosis and subsequent determination of the fate of these cells and their progeny irrespective of continued expression of MYH11 or other SMC marker genes. Remarkably, our results reveal that 86% of SMCs cannot be identified using traditional SMC markers, such as ACTA2, and 23% of presumed macrophages (LGALS3+ cells) are derived from SMC origins. The last finding was confirmed in human coronary atheromas using the ISH-PLA approach. SMC specific knockout (KO) of the pluripotency factor Klf4 in Myh11 YFP ApoE-/- mice did not alter the frequency of phenotypically modulated (ACTA2-eYFP+) SMCs within atherosclerotic lesions of mice fed a high fat diet for 18 weeks, however, decreased the number of ACTA2-eYFP+ SMCs that expressed LGALS3, and increased several indices of plaque stability, suggesting a detrimental role for KLF4 in SMCs within atherosclerotic lesions. Conversely, SMC specific Oct4 KO resulted in a dramatic reduction in the number of ACTA2-eYFP+ SMCs within the lesion with marked decreases in indices of plaque stability. In summary results show that the majority of SMC-derived cells within advanced atherosclerotic lesions cannot be identified using conventional SMC marker genes, and that phenotypic switching of SMC during atherogenesis is differentially regulated by the pluripotency factors KLF4 and OCT4.

scholarly journals Two microRNAs, miR-330 and miR-125b-5p, mark the juxtaglomerular cell and balance its smooth muscle phenotype

2012 ◽  
Vol 302 (1) ◽  
pp. F29-F37 ◽  
Author(s):  
Silvia Medrano ◽  
Maria C. Monteagudo ◽  
Maria Luisa S. Sequeira-Lopez ◽  
Ellen S. Pentz ◽  
R. Ariel Gomez
Keyword(s):  
Smooth Muscle ◽  
In Situ Hybridization ◽  
In Silico ◽  
Smooth Muscle Actin ◽  
In Silico Analysis ◽  
Screening Strategy ◽  
The Other ◽  
Kidney Cortex

We have shown that microRNAs (miRNAs) are necessary for renin cell specification and kidney vascular development. Here, we used a screening strategy involving microarray and in silico analyses, along with in situ hybridization and in vitro functional assays to identify miRNAs important for renin cell identity. Microarray studies using vascular smooth muscle cells (SMCs) of the renin lineage and kidney cortex under normal conditions and after reacquisition of the renin phenotype revealed that of 599 miRNAs, 192 were expressed in SMCs and 234 in kidney cortex. In silico analysis showed that the highly conserved miR-330 and miR-125b-5p have potential binding sites in smoothelin ( Smtn), calbindin 1, smooth muscle myosin heavy chain, α-smooth muscle actin, and renin genes important for the myoepithelioid phenotype of the renin cell. RT-PCR studies confirmed miR-330 and miR-125b-5p expression in kidney and SMCs. In situ hybridization revealed that under normal conditions, miR-125b-5p was expressed in arteriolar SMCs and in juxtaglomerular (JG) cells. Under conditions that induce reacquisition of the renin phenotype, miR-125b-5p was downregulated in arteriolar SMCs but remained expressed in JG cells. miR-330, normally absent, was expressed exclusively in JG cells of treated mice. In vitro functional studies showed that overexpression of miR-330 inhibited Smtn expression in SMCs. On the other hand, miR-125b-5p increased Smtn expression, whereas its inhibition reduced Smtn expression. Our results demonstrate that miR-330 and miR-125b-5p are markers of JG cells and have opposite effects on renin lineage cells: one inhibiting and the other favoring their smooth muscle phenotype.

scholarly journals RIPK1 Expression Associates with Inflammation in Early Atherosclerosis in Humans and Can be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice

Circulation ◽  
2020 ◽  
Author(s):  
Denuja Karunakaran ◽  
My-Anh Nguyen ◽  
Michele Geoffrion ◽  
Dianne Vreeken ◽  
Zachary Lister ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Activation ◽  
Inflammatory Responses ◽  
Early Stage ◽  
Oxidized Ldl ◽  
Loss Of Function ◽  
Aortic Sinus ◽  
Atherosclerotic Lesions

Background: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. Previously, we showed that macrophages in the atherogenic plaque undergo RIPK3-MLKL-dependent programmed necroptosis in response to sterile ligands such as oxidized LDL and damage-associated patterns (DAMPs) and necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1, which acts as a master switch that controls whether the cell undergoes NFκB-dependent inflammation, caspase-dependent apoptosis or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is largely driven by NFκB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NFκB-dependent inflammation in early atherogenic lesions and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. Methods: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and using loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 anti-sense oligonucleotides (ASO) to Apoe -/- mice fed a cholesterol-rich (Western) diet for 8 weeks. Results: We find RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 ASOs led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, p<0.01) and plasma inflammatory cytokines (IL-1α, IL-17A, p<0.05) compared to controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NFκB, TNFα, IL-1α) and in vivo LPS- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin and monocyte attachment. Conclusions: We have identified RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

Abstract 1079: Modulation of Atherosclerotic Plaque Characteristics by Minocycline: An Evaluation by Molecular Imaging of Matrix Metalloprotainase (MMP) Expression

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Satoru Ohshima ◽  
Shinichiro Fujimoto ◽  
Hironori Nakagami ◽  
Nezam Haider ◽  
Jun Zhou ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Molecular Imaging ◽  
Low Dose ◽  
High Cholesterol Diet ◽  
High Dose ◽  
Targeted Interventions ◽  
Mmp Inhibitor ◽  
Atherosclerotic Lesions

MMP activation in atherosclerotic lesions plays an important role in plaque progression and vulnerability, and is a potential target for plaque stabilization. We evaluated the effectiveness of minocycline (MC) in modulation of plaque characteristics using molecular imaging of MMP expression, compared with fluvastatin (FS). 38 NZW rabbits with experimental atherosclerotic lesions were subjected to in vivo micro SPECT/CT imaging for the assessment of MMP activity using 99mTc-labeled broad spectrum MMP inhibitor (MPI). Atherosclerotic lesions were produced by balloon deendotheliazation of abdominal aorta, and high cholesterol diet for 4 months. Of these, 7 received low dose MC (1.5mg/kg), 7 high dose MC (3mg/kg, N=7), 6 FS (1mg/kg), and 6 combination of low dose MC and FS (MC+FS) in the fourth month, 12 received no intervention (Rx-control). 8 unmanipulated normal chow-fed rabbits were used as disease controls (Dz-control). After in vivo imaging, aortas were explanted for %ID/g MPI uptake, histological characterization, and MMP activity assays. Parallel in vitro studies were performed for the effect of MC and FS on NF-κB, VCAM-1 and MMP-9 expression from cultured TNF-α-stimulated smooth muscle cells and macrophages. Target accumulation of MPI was best visualized in atherosclerotic aorta in Rx-control atherosclerotic rabbits. % ID/g MPI in Rx-control (.10±.04%) was significantly higher than Dz-control (.016±.004%), MC-low (.081±.02%) and high (.045±.01%) dose, FS (.056±.011%), and MC+FS (.049±.005%) rabbits, and showed a significant correlation with histologically and biochemically-verified MMP-2, -9 activity. MC-treated plaques showed marked reduction in inflammation and increase in smooth muscle content. In vitro cell culture confirmed that NF-κB and VCAM-1 expression in activated VSMC were reduced by MC, and that MMP-9 expression in activated macrophage was also inhibited by MC. The present study demonstrates that MC is equally effective as FS in modulation of plaque characteristics, and that molecular imaging can characterize various biologic processes in vivo and allows for the study of the efficacy of targeted interventions.

Abstract 17038: Twist1 Modulates Smooth Muscle Cell Phenotypes in Atherosclerosis

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Marie A Guerraty ◽  
Sylvia T Nurnberg ◽  
Vraj Shah ◽  
Daniel J Rader
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Risk Allele ◽  
Muscle Cells ◽  
Chromosome 9 ◽  
Alizarin Red ◽  
Plaque Stability ◽  
Atherosclerotic Lesions ◽  
Mesenchymal Transformation

Introduction: Genome-wide association studies have identified rs2107595, a non-coding locus on chromosome 9 between HDAC9 and Twist1 genes, as a risk allele for several vascular phenotypes, including Coronary Artery Disease (CAD). Rs2107595 has, more specifically, been associated with stable CAD over myocardial infarction phenotypes. Recent work has shown that rs2107595 risk allele increases Twist1 expression in smooth muscle cells (SMCs) by creating an RBPJ binding site. In other cell types, Twist1 is known to maintain cells in a de-differentiated state and to promote epithelial to mesenchymal transformation, driving tumor progression and metastasis. Hypothesis: Twist1 modulates SMC differentiation to promote an immature proliferative state over a differentiated (osteoblastic) state. This shift in phenotype promotes features of plaque stability in vivo . Methods: Twist1 expression plasmid (pCMV6-TWIST1) was transfected into A7r5 rat smooth muscle cells. To assess proliferation, cells were counted at 24, 48, 72, and 96 hours. To assess calcification, A7r5 cells were cultured in calcification media (2mM NaPhos) for 10 days and stained with Alizarin Red. In vivo studies were performed in Twist1 fl/fl tamoxifen-inducible MYH11-Cre C57BL/6 mice on ApoE-/- background fed a Western diet for 16 weeks to induce atherosclerotic lesions. Immunohistochemistry with SM22a identified lesion SMCs, and alizarin red was used to identify calcifications. Results: Ectopic overexpression of Twist1 in A7r5 SMCs decreased proliferation at 48h and 72h (80%, p=0.014). Twist 1 overexpression also decreased the total area of calcification (33% reduction, p=0.007). In vivo , both control and Twist 1 KO mice show similar burden of atherosclerosis. However, there is a decrease in sub-endothelial SMCs in atherosclerotic lesions by SM22a staining in the Twist1 KO. Additionally, Twist1 KO mice have more prominent and larger focal calcifications. Conclusions: Twist1 promotes SMC proliferation and decreases calcification in vitro , and may affect the presence of subendothelial SMCs and calcification in vivo . This provides a compelling link that rs2107595 may promote plaque stability in CAD by increasing Twist1 to modulate SMC phenotypes.

KNOCK DOWN PDE5 GENE OF HUMAN CORPUS CAVERNOSUM SMOOTH MUSCLE CELLS BY SIRNA IN VITRO

2009 ◽  
Vol 181 (4S) ◽  
pp. 302-302
Author(s):  
Jihong Liu ◽  
Ying Zhan ◽  
Tao Wang ◽  
Shaogang Wang ◽  
Zhang-Qun Ye
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Corpus Cavernosum ◽  
Muscle Cells ◽  
Knock Down
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