Human Carotid Plaques Research Articles

Arachidonate 15-Lipoxygenase Enzyme Products Increase Platelet Aggregation and Thrombin Generation

Atherosclerotic cardiovascular diseases are the leading causes of morbidity and mortality worldwide. We have previously shown that arachidonate 15-lipoxygenase B (ALOX15B) is highly expressed in atherosclerotic carotid plaques, and elucidation of mechanisms downstream of activated lipoxygenases may be relevant to our understanding of the genesis of atherosclerotic diseases. We examined 120 carotid plaques from patients with symptomatic carotid artery stenosis and showed that the extent of ALOX15B staining was significantly increased in carotid plaques with thrombosis. Impedance aggregometry analyses showed that the ALOX15B enzyme products 15-HETE and 15-HPETE increased platelet aggregation. By using a calibrated automatic thrombin assay, we showed that the ALOX15B products also increased both peak levels of thrombin and the total endogenous thrombin potential. Moreover, platelet aggregation was increased by addition of cell lysates from ischemic human macrophages, whereas platelet aggregation was reduced after knockdown of ALOX15B in human macrophages. Our data show that ALOX15B expression in human carotid plaques is associated with thrombus formation and that enzyme products of ALOX15B increase platelet aggregation and thrombin generation. We therefore propose that activated ALOX15B in macrophages may play a role in the induction of atherothrombotic events by increasing platelet aggregation and thrombin generation.

MMP-1 and MMP-9 regulate epidermal growth factor-dependent collagen loss in human carotid plaque smooth muscle cells.

Mechanisms underlying the rupture of atherosclerotic plaque, a crucial factor in the development of myocardial infarction and stroke, are not well defined. Here, we examined the role of epidermal growth factor (EGF)‐mediated matrix metalloproteinases (MMP) on the stability of interstitial collagens in vascular smooth muscle cells (VSMCs) isolated from carotid endarterectomy tissues of symptomatic and asymptomatic patients with carotid stenosis. VSMCs isolated from the carotid plaques of both asymptomatic and symptomatic patients were treated with EGF. The MMP‐9 activity was quantified by gelatin zymography and the analysis of mRNA transcripts and protein for MMP‐9, MMP‐1, EGFR and collagen types I, Col I(α1) and collagen type III, Col III(α1) were analyzed by qPCR and immunofluorescence, respectively. The effect of EGF treatment to increase MMP‐9 activity and mRNA transcripts for MMP‐9, MMP‐1, and EGFR and to decrease mRNA transcripts for Col I(α1) and Col III(α1) was threefold to fourfold greater in VSMCs isolated from the carotid plaques of symptomatic than asymptomatic patients. Inhibitors of EGFR (AG1478) and a small molecule inhibitor of MMP‐9 decreased the MMP9 expression and upregulated Col I(α1) and Col III(α1) in EGF‐treated VSMCs of both groups. Additionally, the magnitude in decreased MMP‐9 mRNA and increased Col I(α1) and Col III(α1) due to knockdown of MMP‐9 gene with siRNA in EGF‐treated VSMCs was significantly greater in the symptomatic group than the asymptomatic group. Thus, a selective blockade of both EGFR and MMP‐9 may be a novel strategy and a promising target for stabilizing vulnerable plaques in patients with carotid stenosis.

Local carotid atherosclerotic plaque proteins for the identification of circulating biomarkers in coronary patients

ObjectiveTo identify circulating biomarkers that originate from atherosclerotic vulnerable plaques and that could predict future cardiovascular events. MethodsAfter a protein enrichment step (combinatorial peptide ligand library approach), we performed a two-dimensional electrophoresis comparative analysis on human carotid plaque protein extracts (fibrotic and hemorrhagic atherosclerotic plaques). In silico analysis of the biological processes was applied on proteomic data. Luminex xMAP assays were used to quantify inflammatory components in carotid plaques. The systemic quantification of proteins originating from vulnerable plaques in blood samples from patients with stable and unstable coronary disease was evaluated. ResultsA total of 118 proteins are differentially expressed in fibrotic and hemorrhagic plaques, and allowed the identification of three biological processes related to atherosclerosis (platelet degranulation, vascular autophagy and negative regulation of fibrinolysis). The multiplex assays revealed an increasing expression of VEGF, IL-6, IL-8, IP-10 and RANTES in hemorrhagic as compared to fibrotic plaques (p < 0.05). Measurement of protein expressions in plasmas from patients with stable and unstable coronary disease identified a combination of biomarkers, including proteins of the smooth muscle cell integrity (Calponin-1), oxidative stress (DJ-1) and inflammation (IL-8), that allows the accurate classification of patients at risk (p = 0.0006). ConclusionUsing tissue protein enrichment technology, we validated proteins that are differentially expressed in hemorrhagic plaques as potential circulating biomarkers of coronary patients. Combinations of such circulating biomarkers could be used to stratify coronary patients.

Image-based modeling for better understanding and assessment of atherosclerotic plaque progression and vulnerability: Data, modeling, validation, uncertainty and predictions

Medical imaging and image-based modeling have made considerable progress in recent years in identifying atherosclerotic plaque morphological and mechanical risk factors which may be used in developing improved patient screening strategies. However, a clear understanding is needed about what we have achieved and what is really needed to translate research to actual clinical practices and bring benefits to public health. Lack of in vivo data and clinical events to serve as gold standard to validate model predictions is a severe limitation. While this perspective paper provides a review of the key steps and findings of our group in image-based models for human carotid and coronary plaques and a limited review of related work by other groups, we also focus on grand challenges and uncertainties facing the researchers in the field to develop more accurate and predictive patient screening tools.

The rhythmic expression of clock genes attenuated in human plaque-derived vascular smooth muscle cells

BackgroundAcute myocardial infarction and stroke are more likely to occur in the early morning. Circadian pacemakers are considered to be involved in the process. Many peripheral tissues and cells also contain clock systems. In this study, we examined whether the primary cultured human plaque-derived vascular smooth muscle cells (VSMCs) process circadian rhythmicity; furthermore, we investigated the expression difference of clock genes between normal human carotid VSMCs and human plaque-derived VSMCs.MethodsFifty-six human carotid plaques provided the atherosclerotic tissue, and 21 samples yielded viable cultured primary VSMCs. The normal carotid VSMCs were cultured from donors’ normal carotids. The mRNA levels of the target genes were measured by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).ResultsAfter serum shock, both types of cells showed clear circadian expressions of Bmal1, Cry1, Cry2, Per1, Per2, Per3 and Rev-erbα mRNA; meanwhile the Clock mRNA show a rhythmic expression in plaque-derived SMCs but not in normal carotid VSMCs. The expression levels of these main clock genes were significantly attenuated in human plaque-derived VSMCs compared with normal human carotid VSMCs. The rhythm of Bmal1 mRNA in plaque-derived VSMCs was changed.ConclusionThe present results demonstrate that the human plaque-derived VSMCs possess different circadian rhythmicity from that of normal carotid VSMCs. The rhythm changes of clock genes in plaque-derived VSMCs may be involved in the process of atherosclerosis and finally promote the rupture of plaque.

PET/CT Imaging of Integrin αvβ3 Expression in Human Carotid Atherosclerosis

The goal of this study was to evaluate the feasibility of [(18)F]Galacto-RGD positron emission tomography (PET)/computed tomography (CT) imaging of αvβ3 expression in human carotid plaques. The integrin αvβ3 is expressed by macrophages and angiogenic endothelial cells in atherosclerotic lesions and thus is a marker of plaque inflammation and, potentially, of plaque vulnerability. [(18)F]Galacto-RGD is a PET tracer binding specifically to αvβ3. Therefore, [(18)F]Galacto-RGD PET/CT imaging of αvβ3 expression in human carotid plaques might provide a novel noninvasive biomarker of plaque vulnerability. [(18)F]Galacto-RGD PET/CT imaging was performed in 10 patients with high-grade carotid artery stenosis scheduled for carotid endarterectomy. Tracer uptake was measured in the stenotic areas of the carotid arteries, as well as on the contralateral side, and was corrected for blood pool activity, measured in the distal common carotid artery (target-to-background [TB] ratio). TB ratio was correlated with immunohistochemistry of αvβ3 expression (LM609), macrophage density (CD68), and microvessel density (CD31) of the surgical specimen. In addition, ex vivo autoradiography of the surgical specimen with [(18)F]Galacto-RGD and competition experiments with an unlabeled αvβ3-specific RGD peptide were performed. [(18)F]Galacto-RGD PET/CT showed significantly higher TB ratios in stenotic areas compared with nonstenotic areas (p = 0.01). TB ratios correlated significantly with αvβ3 expression (R = 0.787, p = 0.026) and intensity of ex vivo autoradiography (R = 0.733, p = 0.038). Binding to atherosclerotic plaques was efficiently blocked in ex vivo competition experiments. A weak-to-moderate correlation was found with macrophage density (R = 0.367, p = 0.299) and microvessel density (R = 0.479, p = 0.176), which did not reach statistical significance. [(18)F]Galacto-RGD PET/CT shows specific tracer accumulation in human atherosclerotic carotid plaques, which correlates with αvβ3 expression. Based on these initial data, larger prospective studies are now warranted to evaluate the potential of molecular imaging of αvβ3 expression for assessment of plaque inflammation in patients.

Treatment with Angiotensin-(1–7) reduces inflammation in carotid atherosclerotic plaques

Angiotensin (Ang)-(1-7), acting through the receptor Mas, has atheroprotective effects; however, its role on plaque vulnerability has been poorly studied. Here, we investigated the expression of the renin-angiotensin system (RAS) components in stable and unstable human carotid plaques. In addition, we evaluated the effects of the chronic treatment with an oral formulation of Ang-(1-7) in a mouse model of shear stress-determined carotid atherosclerotic plaque. Upstream and downstream regions of internal carotid plaques were obtained from a recently published cohort of patients asymptomatic or symptomatic for ischaemic stroke. Angiotensinogen and renin genes were strongly expressed in the entire cohort, indicating an intense intraplaque modulation of the RAS. Intraplaque expression of the Mas receptor mRNA was increased in the downstream portion of asymptomatic patients as compared to corresponding region in symptomatic patients. Conversely, AT1 receptor gene expression was not modified between asymptomatic and symptomatic patients. Treatment with Ang-(1-7) in ApoE-/- mice was associated with increased intraplaque collagen content in the aortic root and low shear stress-induced carotid plaques, and a decreased MMP-9 content and neutrophil and macrophage infiltration. These beneficial effects were not observed in the oscillatory shear stress-induced plaque. In vitro incubation with Ang-(1-7) did not affect ICAM-1 expression and apoptosis on cultured endothelial cells. In conclusion, Mas receptor is up regulated in the downstream portions of human stable carotid plaques as compared to unstable lesions. Treatment with the oral formulation of Ang-(1-7) enhances a more stable phenotype in atherosclerotic plaques, depending on the local pattern of shear stress forces.

Matrix metalloproteinase-2 of human carotid atherosclerotic plaques promotes platelet activation

Purified active matrix metalloproteinase-2 (MMP-2) is able to promote platelet aggregation. We aimed to assess the role of MMP-2 expressed in atherosclerotic plaques in the platelet-activating potential of human carotid plaques and its correlation with ischaemic events. Carotid plaques from 81 patients undergoing endarterectomy were tested for pro-MMP-2 and TIMP-2 content by zymography and ELISA. Plaque extracts were incubated with gel-filtered platelets from healthy volunteers for 2 minutes before the addition of a subthreshold concentration of thrombin receptor activating peptide-6 (TRAP-6) and aggregation was assessed. Moreover, platelet deposition on plaque extracts immobilised on plastic coverslips under high shear-rate flow conditions was measured. Forty-three plaque extracts (53%) potentiated platelet aggregation (+233 ± 26.8%), an effect prevented by three different specific MMP-2 inhibitors (inhibitor II, TIMP-2, moAb anti-MMP-2). The pro-MMP-2/TIMP-2 ratio of plaques potentiating platelet aggregation was significantly higher than that of plaques not potentiating it (3.67 ± 1.21 vs 1.01 ± 0.43, p<0.05). Moreover, the platelet aggregation-potentiating effect, the active-MMP-2 content and the active MMP-2/pro-MMP-2 ratio of plaque extracts were significantly higher in plaques from patients who developed a subsequent major cardiovascular event. In conclusion, atherosclerotic plaques exert a prothrombotic effect by potentiating platelet activation due to their content of MMP-2; an elevated MMP-2 activity in plaques is associated with a higher rate of subsequent ischaemic cerebrovascular events.

Abstract 18028: [18F]-fluorodeoxyglucose is a Stronger Correlate of Intraplaque Inflammatory Burden in Human Carotid Plaque Than C-Reactive Protein: A Sub-Study of the Canadian Atherosclerosis Imaging Network (CAIN)

Background and Purpose: Inflammation triggers and contributes to the progression of atherosclerosis. C-reactive protein (CRP), a circulating marker of inflammation, is a putative risk factor for cardiovascular disease, but this is not borne out in Mendelian randomization studies. Inflammation in carotid arteries can be imaged with hybrid [18F]-fluorodeoxyglucose (18FDG) positron emission tomography (PET) computed tomography (CT) imaging. In this investigation, circulating levels of CRP and 18FDG uptake in carotid vasculature were directly compared with intraplaque inflammatory burden, using macrophage-specific CD68 immunohistology. Methods: Nineteen prospectively recruited patients (66 ± 11 years, 15 male) scheduled for carotid endarterectomy underwent 18FDG-PET and CT angiography of carotids. CRP levels were assessed. Maximum 18FDG uptake in both internal carotids was quantified and normalized to blood (tissue:blood ratio, TBR). Following endarterectomy, excised plaque was fixed, sectioned and immunostained for CD68. CD68 expression was quantified. Results: Carotid endarterectomy was performed in 19 patients; one received a 2 nd carotid endarterectomy due to bilateral disease. The direct burden of inflammation, as quantified by CD68 immunohistology, correlated with maximum 18FDG uptake (r=0.716, p&lt;0.001). There was no evidence of a correlation between CRP and CD68 (r=0.159, p=0.50), nor with maximum 18FDG uptake (r=0.238, p=0.31). Furthermore, 18FDG and CRP are different correlations where FDG is more strongly correlated with CD68 than CRP (p=0.02, Hoteling Williams test). Conclusion: 18FDG uptake is more strongly related to the extent of inflammatory burden within high-risk carotid plaque than CRP. CRP may not be an accurate criterion for assessing vulnerable carotid plaque (based on histopathology nor 18FDG uptake). Prospective outcomes-based trials are needed to establish 18FDG as a direct biomarker of vulnerable carotid plaque.

Phage Display Identification of CD100 in Human Atherosclerotic Plaque Macrophages and Foam Cells

Atherosclerosis is a complex disease in which vessels develop plaques comprising dysfunctional endothelium, monocyte derived lipid laden foam cells and activated lymphocytes. Considering that humans and animal models of the disease develop quite distinct plaques, we used human plaques to search for proteins that could be used as markers of human atheromas. Phage display peptide libraries were probed to fresh human carotid plaques, and a bound phage homologous to plexin B1, a high affinity receptor for CD100, was identified. CD100 is a member of the semaphorin family expressed by most hematopoietic cells and particularly by activated T cells. CD100 expression was analyzed in human plaques and normal samples. CD100 mRNA and protein were analyzed in cultured monocytes, macrophages and foam cells. The effects of CD100 in oxLDL-induced foam cell formation and in CD36 mRNA abundance were evaluated. Human atherosclerotic plaques showed strong labeling of CD100/SEMA4D. CD100 expression was further demonstrated in peripheral blood monocytes and in in vitro differentiated macrophages and foam cells, with diminished CD100 transcript along the differentiation of these cells. Incubation of macrophages with CD100 led to a reduction in oxLDL-induced foam cell formation probably through a decrease of CD36 expression, suggesting for the first time an atheroprotective role for CD100 in the human disease. Given its differential expression in the numerous foam cells and macrophages of the plaques and its capacity to decrease oxLDL engulfment by macrophages we propose that CD100 may have a role in atherosclerotic plaque development, and may possibly be employed in targeted treatments of these atheromas.

Nanolipoblockers: Ex Vivo Human Plaque Interaction for Therapeutic Management for Atherosclerosis

Atherosclerosis (AthSc) is escalated by the excessive accumulation of oxidized low density lipoprotein (OxLDL). A new class of nanopolymers, termed nanolipoblockers (NLB), has previously demonstrated inhibition of OxLDL uptake by macrophages. Using an ex vivo human carotid plaque model for targeted intervention with NLB offers potential new mechanisms of AthSc treatment. Carotid plaques were sectioned and incubated in culture medium at 37°C. Samples were treated with the NLBs of different polymer chemistries and 5 μg/mL oxLDL to evaluate NLB binding to plaque and to assess inhibition of plaque recruitment of OxLDL and effect on inflammation. Samples were digested, homogenized, filtered, and fluorescence quantified by flow cytometry and confocal microscopy to identify plaque composition, NLB, and oxLDL uptake. Plaque cells were identified in the specimen showing high levels of NLB binding, merely within 5 minutes, with an increase in uptake in a chemistry-dependent manner after 24 hours (Fig 1). NLBs lowered inflammatory markers in the presence of oxLDL. Furthermore, NLBs caused a marked decrease in the uptake of OxLDL in plaque (Fig 2). AthSc plaques could be targeted through NLB intervention via a reduction in inflammatory substrates and multicellular OxLDL uptake inhibition. This ex vivo model and the NLBs provide a promising therapeutic strategy for the management of active AthSc.

The galectin-3/RAGE dyad modulates vascular osteogenesis in atherosclerosis

Vascular calcification correlates with inflammation and plaque instability in a dual manner, depending on the spotty/granular (micro) or sheet-like/lamellated (macro) pattern of calcification. Modified lipoproteins trigger both inflammation and calcification via receptors for advanced lipoxidation/glycation endproducts (ALEs/AGEs). This study compared the roles of galectin-3 and receptor for AGEs (RAGE), two ALEs/AGEs-receptors with diverging effects on inflammation and bone metabolism, in the process of vascular calcification. We evaluated galectin-3 and RAGE expression/localization in 62 human carotid plaques and its relation to calcification pattern, plaque phenotype, and markers of inflammation and vascular osteogenesis; and the effect of galectin-3 ablation and/or exposure to an ALE/AGE on vascular smooth muscle cell (VSMC) osteogenic differentiation. While RAGE co-localized with inflammatory cells in unstable regions with microcalcification, galectin-3 was expressed also by VSMCs, especially in macrocalcified areas, where it co-localized with alkaline phosphatase. Expression of galectin-3 and osteogenic markers was higher in macrocalcified plaques, whereas the opposite occurred for RAGE and inflammatory markers. Galectin-3-deficient VSMCs exhibited defective osteogenic differentiation, as shown by altered expression of osteogenic transcription factors and proteins, blunted activation of pro-osteoblastogenic Wnt/β-catenin signalling and proliferation, enhanced apoptosis, and disorganized mineralization. These abnormalities were associated with RAGE up-regulation, but were only in part prevented by RAGE silencing, and were partially mimicked or exacerbated by treatment with an AGE/ALE. These data indicate a novel molecular mechanism by which galectin-3 and RAGE modulate in divergent ways, not only inflammation, but also vascular osteogenesis, by modulating Wnt/β-catenin signalling, and independently of ALEs/AGEs.

RGS5 is decreased in unstable human carotid plaque, and plays a role in the vascular smooth muscle cell/endothelial cell recruitment in vitro

Purpose: Plaque stability is governed by it's collagen content and VSMC phenotype1. RGS5, a GPCR modulator, has been shown to be associated with plaque stability3. RGS5 may moderate VSMC/endothelial cell interactions4. We examined if RGS5 expression is associated with plaque stability and detailed its role in VSMC/HUVEC behavior in vitro. Methods: With IRB permission, samples from 91 patients undergoing carotid thrombendarterectomy were collected and RNA was extracted. Target-PCRs for RGS5 were normalized for PCR efficiency and 3 housekeeping genes (GAPDH, ATPsynthase, Tubulin) using qBase software. RGS5 was downregulated in human VSMC or HUVEC by siRNA. After 24h, cells were incubated with mytomicin-C for 1 hour, and the confluent monolayer was wounded. Wound closure percentage after 20h, of scrambled vs. RGS5 siRNA transfected VSMCs was measured (ImageJ). HUVECs conditioned medium was used to perform VSMC scratch assays. RGS5 expression in resident vs. migrated VSMCs was assessed by collecting the cells that had migrated off a confluent coverslip. RNA was extracted for qRT-PCR. RGS5 expression was normalized by HPRT using the Δ(ΔCT) method. Scrambeled and RGS5 siRNA transfected HUVECs were cocultured with VSMCs to produce spheroids in a 96 well plate (1:1 ratio, 3000 cell/well). Spheroids structure was scored 24 hours later for HUVEC coverage, spheroid shape and size. Results: RGS5-RNA was increased in stable compared to unstable plaques (3.1±0.5 vs 1.6±0.3 CNRQ, n=91, p<0.05). Consistent with previous reports5, RGS5 downregulation in VSMCs enhanced cell migration (40.9±5.7 vs 23.3±3.9, n=7, p<0.05). RGS5 was significantly downregulated in migrated compared to resident VSMCs (1.6±0.6 vs 0.17±0.1, n=6, p<0.05). Supernatant from RGS5- vs scrambeled siRNA transfected HUVECs significantly enhanced VSMC migration (66±2.4 vs 53±1.8, n=4, p<0.05). RGS5 knockdown HUVECs yielded more organized spheroids compared to scrambled siRNA control (9.5 vs 11, n=2). Conclusion: RGS5 plays a pivotal role in VSMC migration. RGS5 is downregulated in migrating cells while downregulation induces a migratory phenotype. RGS5-dependent soluble factors from endothelial cells can exert paracrine effects. The increased RGS5 expression in stable plaques may therefore suggest that stabilization of the plaque cap could be accessible through the RGS5 and GPCR pathway.

Mechanical, biological and structural characterization of in vitro ruptured human carotid plaque tissue

Recent experimental studies performed on human carotid plaques have focused on mechanical characterization for the purpose of developing material models for finite-element analysis without quantifying the tissue composition or relating mechanical behaviour to preoperative classification. This study characterizes the mechanical and biological properties of 25 human carotid plaques and also investigates the common features that lead to plaque rupture during mechanical testing by performing circumferential uniaxial tests, Fourier transform infrared (FTIR) and scanning electron microscopy (SEM) on each specimen to relate plaque composition to mechanical behaviour. Mechanical results revealed large variations between plaque specimen behaviour with no correlation to preoperative ultrasound prediction. However, FTIR classification demonstrated a statistically significant relationship between stress and stretch values at rupture and the level of calcification (P=0.002 and P=0.009). Energy-dispersive X-ray spectroscopy was carried out to confirm that the calcium levels observed using FTIR analysis were accurate. This work demonstrates the potential of FTIR as an alternative method to ultrasound forpredicting plaque mechanical behaviour. SEM imaging at the rupture sites of each specimen highlighted voids created by the nodes of calcifications in the tissue structure which could lead to increased vulnerability of the plaque.

NOD2-Mediated Innate Immune Signaling Regulates the Eicosanoids in Atherosclerosis

The activity of eicosanoid pathways is critical to the inflammatory and immune responses that are associated with the progression of atherosclerosis. Yet, the signals that regulate these pathways are poorly understood. Here, we address whether the innate immune signals of nucleotide-binding oligomerization domain-containing protein (NOD) 2 affect eicosanoids metabolism in atherosclerosis. Analysis of human carotid plaques revealed that NOD2 was abundantly expressed at both mRNA and protein levels by endothelial cells and macrophages. Stimulation of NOD2 in ex vivo-cultured carotid plaques by muramyl dipeptide, an extrinsic ligand of NOD2, led to release of prostaglandin E2, upregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, and to downregulation of cyclooxygenase-1. NOD2 was coexpressed with cyclooxygenase-2 in lesional macrophages. NOD2-induced cyclooxygenase-2 expression in macrophages was dependent on p38 mitogen-activated protein kinase activation and was mediated by interleukin-1β and tumor necrosis factor-α. Selective lipidomic analysis of the eicosanoids released by the carotid plaques characterized the metabolites of 12-, 5-, and 15-lipoxygenase as the predominant eicosanoids that were produced by the atherosclerotic lesion in the absence of additional stimuli. Unlike the prostaglandin E2 pathway, metabolic activity of the lipoxygenase pathways was not altered on the short-term activation of NOD2 in carotid plaques. These results suggest that atherosclerosis may involve enhanced NOD2-mediated innate immunity. Activation of NOD2 preferentially upregulates the prostaglandin E2 pathway. Nevertheless, lipoxygenase pathways, such as 12-lipoxygenase, predominate the basal synthesis and metabolism of eicosanoids in atherosclerotic plaques. These findings provide new insights into the regulation of eicosanoids in atherosclerosis.

Fast LC–MS/MS analysis of free oxysterols derived from reactive oxygen species in human plasma and carotid plaque

BackgroundA rapid liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the quantification of reactive oxygen species (ROS) derived free oxysterols and cholesterol in human plasma and atherosclerotic plaque. MethodIn vitro autoxidation of cholesterol during sample pretreatment was avoided by applying only one protein precipitation and re-concentration step using 80μl plasma. For preparation of 10mg atherosclerotic plaques an additional liquid–liquid extraction was included. Free 7-keto-, 7-α/ß-hydroxy-, 5,6-α-epoxy-, 5,6-β-epoxycholesterol, cholestane-3ß,5α,6ß-triol and cholesterol were separated within 7min on a monolithic column. An API 4000 tandem mass spectrometer was applied in positive ionization mode using atmospheric pressure chemical ionization. ResultsThe detection limit was 0.1ng/ml and the linearity ranged from 0.5 to 0.75 to 2000ng/ml for the oxysterols and from 50 to 1000μg/ml for cholesterol. Recovery was between 80.9 and 107.9%. Between-run imprecision ranged from 7.9 to 11.7%. Analysis of plasma samples from additional 50 middle-aged volunteers revealed a large inter-individual variability (e.g. 7-ketocholesterol 2.63-30.47ng/ml). Oxysterol concentrations normalized to cholesterol were about 43 times higher in carotid plaque compared to plasma (n=5). ConclusionThis rapid LC–MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions.

Macrophage-derived matrix vesicles: an alternative novel mechanism for microcalcification in atherosclerotic plaques.

We previously showed that early calcification of atherosclerotic plaques associates with macrophage accumulation. Chronic renal disease and mineral imbalance accelerate calcification and the subsequent release of matrix vesicles (MVs), precursors of microcalcification. We tested the hypothesis that macrophage-derived MVs contribute directly to microcalcification. Macrophages associated with regions of calcified vesicular structures in human carotid plaques (n=136 patients). In vitro, macrophages released MVs with high calcification and aggregation potential. MVs expressed exosomal markers (CD9 and TSG101) and contained S100A9 and annexin V. Silencing S100A9 in vitro and genetic deficiency in S100A9-/- mice reduced MV calcification, whereas stimulation with S100A9 increased calcification potential. Externalization of phosphatidylserine after Ca/P stimulation and interaction of S100A9 and annexin V indicated that a phosphatidylserine-annexin V-S100A9 membrane complex facilitates hydroxyapatite nucleation within the macrophage-derived MV membrane. Our results support the novel concept that macrophages release calcifying MVs enriched in S100A9 and annexin V, which contribute to accelerated microcalcification in chronic renal disease.

Treatment with beta-blockers is associated with lower levels of Lp-PLA2 and suPAR in carotid plaques

To determine whether a long-term treatment with beta-blockers influences the inflammatory activity in carotid artery disease by reducing the carotid plaque levels of lipoprotein-associated phospholipase A2 (Lp-PLA2), its enzymatic products lysophosphatidylcholine (lysoPCs), and of soluble urokinase plasminogen activator receptor (suPAR). One hundred and thirty-four patients with significant symptomatic or asymptomatic carotid stenosis undergoing surgery were prospectively included and divided into two groups (Group A or B) based on the absence or presence of an on-going long-term oral treatment with beta-blockers. The harvested carotid plaques were analyzed for the levels of lysoPCs using mass spectrometry and Lp-PLA2 and suPAR by Enzyme-linked immunosorbent assay (ELISA). Plaques of patients on long-term treatment with beta-blockers revealed lower levels of Lp-PLA2 (Group A 0.752 ± 0.393 ug/g vs. Group B 0.644 ± 0.445 ug/g, P=.049) as well as suPAR (Group A 0.044 ± 0.024 μg/g vs. Group B 0.036 ± 0.025 μg/g, P=.028). Levels of Lp-PLA2 and suPAR were positively correlated (r=.637, P<.0001). Lp-PLA2 and suPAR levels were also correlated (P<.0001) with the three lysoPC species tested (lysoPC 16:0, lysoPC 18:0. lysoPC 18:1). All the above-mentioned findings were confirmed after correction for age, gender, hypertension, coronary artery disease, and statin usage. The reduced levels of Lp-PLA2 and suPAR in human carotid plaques of subjects on long-term treatment with beta-blockers suggest their possible protective role in plaque inflammation. Our findings support an even more selective Lp-PLA2 and suPAR inhibition as a possible strategy for the prevention of cardiovascular disease.

Plasmatic Level of Leukocyte-Derived Microparticles Is Associated With Unstable Plaque in Asymptomatic Patients With High-Grade Carotid Stenosis

This study sought to analyze whether the plasmatic level of leukocyte-derived microparticles (LMP) is associated with unstable plaques in patients with high-grade carotid stenosis. Preventive carotid surgery in asymptomatic patients is currently debated given the improvement of medical therapy. Therefore, noninvasive biomarkers that can predict plaque instability are needed. The LMPs, originating from activated or apoptotic leukocytes, are the major microparticle (MP) subset in human carotid plaque extracts. Forty-two patients with >70% carotid stenosis were enrolled. Using a new standardized high-sensitivity flow cytometry assay, LMPs were measured before thromboendarterectomy. The removed plaques were characterized as stable or unstable using histological analysis according to the American Heart Association criteria. The LMP levels were analyzed according to the plaque morphology. The median LMP levels were significantly higher in patients with unstable plaque (n = 28; CD11bCD66b+ MP/μl 240 [25th to 75th percentile: 147 to 394], and CD15+ MP/μl 147 [60 to 335]) compared to patients with stable plaque (16 [0 to 234] and 55 [36 to 157]; p < 0.001 and p < 0.01, respectively). The increase in LMP levels was also significant when considering only the group of asymptomatic patients with unstable plaque (n = 10; CD11bCD66b+ MP/μl 199 [153 to 410] and CD15+ MP/μl 78 [56 to 258] compared with patients with stable plaque (n = 14; 20 [0 to 251] and 55 [34 to 102]; p < 0.05 and p < 0.05, respectively). After logistic regression, the neurologic symptoms (odds ratio: 48.7, 95% confidence interval: 3.0 to 788, p < 0.01) and the level of CD11bCD66b+ MPs (odds ratio: 24.4, 95% confidence interval: 2.4 to 245, p < 0.01) independently predicted plaque instability. LMP constitute a promising biomarker associated with plaque vulnerability in patients with high-grade carotid stenosis. These data provide clues for identifying asymptomatic subjects that are most at risk of neurologic events.

Comparison of grey scale median (GSM) measurement in ultrasound images of human carotid plaques using two different softwares

Grey scale median (GSM) measured on ultrasound images of carotid plaques has been used for several years now in research to find the vulnerable plaque. Centres have used different software and also different methods for GSM measurement. This has resulted in a wide range of GSM values and cut-off values for the detection of the vulnerable plaque. The aim of this study was to compare the values obtained with two different softwares, using different standardization methods, for the measurement of GSM on ultrasound images of carotid human plaques. GSM was measured with Adobe Photoshop(®) and with Artery Measurement System (AMS) on duplex ultrasound images of 100 consecutive medium- to large-sized carotid plaques of the Beta-blocker Cholesterol-lowering Asymptomatic Plaque Study (BCAPS). The mean values of GSM were 35·2±19·3 and 55·8±22·5 for Adobe Photoshop(®) and AMS, respectively. Mean difference was 20·45 (95% CI: 19·17-21·73). Although the absolute values of GSM differed, the agreement between the two measurements was good, correlation coefficient 0·95. A chi-square test revealed a kappa value of 0·68 when studying quartiles of GSM. The intra-observer variability was 1·9% for AMS and 2·5% for Adobe Photoshop. The difference between softwares and standardization methods must be taken into consideration when comparing studies. To avoid these problems, researcher should come to a consensus regarding software and standardization method for GSM measurement on ultrasound images of plaque in the arteries.