Unstable Carotid Atherosclerosis Research Articles

MiR-223 Exerts Translational Control of Proatherogenic Genes in Macrophages.

Background:A significant burden of atherosclerotic disease is driven by inflammation. Recently, microRNAs (miRNAs) have emerged as important factors driving and protecting from atherosclerosis. miR-223 regulates cholesterol metabolism and inflammation via targeting both cholesterol biosynthesis pathway and NFkB signaling pathways; however, its role in atherosclerosis has not been investigated. We hypothesize that miR-223 globally regulates core inflammatory pathways in macrophages in response to inflammatory and atherogenic stimuli thus limiting the progression of atherosclerosis.Methods and Results:Loss of miR-223 in macrophages decreases Abca1 gene and protein expression as well as cholesterol efflux to apoA1 (Apolipoprotein A1) and enhances proinflammatory gene expression. In contrast, overexpression of miR-223 promotes the efflux of cholesterol and macrophage polarization toward an anti-inflammatory phenotype. These beneficial effects of miR-223 are dependent on its target gene, the transcription factor Sp3. Consistent with the antiatherogenic effects of miR-223 in vitro, mice receiving miR223−/− bone marrow exhibit increased plaque size, lipid content, and circulating inflammatory cytokines (ie, IL-1β). Deficiency of miR-223 in bone marrow–derived cells also results in an increase in circulating pro-atherogenic cells (total monocytes and neutrophils) compared with control mice. Furthermore, the expression of miR-223 target gene (Sp3) and pro-inflammatory marker (Il-6) are enhanced whereas the expression of Abca1 and anti-inflammatory marker (Retnla) are reduced in aortic arches from mice lacking miR-223 in bone marrow–derived cells. In mice fed a high-cholesterol diet and in humans with unstable carotid atherosclerosis, the expression of miR-223 is increased. To further understand the molecular mechanisms underlying the effect of miR-223 on atherosclerosis in vivo, we characterized global RNA translation profile of macrophages isolated from mice receiving wild-type or miR223−/− bone marrow. Using ribosome profiling, we reveal a notable upregulation of inflammatory signaling and lipid metabolism at the translation level but less significant at the transcription level. Analysis of upregulated genes at the translation level reveal an enrichment of miR-223-binding sites, confirming that miR-223 exerts significant changes in target genes in atherogenic macrophages via altering their translation.Conclusions:Our study demonstrates that miR-223 can protect against atherosclerosis by acting as a global regulator of RNA translation of cholesterol efflux and inflammation pathways.

Carotid Plaque Phenotyping by Correlating Plaque Morphology from Computed Tomography Angiography with Transcriptional Profiling

<h3>Objective</h3> Ischaemic strokes can be caused by unstable carotid atherosclerosis, but methods for identification of high risk lesions are lacking. Carotid plaque morphology imaging using software for visualisation of plaque components in computed tomography angiography (CTA) may improve assessment of plaque phenotype and stroke risk, but it is unknown if such analyses also reflect the biological processes related to lesion stability. Here, we investigated how carotid plaque morphology by image analysis of CTA is associated with biological processes assessed by transcriptomic analyses of corresponding carotid endarterectomies (CEAs). <h3>Methods</h3> Carotid plaque morphology was assessed in patients undergoing CEA for symptomatic or asymptomatic carotid stenosis consecutively enrolled between 2006 and 2015. Computer based analyses of pre-operative CTA was performed to define calcification, lipid rich necrotic core (LRNC), intraplaque haemorrhage (IPH), matrix (MATX), and plaque burden. Plaque morphology was correlated with molecular profiles obtained from microarrays of corresponding CEAs and models were built to assess the ability of plaque morphology to predict symptomatology. <h3>Results</h3> Carotid plaques (<i>n</i> = 93) from symptomatic patients (<i>n</i> = 61) had significantly higher plaque burden and LRNC compared with plaques from asymptomatic patients (<i>n</i> = 32). Lesions selected from the transcriptomic cohort (<i>n</i> = 40) with high LRNC, IPH, MATX, or plaque burden were characterised by molecular signatures coupled with inflammation and extracellular matrix degradation, typically linked with instability. In contrast, highly calcified plaques had a molecular signature signifying stability with enrichment of profibrotic pathways and repressed inflammation. In a cross validated prediction model for symptoms, plaque morphology by CTA alone was superior to the degree of stenosis. <h3>Conclusion</h3> The study demonstrates that CTA image analysis for evaluation of carotid plaque morphology, also reflects prevalent biological processes relevant for assessment of plaque phenotype. The results support the use of CTA image analysis of plaque morphology for risk stratification and management of patients with carotid stenosis.

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Novel Multiomics Profiling of Human Carotid Atherosclerotic Plaques and Plasma Reveals Biliverdin Reductase B as a Marker of Intraplaque Hemorrhage

Clinical tools to identify individuals with unstable atherosclerotic lesions are required to improve prevention of myocardial infarction and ischemic stroke. Here, a systems-based analysis of atherosclerotic plaques and plasma from patients undergoing carotid endarterectomy for stroke prevention was used to identify molecular signatures with a causal relationship to disease. Local plasma collected in the lesion proximity following clamping prior to arteriotomy was profiled together with matched peripheral plasma. This translational workflow identified biliverdin reductase B as a novel marker of intraplaque hemorrhage and unstable carotid atherosclerosis, which should be investigated as a potential predictive biomarker for cardiovascular events in larger cohorts.

The Urotensin II System and Carotid Atherosclerosis: A Role in Vascular Calcification

Background and Aims: The aims of the present study were to determine the expression of urotensin II (UII), urotensin-II related peptide (URP), and their receptor (UT) in stable and unstable carotid atherosclerosis, and determine the effects of UII on human aortic smooth muscle cell (SMCs) calcification.Methods and Results: We examined UII, URP, and UT protein expression in 88 carotid endarterectomy specimens using immunohistochemistry. Expression of UII, URP, and UT was more evident in unstable compared to stable plaques (P < 0.05). Multivariate Spearman correlation analyses revealed significant positive correlations between UII, URP and UT overall staining and presence of calcification, severity of stenosis and inflammation (P < 0.05). Subjects undergoing carotid endarterectomy had significantly higher plasma UII levels, as assessed by ELISA, when compared with normolipidemic healthy control subjects (P < 0.05). Incubation of human aortic SMCs cultured in phosphate media with varying concentrations of UII resulted in a significant increase in calcium deposition and alkaline phosphatase activity. UII also significantly increased β-catenin translocation and expression of ALPL, BMP2, ON, and SOX9 (P < 0.05). Incubation of cells with phosphate medium alone increased the expression of the pre-UT and mature UT (P < 0.01), and addition of UII had a synergistic effect on pre-UT protein expression (P < 0.001) compared to phosphate medium alone.Conclusions: Upregulation of UII, URP, and UT in unstable carotid endarterectomy plaques and plasma, and the stimulatory effect of UII on vascular smooth muscle cell calcification suggest that the UII system may play a role in the pathogenesis of vascular calcification and stability of atherosclerosis.

Abstract 3: Targeting Macrophage Necroptosis for Therapeutic and Diagnostic Interventions to Treat Atherosclerosis

Background: Atherosclerosis results from maladaptive inflammation driven primarily by macrophages, whose recruitment and proliferation drive plaque progression. In advanced plaques, macrophage death contributes centrally to the formation of plaque necrosis, which underlies the instability that promotes plaque rupture and myocardial infarction. As such, targeting macrophage cell death pathways may offer promise for the stabilization of vulnerable plaques. Necroptosis is a recently discovered pathway of programmed cell necrosis regulated by RIP3 and MLKL kinases that in contrast to apoptosis, induces a pro-inflammatory state. We hypothesize that atherogenic ligands within the plaque promote macrophage necroptosis and this process underlies necrotic core formation and drives atherosclerotic plaque instability. Results: In humans with unstable carotid atherosclerosis, expression of RIP3 and MLKL is increased and MLKL phosphorylation, a key step in the commitment to necroptosis, is detected in advanced atheromas. Investigation of the molecular mechanisms underlying plaque necroptosis showed that macrophages treated with oxidized LDL have increased expression of necroptotic genes RIP3 and MLKL through ROS-dependent activation of the promoter region and increased RIP3 and MLKL phosphorylation. Combined treatment with oxLDL and DAMPs (damage associated molecular patterns) amplified macrophage necroptotic cell death, indicating that additional inflammatory stimuli present in the lesion could act synergistically to promote necroptosis. Using a radiotracer developed with the necroptosis inhibitor Nec-1, we show that 123 I-Nec1 localizes specifically to atherosclerotic plaques in Apoe-/- mice, and its uptake is tightly correlated to lesion areas by ex vivo nuclear imaging. Furthermore, treatment of Apoe-/- mice with established atherosclerosis with Nec-1 reduced lesion size and markers of plaque instability, including necrotic core formation. Conclusions: Our findings offer molecular insight into the mechanisms of macrophage cell death that drive necrotic core formation in atherosclerosis and suggest that this pathway can be used as both a diagnostic and therapeutic tool for the treatment of unstable atherosclerosis.

Abstract 26: Who is Going to Stroke? Biomarkers for the Unstable Carotid Atheroma

Unstable carotid stenosis is an important source of extracranial atheroembolism and stroke. Current interventional therapy is based on surrogate markers for stroke risk and no precise diagnostic tool is available for identification of either individuals or lesions at risk. Thus, biomarkers for the unstable carotid atheroma may improve selection of patient for intervention. Here, a high-throughput pipeline for identification of circulating biomarkers for unstable carotid atherosclerosis was developed. Carotid plaques were part of the Biobank for Karolinska Endarterectomies (BiKE) and obtained from patients treated for symptomatic (S) and asymptomatic (AS) carotid stenosis together with peripheral plasma and ‘local’ plasma (n = 47) sampled after clamping prior to arteriotomy. Screening of proteins was processed through Luminex based, suspension-bead assays using over 10,000 antibodies from the Human Protein Resource (HPR) and compared to peripheral plasma (n = 200 total). Plasma analysis identified 150 significantly dysregulated candidates based on three comparisons: proteins enriched in local plasma compared to peripheral plasma; in local plasma from S compared with AS patients; in peripheral plasma from S compared with AS patients. Pathway analysis revealed that detected proteins were related to ossification, lysosomes, magnesium transport, prenylation, collagens, lipid metabolism, and transcription factors. Identified candidate proteins were further filtered using public database mining, comparisons with transcriptomic data from BiKE, and qPCR/IHC in matched endarterectomies. Among the identified candidates, THEMIS2 and BLVRB were examples of proteins successfully validated as upregulated in both plaques and plasma from S patients and localized to CD68+ macrophages. Extensive analysis of plasma and tissue samples from patients undergoing carotid surgery permitted identification of several potential biomarkers for unstable carotid atherosclerosis. Further validation in larger cohorts of unstable atherosclerosis is necessary in order to determine predictive power for the identification of individuals at risk as well as for the development of targeted bioimaging for lesion detection.

Profiling of Atherosclerotic Lesions by Gene and Tissue Microarrays Reveals PCSK6 as a Novel Protease in Unstable Carotid Atherosclerosis

Carotid plaque instability is a major cause of ischemic stroke, but detailed knowledge about underlying molecular pathways is still lacking. Here, we evaluated large-scale transcriptomic and protein expression profiling in a biobank of carotid endarterectomies followed by characterization of identified candidates, as a platform for discovery of novel proteins differentially regulated in unstable carotid lesions. Genes highly upregulated in symptomatic versus asymptomatic plaques were selected from Affymetrix microarray analyses (n=127 plaques), and tissue microarrays constructed from 34 lesions were assayed for 21 corresponding proteins by immunohistochemistry. Quantification of stainings demonstrated differential expression of CD36, CD137, and DOCK7 (P<0.05) in unstable versus stable lesions and the most significant upregulation of a proprotein convertase, PCSK6 (P<0.0001). Increased expression of PCSK6 in symptomatic lesions was verified by quantitative real-time polymerase chain reaction (n=233), and the protein was localized to smooth muscle α-actin positive cells and extracellular matrix of the fibrous cap by immunohistochemistry. PCSK6 expression positively correlated to genes associated with inflammation, matrix degradation, and mitogens in microarrays. Stimulation of human carotid smooth muscle cells in vitro with cytokines caused rapid induction of PCSK6 mRNA. Using a combination of transcriptomic and tissue microarray profiling, we demonstrate a novel approach to identify proteins differentially expressed in unstable carotid atherosclerosis. The proprotein convertase PCSK6 was detected at increased levels in the fibrous cap of symptomatic carotid plaques, possibly associated with key processes in plaque rupture such as inflammation and extracellular matrix remodeling. Further studies are needed to clarify the role of PCSK6 in atherosclerosis.

Intra-plaque production of M1-type cytokines and matrix metalloproteinases differentiate stable from unstable carotid atherosclerosis

Intra-plaque production of M1-type cytokines and matrix metalloproteinases differentiate stable from unstable carotid atherosclerosis