Spatial Metabolomics Identifies LPC(18:0) and LPA(18:1) in Advanced Atheroma With Translation to Plasma for Cardiovascular Risk Estimation.

BACKGROUND: The metabolic alterations occurring within the arterial architecture during atherosclerosis development remain poorly understood, let alone those particular to each arterial tunica. We aimed first to identify, in a spatially resolved manner, the specific metabolic changes in plaque, media, adventitia, and cardiac tissue between control and atherosclerotic murine aortas. Second, we assessed their translatability to human tissue and plasma for cardiovascular risk estimation. METHODS: In this observational study, mass spectrometry imaging (MSI) was applied to identify region-specific metabolic differences between atherosclerotic (n=11) and control (n=11) aortas from low-density lipoprotein receptor-deficient mice, via histology-guided virtual microdissection. Early and advanced plaques were compared within the same atherosclerotic animals. Progression metabolites were further analyzed by MSI in 9 human atherosclerotic carotids and by targeted mass spectrometry in human plasma from subjects with elective coronary artery bypass grafting (cardiovascular risk group, n=27) and a control group (n=27). RESULTS: MSI identified 362 local metabolic alterations in atherosclerotic mice (log2 fold-change ≥1.5; P≤0.05). The lipid composition of cardiac tissue is altered during atherosclerosis development and presents a generalized accumulation of glycerophospholipids, except for lysolipids. Lysolipids (among other glycerophospholipids) were found at elevated levels in all 3 arterial layers of atherosclerotic aortas. LPC(18:0) (lysophosphatidylcholine; P=0.024) and LPA(18:1) (lysophosphatidic acid; P=0.025) were found to be significantly elevated in advanced plaques as compared with mouse-matched early plaques. Higher levels of both lipid species were also observed in fibrosis-rich areas of advanced- versus early-stage human samples. They were found to be significantly reduced in human plasma from subjects with elective coronary artery bypass grafting (P<0.001 and P=0.031, respectively), with LPC(18:0) showing significant association with cardiovascular risk (odds ratio, 0.479 [95% CI, 0.225-0.883]; P=0.032) and diagnostic potential (area under the curve, 0.778 [95% CI, 0.638-0.917]). CONCLUSIONS: An altered phospholipid metabolism occurs in atherosclerosis, affecting both the aorta and the adjacent heart tissue. Plaque-progression lipids LPC(18:0) and LPA(18:1), as identified by MSI on tissue, reflect cardiovascular risk in human plasma.

Protective role of chaperone-mediated autophagy against atherosclerosis.

Chaperone-mediated autophagy (CMA) contributes to regulation of energy homeostasis by timely degradation of enzymes involved in glucose and lipid metabolism. Here, we report reduced CMA activity in vascular smooth muscle cells and macrophages in murine and human arteries in response to atherosclerotic challenges. We show that in vivo genetic blockage of CMA worsens atherosclerotic pathology through both systemic and cell-autonomous changes in vascular smooth muscle cells and macrophages, the two main cell types involved in atherogenesis. CMA deficiency promotes dedifferentiation of vascular smooth muscle cells and a proinflammatory state in macrophages. Conversely, a genetic mouse model with up-regulated CMA shows lower vulnerability to proatherosclerotic challenges. We propose that CMA could be an attractive therapeutic target against cardiovascular diseases.

Human atherosclerotic plaque transcriptomics reveals endothelial beta-2 spectrin as a potential regulator a leaky plaque microvasculature phenotype.

The presence of atherosclerotic plaque vessels is a critical factor in plaque destabilization. This may be attributable to the leaky phenotype of these microvessels, although direct proof for this notion is lacking. In this study, we investigated molecular and cellular patterns of stable and hemorrhaged human plaque to identify novel drivers of intraplaque vessel dysfunction. From transcriptome data of a human atherosclerotic lesion cohort, we reconstructed a co-expression network, identifying a gene module strongly and selectively correlated with both plaque microvascular density and inflammation. Spectrin Beta Non-Erythrocytic 1 (sptbn1) was identified as one of the central hubs of this module (along with zeb1 and dock1) and was selected for further study based on its predominant endothelial expression. Silencing of sptbn1 enhanced leukocyte transmigration and vascular permeability in vitro, characterized by an increased number of focal adhesions and reduced junctional VE-cadherin. In vivo, sptbn1 knockdown in zebrafish impaired the development of the caudal vein plexus. Mechanistically, increased substrate stiffness was associated with sptbn1 downregulation in endothelial cells in vitro and in human vessels. Plaque SPTBN1 mRNA and protein expression were found to correlate with an enhanced presence of intraplaque hemorrhage and future cardiovascular disease (CVD) events during follow-up. In conclusion, we identify SPTBN1 as a central hub gene in a gene program correlating with plaque vascularisation. SPTBN1 was regulated by substrate stiffness in vitro while silencing blocked vascular development in vivo, and compromised barrier function in vitro. Together, SPTBN1 is identified as a new potential regulator of the leaky phenotype of atherosclerotic plaque microvessels.

Genome-scale metabolic network of human carotid plaque reveals the pivotal role of glutamine/glutamate metabolism in macrophage modulating plaque inflammation and vulnerability.

BACKGROUND: Metabolism is increasingly recognized as a key regulator of the function and phenotype of the primary cellular constituents of the atherosclerotic vascular wall, including endothelial cells, smooth muscle cells, and inflammatory cells. However, a comprehensive analysis of metabolic changes associated with the transition of plaque from a stable to a hemorrhaged phenotype is lacking. METHODS: In this study, we integrated two large mRNA expression and protein abundance datasets (BIKE, n = 126; MaasHPS, n = 43) from human atherosclerotic carotid artery plaque to reconstruct a genome-scale metabolic network (GEM). Next, the GEM findings were linked to metabolomics data from MaasHPS, providing a comprehensive overview of metabolic changes in human plaque. RESULTS: Our study identified significant changes in lipid, cholesterol, and inositol metabolism, along with altered lysosomal lytic activity and increased inflammatory activity, in unstable plaques with intraplaque hemorrhage (IPH+) compared to non-hemorrhaged (IPH-) plaques. Moreover, topological analysis of this network model revealed that the conversion of glutamine to glutamate and their flux between the cytoplasm and mitochondria were notably compromised in hemorrhaged plaques, with a significant reduction in overall glutamate levels in IPH+ plaques. Additionally, reduced glutamate availability was associated with an increased presence of macrophages and a pro-inflammatory phenotype in IPH+ plaques, suggesting an inflammation-prone microenvironment. CONCLUSIONS: This study is the first to establish a robust and comprehensive GEM for atherosclerotic plaque, providing a valuable resource for understanding plaque metabolism. The utility of this GEM was illustrated by its ability to reliably predict dysregulation in the cholesterol hydroxylation, inositol metabolism, and the glutamine/glutamate pathway in rupture-prone hemorrhaged plaques, a finding that may pave the way to new diagnostic or therapeutic measures.

CARMN Loss Regulates Smooth Muscle Cells and Accelerates Atherosclerosis in Mice.

[Figure: see text].

The Human-Specific and Smooth Muscle Cell-Enriched LncRNA SMILR Promotes Proliferation by Regulating Mitotic CENPF mRNA and Drives Cell-Cycle Progression Which Can Be Targeted to Limit Vascular Remodeling.

RATIONALE: In response to blood vessel wall injury, aberrant proliferation of vascular smooth muscle cells (SMCs) causes pathological remodeling. However, the controlling mechanisms are not completely understood. OBJECTIVE: We recently showed that the human long noncoding RNA, SMILR, promotes vascular SMCs proliferation by a hitherto unknown mechanism. Here, we assess the therapeutic potential of SMILR inhibition and detail the molecular mechanism of action. METHODS AND RESULTS: We used deep RNA-sequencing of human saphenous vein SMCs stimulated with IL (interleukin)-1α and PDGF (platelet-derived growth factor)-BB with SMILR knockdown (siRNA) or overexpression (lentivirus), to identify SMILR-regulated genes. This revealed a SMILR-dependent network essential for cell cycle progression. In particular, we found using the fluorescent ubiquitination-based cell cycle indicator viral system that SMILR regulates the late mitotic phase of the cell cycle and cytokinesis with SMILR knockdown resulting in ≈10% increase in binucleated cells. SMILR pulldowns further revealed its potential molecular mechanism, which involves an interaction with the mRNA of the late mitotic protein CENPF (centromere protein F) and the regulatory Staufen1 RNA-binding protein. SMILR and this downstream axis were also found to be activated in the human ex vivo vein graft pathological model and in primary human coronary artery SMCs and atherosclerotic plaques obtained at carotid endarterectomy. Finally, to assess the therapeutic potential of SMILR, we used a novel siRNA approach in the ex vivo vein graft model (within the 30 minutes clinical time frame that would occur between harvest and implant) to assess the reduction of proliferation by EdU incorporation. SMILR knockdown led to a marked decrease in proliferation from ≈29% in controls to ≈5% with SMILR depletion. CONCLUSIONS: Collectively, we demonstrate that SMILR is a critical mediator of vascular SMC proliferation via direct regulation of mitotic progression. Our data further reveal a potential SMILR-targeting intervention to limit atherogenesis and adverse vascular remodeling.

Novel Plaque Enriched Long Noncoding RNA in Atherosclerotic Macrophage Regulation (PELATON).

OBJECTIVE: Long noncoding RNAs (lncRNAs) are an emergent class of molecules with diverse functional roles, widely expressed in human physiology and disease. Although some lncRNAs have been identified in cardiovascular disease, their potential as novel targets in the prevention of atherosclerosis is unknown. We set out to discover important lncRNAs in unstable plaque and gain insight into their functional relevance. Approach and Results: Analysis of RNA sequencing previously performed on stable and unstable atherosclerotic plaque identified a panel of 47 differentially regulated lncRNAs. We focused on LINC01272, a lncRNA upregulated in unstable plaque previously detected in inflammatory bowel disease, which we termed PELATON (plaque enriched lncRNA in atherosclerotic and inflammatory bowel macrophage regulation). Here, we demonstrate that PELATON is highly monocyte- and macrophage-specific across vascular cell types, and almost entirely nuclear by cellular fractionation (90%-98%). In situ hybridization confirmed enrichment of PELATON in areas of plaque inflammation, colocalizing with macrophages around the shoulders and necrotic core of human plaque sections. Consistent with its nuclear localization, and despite containing a predicted open reading frame, PELATON did not demonstrate any protein-coding potential in vitro. Functionally, knockdown of PELATON significantly reduced phagocytosis, lipid uptake and reactive oxygen species production in high-content analysis, with a significant reduction in phagocytosis independently validated. Furthermore, CD36, a key mediator of phagocytic oxLDL (oxidized low-density lipoprotein) uptake was significantly reduced with PELATON knockdown. CONCLUSIONS: PELATON is a nuclear expressed, monocyte- and macrophage-specific lncRNA, upregulated in unstable atherosclerotic plaque. Knockdown of PELATON affects cellular functions associated with plaque progression.

Fibroblasts in atherosclerosis: heterogeneous and plastic participants.

PURPOSE OF REVIEW: Fibroblasts are very heterogeneous and plastic cells in the vasculature. A growing interest in fibroblasts in healthy and atherosclerotic vasculature is observed, next to macrophages, endothelial cells, and smooth muscle cells (SMCs). In this review, we discuss fibroblast presence, heterogeneity, origin, and plasticity in health and atherosclerosis based on latest literature. RECENT FINDINGS: With help of single cell sequencing (SCS) techniques, we have gained more insight into presence and functions of fibroblasts in atherosclerosis. Next to SMCs, fibroblasts are extracellular matrix-producing cells abundant in the vasculature and involved in atherogenesis. Fibroblasts encompass a heterogeneous population and SCS data reveal several fibroblast clusters in healthy and atherosclerotic tissue with varying gene expression and function. Moreover, recent findings indicate interesting similarities between adventitial stem and/or progenitor cells and fibroblasts. Also, communication with inflammatory cells opens up a new therapeutic avenue. SUMMARY: Because of their highly plastic and heterogeneous nature, modulating fibroblast cell function and communication in the atherosclerotic vessel might be useful in battling atherosclerosis from within the plaque.

Differential regulation of angiogenic cellular processes and claudin-5 by histamine and VEGF via PI3K-signaling, transcription factor SNAI2 and interleukin-8.

AIMS: Histamine and vascular endothelial growth factor A (VEGF) are central regulators in vascular pathologies. Their gene regulation leading to vascular remodeling has remained obscure. In this study, EC regulation mechanisms of histamine and VEGF were compared by RNA sequencing of primary endothelial cells (ECs), functional in vitro assays and in vivo permeability mice model. METHODS AND RESULTS: By RNA sequencing, similar transcriptional alterations of genes involved in activation of primary ECs, cell proliferation and adhesion were observed between histamine and VEGF. Seventy-six commonly regulated genes were found, representing ~53% of all VEGF-regulated transcripts and ~26% of all histamine-regulated transcripts. Both factors regulated tight junction formation and expression of pro-angiogenic transcription factors (TFs) affecting EC survival, migration and tube formation. Novel claudin-5 upstream regulatory genes were identified. VEGF was demonstrated to regulate expression of SNAI2, whereas pro-angiogenic TFs NR4A1, MYCN and RCAN1 were regulated by both histamine and VEGF. Claudin-5 was shown to be regulated VEGFR2/PI3K-Akt dependently by VEGF and PI3K-Akt independently by histamine. Interleukin-8 was shown to downregulate claudin-5 by histamine. Additionally, SNAI2, NR4A1 and MYCN were shown to mediate EC survival, migration and tube formation and to regulate expression of claudin-5. Further systemic delivery of VEGF and histamine was shown to induce a fast vascular hyperpermeability response in intact vasculature of C57/Bl6 mice followed by regulation of NR4A1 and MYCN. CONCLUSIONS: Our study identifies novel claudin-5 upstream regulatory genes of histamine and VEGF that induce cellular angiogenic processes. Our results increase knowledge of angiogenic EC phenotype and provide novel treatment targets for vascular pathologies.

Feasibility of in vivo 18F-florbetaben PET/MR imaging of human carotid amyloid-ß.

PURPOSE: Amyloid-beta (Aß) peptides are involved in the inflammatory pathology of atherosclerosis. 18F-Florbetaben is a PET tracer for clinical imaging of cerebral Aß plaques in Alzheimer's disease (AD). We sought to determine whether specific uptake of 18F-florbetaben in the carotid arteries can be identified using a fully integrated hybrid PET/MRI system and whether this uptake is associated with clinical cardiovascular disease (CVD) risk factors. METHODS: Carotid 18F-florbetaben uptake was quantified as the mean of the maximum target-to-background ratio (meanTBRmax) in 40 cognitively impaired subjects (age 68.2 ± 9.5 years) undergoing 18F-florbetaben PET/MRI to diagnose AD. Associations between carotid 18F-florbetaben uptake and several CVD risk factors were assessed by univariate analysis followed by a multivariate linear regression analysis. Furthermore, carotid 18F-florbetaben uptake was compared between patients with and without a positive cerebral Aß PET scan. RESULTS: 18F-Florbetaben uptake was clearly visualized in the carotid arteries. Values of meanTBRmax corrected for the blood pool activity of the tracer showed specific 18F-florbetaben uptake in the carotid wall. Male gender was associated with carotid 18F-florbetaben uptake in the univariate analysis, and was found to be an independent predictor of 18F-florbetaben uptake in the multivariate regression analysis (standardized regression coefficient ß = 0.407, p = 0.009). Carotid 18F-florbetaben meanTBRmax in patients with a positive cerebral Aß scan did not differ from that in patients without cerebral Aß deposits. CONCLUSION: Specific 18F-florbetaben uptake in human carotid arteries was detected. Male gender was identified as an independent clinical risk factor. Therefore, 18F-florbetaben PET/MRI might provide new insights into the pathophysiological process in atherosclerosis.

Vessel wall and adventitial DCE-MRI parameters demonstrate similar correlations with carotid plaque microvasculature on histology.

PURPOSE: To assess parameter agreement of volume transfer coefficient (Ktrans ) between two vascular regions and to study the correlation with microvessel density on histology. The dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) parameter Ktrans is frequently used to study atherosclerotic plaque microvasculature. Ktrans has been reported using different descriptive statistics (mean, median, 75th percentile) either for the whole vessel wall or the adventitia in previous studies. MATERIALS AND METHODS: DCE-MRI parameter agreement was analyzed in 110 symptomatic patients with ≥2 mm carotid plaque that underwent a 3T carotid DCE-MRI examination. Ktrans was estimated in the entire vessel wall and adventitia. Twenty-three patients underwent carotid endarterectomy and were used for comparison with histological quantification of microvessel density of the plaque using CD31 immunohistochemistry. DCE-MRI parameters in the vessel wall regions were compared using Pearson's correlation coefficient, Bland-Altman analysis, and a two-sided paired samples t-test. Correlation of the DCE-MRI parameters with histology was studied using the Pearson's correlation coefficient. RESULTS: Median adventitial Ktrans was 5% higher (P = 0.003) than entire vessel wall Ktrans , with no differences for other descriptive statistics. Vessel wall and adventitial Ktrans showed similar moderately strong correlations with plaque microvessel density on histology (Pearson's ρ: 0.59-0.65 [P < 0.003] and 0.52-0.64 [P < 0.011], respectively). CONCLUSION: The similar moderately strong correlations for vessel wall and adventitial Ktrans with microvessel density on histology suggested that both regions reflected plaque microvessel density. Care should to be taken when comparing absolute values between studies. Future studies incorporating thresholds for risk stratification need to agree upon standardization of DCE-MRI parameters. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2017;46:1053-1059.

Deficiency of the oxygen sensor prolyl hydroxylase 1 attenuates hypercholesterolaemia, atherosclerosis, and hyperglycaemia.

AIMS: Normalization of hypercholesterolaemia, inflammation, hyperglycaemia, and obesity are main desired targets to prevent cardiovascular clinical events. Here we present a novel regulator of cholesterol metabolism, which simultaneously impacts on glucose intolerance and inflammation. METHODS AND RESULTS: Mice deficient for oxygen sensor HIF-prolyl hydroxylase 1 (PHD1) were backcrossed onto an atherogenic low-density lipoprotein receptor (LDLR) knockout background and atherosclerosis was studied upon 8 weeks of western-type diet. PHD1-/-LDLR-/- mice presented a sharp reduction in VLDL and LDL plasma cholesterol levels. In line, atherosclerotic plaque development, as measured by plaque area, necrotic core expansion and plaque stage was hampered in PHD1-/-LDLR-/- mice. Mechanistically, cholesterol-lowering in PHD1 deficient mice was a result of enhanced cholesterol excretion from blood to intestines and ultimately faeces. Additionally, flow cytometry of whole blood of these mice revealed significantly reduced counts of leucocytes and particularly of Ly6Chigh pro-inflammatory monocytes. In addition, when studying PHD1-/- in diet-induced obesity (14 weeks high-fat diet) mice were less glucose intolerant when compared with WT littermate controls. CONCLUSION: Overall, PHD1 knockout mice display a metabolic phenotype that generally is deemed protective for cardiovascular disease. Future studies should focus on the efficacy, safety, and gender-specific effects of PHD1 inhibition in humans, and unravel the molecular actors responsible for PHD1-driven, likely intestinal, and regulation of cholesterol metabolism.

Deficiency of HIF1α in Antigen-Presenting Cells Aggravates Atherosclerosis and Type 1 T-Helper Cell Responses in Mice.

OBJECTIVE: Although immune responses drive the pathogenesis of atherosclerosis, mechanisms that control antigen-presenting cell (APC)-mediated immune activation in atherosclerosis remain elusive. We here investigated the function of hypoxia-inducible factor (HIF)-1α in APCs in atherosclerosis. APPROACH AND RESULTS: We found upregulated HIF1α expression in CD11c(+) APCs within atherosclerotic plaques of low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Conditional deletion of Hif1a in CD11c(+) APCs in high-fat diet-fed Ldlr(-/-) mice accelerated atherosclerotic plaque formation and increased lesional T-cell infiltrates, revealing a protective role of this transcription factor. HIF1α directly controls Signal Transducers and Activators of Transcription 3 (Stat3), and a reduced STAT3 expression was found in HIF1α-deficient APCs and aortic tissue, together with an upregulated interleukin-12 expression and expansion of type 1 T-helper (Th1) cells. Overexpression of STAT3 in Hif1a-deficient APCs in bone marrow reversed enhanced atherosclerotic lesion formation and reduced Th1 cell expansion in chimeric Ldlr(-/-) mice. Notably, deletion of Hif1a in LysM(+) bone marrow cells in Ldlr(-/-) mice did not affect lesion formation or T-cell activation. In human atherosclerotic lesions, HIF1α, STAT3, and interleukin-12 protein were found to colocalize with APCs. CONCLUSIONS: Our findings identify HIF1α to antagonize APC activation and Th1 T cell polarization during atherogenesis in Ldlr(-/-) mice and to attenuate the progression of atherosclerosis. These data substantiate the critical role of APCs in controlling immune mechanisms that drive atherosclerotic lesion development.

Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death.

AIMS: There is a need for animal models of plaque rupture. We previously reported that elastin fragmentation, due to a mutation (C1039G(+/-)) in the fibrillin-1 (Fbn1) gene, promotes atherogenesis and a highly unstable plaque phenotype in apolipoprotein E deficient (ApoE(-/-)) mice on a Western-type diet (WD). Here, we investigated whether plaque rupture occurred in ApoE(-/-)Fbn1(C1039G+/-) mice and was associated with myocardial infarction, stroke, and sudden death. METHODS AND RESULTS: Female ApoE(-/-)Fbn1(C1039G+/-) and ApoE(-/-) mice were fed a WD for up to 35 weeks. Compared to ApoE(-/-) mice, plaques of ApoE(-/-)Fbn1(C1039G+/-) mice showed a threefold increase in necrotic core size, augmented T-cell infiltration, a decreased collagen I content (70 ± 10%), extensive neovascularization, intraplaque haemorrhage, and a significant increase in matrix metalloproteinase-2, -9, -12, and -13 expression or activity. Plaque rupture was observed in 70% of ascending aortas and in 50% of brachiocephalic arteries of ApoE(-/-)Fbn1(C1039G+/-) mice. In ApoE(-/-) mice, plaque rupture was not seen in ascending aortas and only in 10% of brachiocephalic arteries. Seventy percent of ApoE(-/-)Fbn1(C1039G+/-) mice died suddenly, whereas all ApoE(-/-) mice survived. ApoE(-/-)Fbn1(C1039G+/-) mice showed coronary plaques and myocardial infarction (75% of mice). Furthermore, they displayed head tilt, disorientation, and motor disturbances (66% of cases), disturbed cerebral blood flow (73% of cases; MR angiograms) and brain hypoxia (64% of cases), indicative of stroke. CONCLUSIONS: Elastin fragmentation plays a key role in plaque destabilization and rupture. ApoE(-/-)Fbn1(C1039G+/-) mice represent a unique model of acute plaque rupture with human-like complications.

Reversal of hypoxia in murine atherosclerosis prevents necrotic core expansion by enhancing efferocytosis.

OBJECTIVE: Advanced murine and human plaques are hypoxic, but it remains unclear whether plaque hypoxia is causally related to atherogenesis. Here, we test the hypothesis that reversal of hypoxia in atherosclerotic plaques by breathing hyperoxic carbogen gas will prevent atherosclerosis. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice (LDLR(-/-)) were fed a Western-type diet, exposed to carbogen (95% O2, 5% CO2) or air, and the effect on plaque hypoxia, size, and phenotype was studied. First, the hypoxic marker pimonidazole was detected in murine LDLR(-/-) plaque macrophages from plaque initiation onwards. Second, the efficacy of breathing carbogen (90 minutes, single exposure) was studied. Compared with air, carbogen increased arterial blood pO2 5-fold in LDLR(-/-) mice and reduced plaque hypoxia in advanced plaques of the aortic root (-32%) and arch (-84%). Finally, the effect of repeated carbogen exposure on progression of atherosclerosis was studied in LDLR(-/-) mice fed a Western-type diet for an initial 4 weeks, followed by 4 weeks of diet and carbogen or air (both 90 min/d). Carbogen reduced plaque hypoxia (-40%), necrotic core size (-37%), and TUNEL(+) (terminal uridine nick-end labeling positive) apoptotic cell content (-50%) and increased efferocytosis of apoptotic cells by cluster of differentiation 107b(+) (CD107b, MAC3) macrophages (+36%) in advanced plaques of the aortic root. Plaque size, plasma cholesterol, hematopoiesis, and systemic inflammation were unchanged. In vitro, hypoxia hampered efferocytosis by bone marrow-derived macrophages, which was dependent on the receptor Mer tyrosine kinase. CONCLUSIONS: Carbogen restored murine plaque oxygenation and prevented necrotic core expansion by enhancing efferocytosis, likely via Mer tyrosine kinase. Thus, plaque hypoxia is causally related to necrotic core expansion.

Higher levels of advanced glycation endproducts in human carotid atherosclerotic plaques are associated with a rupture-prone phenotype.

AIMS: Rupture-prone atherosclerotic plaques are characterized by inflammation and a large necrotic core. Inflammation is linked to high metabolic activity. Advanced glycation endproducts (AGEs) and their major precursor methylglyoxal are formed during high metabolic activity and can have detrimental effects on cellular function and may induce cell death. Therefore, we investigated whether plaque AGEs are increased in human carotid rupture-prone plaques and are associated with plaque inflammation and necrotic core formation. METHODS AND RESULTS: The protein-bound major methylglyoxal-derived AGE 5-hydro-5-methylimidazolone (MG-H1) and N(ε)-(carboxymethyl)lysine (CML) were measured in human carotid endarterectomy specimens (n = 75) with tandem mass spectrometry. MG-H1 and CML levels were associated with rupture-prone plaques, increased protein levels of the inflammatory mediators IL-8 and MCP-1 and with higher MMP-9 activity. Immunohistochemistry showed that AGEs accumulated predominantly in macrophages surrounding the necrotic core and co-localized with cleaved caspase-3. Intra-plaque comparison revealed that glyoxalase-1 (GLO-1), the major methylglyoxal-detoxifying enzyme, mRNA was decreased (-13%, P < 0.05) in ruptured compared with stable plaque segments. In line, in U937 monoctyes, we found reduced (GLO-1) activity (-38%, P < 0.05) and increased MGO (346%, P < 0.05) production after stimulation with the inflammatory mediator TNF. Direct incubation with methylglyoxal increased apoptosis up to two-fold. CONCLUSION: This is the first study showing that AGEs are associated with human rupture-prone plaques. Furthermore, this study suggests a cascade linking inflammation, reduced GLO-1, methylglyoxal- and AGE-accumulation, and subsequent apoptosis. Thereby, AGEs may act as mediators of the progression of stable to rupture-prone plaques, opening a window towards novel treatments and biomarkers to treat cardiovascular diseases.

CXCR4 blockade induces atherosclerosis by affecting neutrophil function.

AIMS: The SDF-1α/CXCR4 dyad was previously shown by us and others to be instrumental in intimal hyperplasia as well as early stage atherosclerosis. We here sought to investigate its impact on clinically relevant stages of atherosclerosis in mouse and man. METHODS AND RESULTS: Immunohistochemical analysis of CXCR4 expression in human atherosclerotic lesions revealed a progressive accumulation of CXCR4(+) cells during plaque progression. To address causal involvement of CXCR4 in advanced stages of atherosclerosis we reconstituted LDLr(-/-) mice with autologous bone marrow infected with lentivirus encoding SDF-1α antagonist or CXCR4 degrakine, which effects proteasomal degradation of CXCR4. Functional CXCR4 blockade led to progressive plaque expansion with disease progression, while also promoting intraplaque haemorrhage. Moreover, CXCR4 knockdown was seen to augment endothelial adhesion of neutrophils. Concordant with this finding, inhibition of CXCR4 function increased adhesive capacity and reduced apoptosis of neutrophils and resulted in hyperactivation of circulating neutrophils. Compatible with a role of the neutrophil CXCR4 in end-stage atherosclerosis, CXCR4 expression by circulating neutrophils was lowered in patients with acute cardiovascular syndromes. CONCLUSION: In conclusion, CXCR4 contributes to later stages of plaque progression by perturbing neutrophil function.

Plaque-associated vasa vasorum in aged apolipoprotein E-deficient mice exhibit proatherogenic functional features in vivo.

OBJECTIVE: Neovascularization of human atherosclerotic plaques is implicated in plaque progression and destabilization, although its functional implications are yet unresolved. Here, we aimed to elucidate functional and morphological properties of plaque microvessels in mice in vivo. METHODS AND RESULTS: Atherosclerotic carotid arteries from aged (>40 weeks) apolipoprotein E-deficient mice were imaged in vivo using multiphoton laser scanning microscopy. Two distinct groups of vasa vasorum microvessels were observed at sites of atherosclerosis development (median diameters of 18.5 and 5.9 µm, respectively), whereas microvessels within the plaque could only rarely be found. In vivo imaging showed ongoing angiogenic activity and injection of fluorescein isothiocyanate-dextran confirmed active perfusion. Plaque vasa vasorum showed increased microvascular leakage, combined with a loss of endothelial glycocalyx. Mean blood flow velocity in plaque-associated vasa vasorum was reduced by ±50% compared with diameter-matched control capillaries, whereas mean blood flow was reduced 8-fold. Leukocyte adhesion and extravasation were increased 6-fold in vasa vasorum versus control capillaries. CONCLUSIONS: Using a novel in vivo functional imaging strategy, we showed that plaque-associated vasa vasorum were angiogenically active and, albeit poorly, perfused. Moreover, plaque-associated vasa vasorum showed increased permeability, reduced blood flow, and increased leukocyte adhesion and extravasation (ie, characteristics that could contribute to plaque progression and destabilization).