Atherosclerosis

Background and aimsLipids play a critical role in atherosclerosis. Low-density lipoprotein (LDL)-cholesterol and certain lipid classes like sphingomyelins are associated with inflammation and poor cardiovascular outcomes. Phosphatidylserine (PS), on the other hand, is a negatively charged anti-inflammatory phospholipid class involved in efferocytosis. In this study, we sought to investigate its anti-atherosclerotic properties through a combination of complementary human lipidomics analyses, in vitro assays and in vivo experiments in Apoe-/- mice. MethodsHuman lipidomics studies were performed on the 300OB cohort comprising 300 obese and overweight individuals at risk of cardiovascular disease. In vitro assays were carried out using human monocytes and macrophages, and in vivo experiments included histopathological, immunophenotyping and single-cell transcriptomic analyses. ResultsIn humans, we identified PS as an anti-inflammatory and atheroprotective biomarker. Hence, we developed a high-density lipoprotein (HDL)-like formulation enriched in PS to exploit its properties in a targeted fashion in mice. In vitro, this formulation potently inhibited inflammatory cytokine production on human myeloid cells. Our in-depth in vivo experiments provided evidence of the formulations potent plaque-stabilizing and anti-inflammatory actions. These effects were mediated by a shift in the monocyte/macrophage compartment toward homeostatic/repairing phenotypes. ConclusionsCollectively, our results demonstrate that HDL-associated PS potently suppresses inflammation and atheroprogression, and holds promise as a viable approach to improve immunomodulatory therapies.

Although the cannabinoid CB1 receptor has been implicated in atherosclerosis, its cell-specific effects in this disease are not well understood. Here, we report that male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores than controls, while only minor genotype differences were observed in females. Male Cnr1 deficient mice showed reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences were reproducible in bone marrow derived macrophages and blunted by estradiol. Kinase activity profiling revealed a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling further unveiled chromatin modifications, mRNA processing and mitochondrial respiration among the key processes affected by CB1 signaling, which was supported by metabolic flux assays. Chronic administration of the peripherally-restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, but only in male mice. Finally, CNR1 expression was detectable in human carotid endarterectomy plaques and inversely correlated with proliferation, oxidative metabolism and inflammatory markers, hinting to a possible implication of CB1-dependent regulation in human pathophysiology. In conclusion, impaired CB1 signaling in macrophages is atheroprotective by limiting their arterial recruitment, proliferation and inflammatory reprogramming. The importance of macrophage CB1 signaling seems to be more pronounced in male mice. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/535832v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1d0b2f4org.highwire.dtl.DTLVardef@94f1b4org.highwire.dtl.DTLVardef@8868edorg.highwire.dtl.DTLVardef@1a0b151_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical summary (created with BioRender.com)

BackgroundAtherosclerosis is characterized by the buildup of fatty plaques that thicken and stiffen arterial walls. Macrophages (M{varphi}s) significantly contribute to this process through their scavenger receptor CD36. PIM1 is a serine/threonine kinase known to modulate immune responses and cell metabolism. However, its role in M{varphi} lipid handling and atherogenesis is not well defined. This study examines the role of PIM1 in regulating CD36 expression and function in M{varphi}s during foam cell formation and atherosclerosis progression. MethodsWe performed in vitro studies by treating murine peritoneal M{varphi}s from Pim1-/- and wild-type (WT) mice with oxidized low-density lipoprotein (oxLDL). We measured CD36, PIM1, and plaque-associated proteins and mRNA levels, oxLDL binding and uptake rates, as well as foam cell formation. For in vivo studies, we fed M{varphi}-specific PIM1-deficient (Apoe-/- Lyz2Cre/+Pim1fl/fl) and their littermate control (Apoe-/-Pim1fl/fl) mice a high-fat diet for 12 weeks. We then evaluated the plaque formation in their aortic sinuses and arches. ResultsDeletion of Pim1 in M{varphi}s reduced CD36 protein expression by up to 96.7% compared to WT controls. This led to a 49.6% decrease in foam cell formation and a 25.5% reduction in cellular cholesterol after oxLDL treatment. Pharmacological inhibition of PIM kinase activity in WT M{varphi}s also impaired oxLDL handling, with a 64.5% reduction in binding and a 57.9% in uptake. Bulk RNA-seq revealed that Pim1 deficiency downregulated PPAR{gamma} signaling. Treatment with a PPAR{gamma} agonist restored CD36 levels in the PIM1 knockdown M{varphi}s, suggesting that PIM1 regulates CD36 through PPAR{gamma}. Moreover, PIM1 M{varphi}-specific deficiency caused a 69.4% reduction in atherosclerotic plaque formation. ConclusionPIM1 acts as a key upstream regulator of CD36 by enhancing PPAR{gamma} activity in M{varphi}s. The PIM1-CD36 axis promotes oxLDL binding, uptake, and foam cell formation. Targeting the PIM1/PPAR{gamma}/CD36 pathway could offer new ways to modulate M{varphi} lipid metabolism and reduce atherosclerotic plaque progression. Non-standard Abbreviations and AcronymsELISA: enzyme-linked immunosorbent assay; HFD: high-fat diet; M{varphi}s: macrophages; MCP-1: monocyte chemoattractant protein-1; ORO: oil red O; oxLDL: oxidized low-density lipoprotein; PBS: phosphate-buffered saline; PPAR{gamma}: peroxisome proliferator-activated receptor gamma; WT: wild type.

RationaleThe CC-chemokine ligand-2 (CCL2)/ CC-chemokine receptor-2 (CCR2) axis governs monocyte recruitment to atherosclerotic lesions. Coherent evidence from experimental studies employing genetic deletion of CCL2 or CCR2 and human epidemiological studies support a causal involvement of the CCL2/CCR2 axis in atherosclerosis. Still, preclinical studies testing pharmacological inhibition of CCL2 or CCR2 in atheroprone mice apply widely different approaches and report inconsistent results, thus halting clinical translation. ObjectiveTo systematically review and meta-analyze preclinical studies pharmacologically targeting the CCL2/CCR2 axis in atherosclerosis in an effort to inform the design of future trials. Methods and ResultsWe identified 14 studies testing CCL2/CCR2 inhibition using 11 different pharmacological agents in mouse models of atherosclerosis. In meta-analyses, blockade of CCL2 or CCR2 attenuated atherosclerotic lesion size in the aortic root or arch (g=-0.75 [-1.17 to -0.32], p=6x10-4; N=171/171 mice in experimental/control group), the carotid (g=-2.39 [-4.23 to -0.55], p=0.01; N=24/25) and the femoral artery (g=-2.38 [-3.50 to -1.26], p=3x10-5; N=10/10). Furthermore, CCL2/CCR2 inhibition reduced intralesional macrophage accumulation and increased smooth muscle cell content and collagen deposition, consistent with a plaque-stabilizing effect. While there was heterogeneity across studies, the effects of CCL2/CCR2 inhibition on lesion size correlated with reductions in plaque macrophage accumulation, in accord with a prominent role of CCL2/CCR2 signaling in monocyte recruitment. Subgroup analyses revealed similar efficacy of both CCL2- and CCR2-inhibiting approaches across different atherosclerosis models in reducing lesion size and intralesional macrophage accumulation, but stronger atheroprotective effects in carotid and femoral arteries, as compared to the aorta. ConclusionsPharmacological targeting of CCL2 or CCR2 lowers atherosclerotic lesion burden and confers plaque stability in mice across different vascular territories, drug candidates, and models of atherosclerosis. Our findings in conjunction with recent human data highlight the translational potential of targeting the CCL2/CCR2 axis in atherosclerosis and can inform future clinical trials. Subject codesatherosclerosis, inflammation, vascular biology, translational studies

Background and AimsMetabolism dictates macrophage function and plays a central role in atherosclerotic plaque progression. The kynurenine pathway, which metabolizes the majority of the essential amino acid tryptophan, plays a pivotal role in regulating immune responses and supporting NAD+ synthesis, essential for cellular energy metabolism. Higher circulating kynurenine levels are associated with cardiovascular disease, yet their role in atherosclerotic plaques is unclear. This study aims to investigate the underlying mechanisms driving increased kynurenine concentrations in plaques and to determine whether kynurenine serves as a mere biomarker of low-grade inflammation or reflects specific macrophage-driven metabolic alterations that could position it as a potential therapeutic target. MethodsWe used histological and transcriptomic data from two biobanks: the Athero Express Biobank (AE; n=91) and Maastricht human plaque study (MaasHPS, n= 26). Macrophages were identified through CD68 staining in AE, and M1/M2-like macrophage subtypes were distinguished by iNOS/CD68 and arginase/CD68 expression in MAASHPS. Primary human monocyte-derived cultured macrophages were polarized into M1- and M2-like phenotypes for using IFN-{gamma} and IL-4, respectively. Tryptophan, kynurenine and/or NAD+ concentrations in plaques were quantified usingliquid chromatography and metabolomics analyses. ResultsKynurenine concentrations were significantly higher in plaques with greater macrophage density (p = 0.023). Transcriptomic analysis in AE revealed upregulation of IDO2, AFMID, and KYNU in plaques with increased macrophage infiltration (p < 0.05), but not IDO1 (p = 0.16). In the MAASHPS biobank, higher IDO1, KYNU, and KMO expression correlated negatively with M2 marker positive macrophages (p < 0.001), while HAAO correlated positively (p < 0.01). In vitro, M1-like macrophages showed increased IDO1 and reduced QPRT expression compared to M2-like macrophages. We found that this disruption in kynurenine pathway gene expression led to decreased NAD+ concentrations in M1-like macrophages compared to M2-like macrophages in vitro. ConclusionHigher kynurenine levels in atherosclerotic plaques are increased by the increased presence of M1 macrophages, likely driven by both an increased IDO1 activity and reduced QPRT gene expression. This leads to decreased concentrations of NAD+, potentially determining the phenotype of the macrophages. Future studies should address whether modulation of the kynurenine pathway restores NAD+ metabolism and leads to a decrease in inflammation and an increased stable plaque phenotype.

Background and aimsOncostatin M (OSM) signaling is implicated in atherosclerosis, however the mechanism remains unclear. We investigated the impact of common genetic variants in OSM and its receptors, OSMR and LIFR, on overall plaque vulnerability, plaque phenotype, intraplaque OSMR and LIFR expression, coronary artery calcification burden and cardiovascular disease susceptibility. Methods and resultsWe queried Genotype-Tissue Expression data and found that rs13168867 (C allele) was associated with decreased OSMR expression and that rs10491509 (A allele) was associated with increased LIFR expression in arterial tissues. No variant was significantly associated with OSM expression. We associated these two variants with plaque characteristics from 1,443 genotyped carotid endarterectomy patients in the Athero-Express Biobank Study. After correction for multiple testing, rs13168867 was significantly associated with an increased overall plaque vulnerability ({beta}=0.118 {+/-} s.e.=0.040, p=3.00x10-3, C allele). Looking at individual plaque characteristics, rs13168867 showed strongest associations with intraplaque fat ({beta}=0.248 {+/-} s.e.=0.088, p=4.66 x 10-3, C allele) and collagen content ({beta}=-0.259 {+/-} s.e.=0.095, p=6.22 x 10-3, C allele), but these associations were not significant after correction for multiple testing. rs13168867 was not associated with intraplaque OSMR expression. Neither was intraplaque OSMR expression associated with plaque vulnerability and no known OSMR eQTLs were associated with coronary artery calcification burden, or cardiovascular disease susceptibility. No associations were found for rs10491509 in the LIFR locus. ConclusionsOur study suggests that rs1316887 in the OSMR locus is associated with increased plaque vulnerability, but not with coronary calcification or cardiovascular disease risk. It remains unclear through which precise biological mechanisms OSM signaling exerts its effects on plaque morphology. However, the OSM-OSMR/LIFR pathway is unlikely to be causally involved in lifetime cardiovascular disease susceptibility.

BackgroundEvolocumab promotes coronary plaque regression in patients with coronary artery disease, but little is known regarding carotid plaques (CP). This study aimed to evaluate the impact of evolocumab on top of lipid-lowering therapy (ELLT) on carotid morphological stabilization (MS) and plaque regression (PR) compared to lipid-lowering therapy (LLT) alone. MethodsAsymptomatic patients with internal carotid stenosis[&ge;]50% and LDL-C[&ge;]100 mg/dL were randomized to ELLT or LLT and monitored by serial duplex ultrasound. The primary endpoint was a composite of 6-month-MS (i.e., switch from morphologic types I-II to III-IV) and/or 12-month-PR (i.e., reduction of carotid stenosis by at least 5% compared to baseline). The secondary endpoint was LDL-C change at 12 months. Major adverse vascular events (MAVE, i.e., cardiac death, stroke, myocardial infarction, carotid or coronary or peripheral revascularization) were recorded. ResultsA total of 170 patients were randomized. Mean carotid stenosis was 57%. At 6 months, MS occurred in the ELLT group (10.3%) only (p=0.29). At 12 months, PR was numerically more frequent in the ELLT group, without reaching statistical significance (43% versus 35.1%, p=0.42). The primary endpoint was met in 44.3% versus 35.1% (p=0.26). As compared to baseline, 6 and 12-month shifts from low to high-risk types were significantly higher in the LLT group (p=0.03). The 12-month LDL-C percentage reduction was -73.5% with ELLT, and -48.3% with LLT (p=0.0001). At 1 year, MAVE were significantly more frequent with LLT (14.6% versus 2.4%, p=0.005), and the absence of evolocumab was the only predictor (OR 7, p=0.014). ConclusionsIn patients with CP[&ge;]50% and LDL-C[&ge;]100 mg/dL, ELLT compared to LLT was associated with numerically but not statistically higher 6-month MS and/or 12-month PR. In the LLT group, 6- and 12-month changes from low to high-risk types, LDL-C, and MAVE were significantly higher. According to these results, evolocumab should be considered standard treatment for patients with CP[&ge;]50%. The study was registered at www.clinicaltrials.gov (NCT04730973) and Eudract (2020-005663-31). SHORT ABSTRACTPatients with carotid stenosis[&ge;]50% and LDL-C[&ge;]100 mg/dL were randomized to evolocumab on top of optimal lipid-lowering therapy (ELLT) or optimal lipid-lowering therapy (LLT) alone to assess the impact of ELLT on carotid plaque morphological stabilization (MS) and plaque regression (PR). At 6 and 12 months, MS and PR occurred in both groups, but were numerically higher in the ELLT group, without reaching statistical significance. In the LLT group, 6- and 12-month changes from low to high-risk types were significantly higher, and the rate of adverse vascular events was sevenfold higher. Evolocumab might become the standard treatment for patients with carotid artery stenosis [&ge;]50%. CLINICAL PERSPECTIVEO_ST_ABSWhat is new?C_ST_ABSO_LIThe CARUSO is the largest randomized trial evaluating the impact of evolocumab on top of lipid-lowering therapy (ELLT) on carotid morphological stabilization (MS) and plaque regression (PR) monitored by serial duplex ultrasound. C_LIO_LIThe primary endpoint was a composite of 6-month-MS (i.e., switch from morphologic types I-II to III-IV) and/or 12-month-PR (i.e., reduction of carotid stenosis by at least 5% compared to baseline) and was numerically higher in the ELLT group compared to lipid-lowering therapy (LLT) alone, without reaching statistical significance. C_LIO_LIThe 1-year rate of major adverse vascular events (MAVE) was sevenfold higher in the LLT group. C_LI What are the clinical implications?O_LICarotid plaque morphology is a dynamic event, and 6 and 12-month shifts from low to high-risk morphological types were significantly higher in the LLT group, thus suggesting that evolocumab added to LLT may prevent morphological deterioration. C_LIO_LIThe absence of evolocumab was the only independent predictor of MAVE; according to our results, ELLT might become the standard treatment for patients with carotid plaques [&ge;]50% and LDL-C not at target. C_LIO_LIFuture larger studies are warranted to validate our findings, assess long-term adherence to therapy, and identify subgroups with higher probability of achieving MS and PR. C_LI

BackgroundInflammation is a precursor to atherosclerotic plaque destabilisation, leading to ischaemic events such as stroke. Macrophage phenotypes can be altered by the microenvironment, and certain anti-inflammatory agents may, therefore, stabilise plaques and reduce the risk of recurrent ischaemic events. MethodsThirteen carotid plaques were obtained from stroke/ Transient Ischaemic Attack (TIA) patients undergoing carotid endarterectomy. An immunofluorescence stain was used to identify common macrophage markers (pan macrophage: CD68, pro-inflammatory: CD86, anti-inflammatory: MRC1), and a novel analysis technique was used to measure the prevalence of macrophage phenotypes in carotid plaques in relation to other histological features of instability. An in vitro model of human blood-derived macrophages was also developed to evaluate the effect of statins and glucocorticoids on macrophage-specific markers using RT-qPCR, Western Blot and immunofluorescence stain. The physiological effect of dexamethasone was further evaluated on macrophages and human carotid plaques cultured ex vivo. ResultsThe macrophage population (CD68+) in the carotid plaques was dominated by "double-positive" (CD86+MRC1+) macrophages (67.8%), followed by "M1-like" (CD86+MRC1-) (16.5%), "M2-like" (CD86-MRC1+) (8.7%) and "double-negative" (CD86-MRC1-) (7.0%) macrophages. M1-like macrophages were more prevalent in unstable plaque sections than stable ones (p=0.0022). Exposure to dexamethasone increased macrophage MRC1 gene expression in vitro and ex vivo. Dexamethasone also reduced Oxidised Low-Density Lipoprotein Receptor 1 (OLR1) gene and protein expression, leading to a decreased ox-LDL uptake in foam cell assays. This was, in turn, associated with reduced lipid uptake in macrophages, as shown by Oil Red O staining. ConclusionsHuman macrophages may be "switched" to a less inflammatory phenotype by exposure to clinically relevant concentrations of glucocorticoid, potentially mediated by a reduction in Oxidised LDL uptake. This effect was not observed following macrophage exposure to statins. Glucocorticoids may have a future role in preventing ischaemic events in patients with advanced atherosclerosis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/598440v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@bb389borg.highwire.dtl.DTLVardef@18151b8org.highwire.dtl.DTLVardef@11c28a1org.highwire.dtl.DTLVardef@1af6acd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA high prevalence (68% in this study) of carotid plaque macrophages express both pro-inflammatory (CD86) and anti-inflammatory (MRC1) markers. These may represent a novel macrophage population. C_LIO_LIHuman macrophages may be "reprogrammed" to a less inflammatory phenotype following exposure to glucocorticoids. C_LIO_LIDexamethasone increased MRC1 and decreased OLR1 expression in macrophages derived from human blood samples in vitro and in cells derived from cultured human carotid plaque tissue ex vivo. This was associated with reduced oxLDL uptake and reduced lipid accumulation in the macrophages. C_LIO_LIDexamethasone has the potential to stabilise carotid atherosclerotic plaques in humans. C_LI

Background and aimsCirculating PCSK9 is a crucial regulator of cholesterol metabolism. Loss-of-function variants in PCSK9 are associated with lower levels of circulating low-density lipoprotein cholesterol (LDL-C) and reduced cardiovascular disease (CVD) risk, while gain-of-function variants correlate with elevated LDL-C concentrations and increased CVD risk. This study investigated whether genetically determined LDL-C levels, proxied by four PCSK9 genetic variants, influence common carotid artery atherosclerosis. MethodsThe analysis included 3,040 European participants (mean age 64.2 {+/-} 5.4 years; 45.8% men) at high cardiovascular risk from the IMPROVE study, alongside 49,088 individuals of white British ancestry (mean age 55.2 {+/-} 7.6 years; 47.9% men) from the UK Biobank (UKB). Ultrasonographic measurements of common carotid intima-media thickness (CC-IMTmean, CC-IMTmax, CC-IMTmean-max) were obtained. Four lipid-level affecting genetic variants in the PCSK9 locus were selected for analysis, both individually and in a standardized polygenic risk score (PRS), to assess their effects on LDL-C and PCSK9 levels in the IMPROVE cohort and on ultrasonographic measures in both IMPROVE and UKB. ResultsIn the IMPROVE cohort, PCSK9 variants (rs11206510, rs2479409, rs11591147, rs11583680) exhibited expected effect directions, albeit not all statistically significant, on LDL-C and PCSK9 levels. The PRS was negatively correlated with CC-IMTmean, CC-IMTmax, and CC-IMTmean-max among women in IMPROVE, and among men and overall in UKB (all P < 0.05). Effect sizes were comparable between cohorts. ConclusionsGenetic variants in the PCSK9 locus influence LDL-C levels and CC-IMT, in keeping with proven benefits of PCSK9 inhibitors on atherosclerotic cardiovascular events.

BACKGROUNDVascular calcification significantly influences the onset and outcome of cardiovascular events, yet no effective treatment currently exists. Dysfunction of osteoclastic macrophages contributes to the formation of calcification. Our previous studies have shown that sonodynamic therapy (SDT) can rapidly reverse atherosclerotic plaques by targeting macrophages. This study aimed to investigate the effect of SDT on reducing early or mild vascular calcification by modulating the function of osteoclastic macrophages. METHODSThirty-two patients with symptomatic femoropopliteal peripheral artery disease (PAD) were recruited to evaluate changes in vessel CT values and the target-to-background ratio (TBR) using positron emission tomography/computed tomography (PET/CT) 30 days post-SDT. An early calcification model was established in ApoE-/- mice, followed by SDT intervention. Frozen plaque sections from the mice were collected for mass spectrometry imaging (MSI)-based spatial metabolic analysis in situ. The NHGRI-EBI GWAS Catalog database and the human single-cell eQTL database (scQTLbase) were employed to analyze the causal relationship between key enzyme genes involved in phosphatidic acid (PA) synthesis in macrophages and vascular calcification using two-sample Mendelian randomization. To investigate cell ossification, calcification, and underlying mechanisms, RAW264.7 mouse macrophages were treated with a medium containing receptor activator of nuclear factor kappa-B ligand (RANKL), while mouse aortic vascular smooth muscle cells (MOVAS cells) were exposed to a calcification medium. RESULTSSDT significantly reduced the number of mildly calcified sites and the target-to-background ratio (TBR) of these sites in patients with femoropopliteal peripheral artery disease (PAD). In ApoE-/- mice, SDT alleviated early calcification of atherosclerotic plaques. MSI revealed that SDT altered the composition and distribution of lipid metabolites in atherosclerotic plaques, notably increasing the content of PA in the early calcified regions. Analysis of single-cell sequencing databases showed that key enzyme genes involved in PA synthesis--PLD1, PLD3, AGPAT4, and diacylglycerol kinase E (DGKE)--were enriched in macrophages of human coronary artery plaques. Mendelian randomization analysis indicated that DGKE negatively regulated coronary artery calcification. In vitro studies demonstrated that PA mediates SDT to promote M1 macrophage fusion and enhance carbonic anhydrase II (CA2) expression, thereby improving osteoclastic function and alleviating early calcification of MOVAS cells via the reactive oxygen species (ROS)-DGKE-PA pathway. In vivo, the CA2 inhibitor acetazolamide impaired the effects of SDT and exacerbated early calcification of atherosclerotic plaques in ApoE-/- mice. CONCLUSIONThis study demonstrates that PA-mediated SDT promotes M1 macrophage fusion and CA2 expression, improving osteoclastic function and alleviating early calcification through the ROS-DGKE-PA pathway. REGISTRATIONURL: https://www.clinicaltrials.gov; Unique identifier: NCT03457662. What Is New?1. SDT reduces early calcification in patients with symptomatic femoropopliteal PAD and in ApoE-/- mouse models of early calcification. 2. SDT upregulates the expression of PA in the early calcified regions of ApoE-/- mouse models. 3. PA-mediated SDT facilitates M1 macrophage fusion and enhances CA2 expression, thereby improving osteoclastic function and alleviating early calcification through the ROS-DGKE-PA pathway. What Are the Clinical Implications?1. Targeting M1 macrophage DGKE-PA may serve as a potential intervention for treating early vascular calcification. 2. The combination of MSI and Mendelian randomization analysis proves to be an effective method for exploring key signaling lipids in disease. 3. PA-mediated SDT represents a promising approach for the effective reduction of early vascular calcification.

The use of senolytic agents to remove senescent cells from atherosclerotic lesions is controversial. A common limitation of previous studies is the failure to rigorously define the effects of senolytic agent ABT-263 (Navitoclax) on smooth muscle cells (SMC) despite studies claiming that they are the major source of senescent cells. Moreover, there are no studies of the effect of ABT-263 on endothelial cells (EC), which along with SMC comprise 90% of -SMA+ myofibroblast-like cells in the protective fibrous cap. Here we tested the hypothesis that treatment of advanced atherosclerotic mice with the ABT-263 will reduce lesion size and increase plaque stability. SMC (Myh11-CreERT2-eYFP) and EC (Cdh5-CreERT2-eYFP) lineage tracing Apoe-/- mice were fed a WD for 18 weeks, followed by ABT-263 100mg/kg/bw for six weeks or 50mg/kg/bw for nine weeks. ABT-263 treatment did not change lesion size or lumen area of the brachiocephalic artery (BCA). However, ABT-263 treatment reduced SMC by 90% and increased EC-contributions to lesions via EC-to-mesenchymal transition (EndoMT) by 60%. ABT-263 treatment also reduced -SMA+ fibrous cap thickness by 60% and increased mortality by >50%. Contrary to expectations, treatment of WD-fed Apoe-/- mice with the senolytic agent ABT-263 resulted in multiple detrimental changes including reduced indices of stability, and increased mortality. Graphical abstractUploaded separately. O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/548696v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@2ca235org.highwire.dtl.DTLVardef@4d864aorg.highwire.dtl.DTLVardef@1ab76e4org.highwire.dtl.DTLVardef@aa2c7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LITreatment of Apoe-/- mice with advanced atherosclerosis with the senolytic agent ABT-263 increased mortality by >50%. C_LIO_LIABT-263 showed a 90% reduction in SMC but a 60% increase in endothelial cell (EC) contributions to lesions via EC to mesenchymal transition (EndoMT) but prevented adaptive increases in investment of EC-derived cells into the fibrous cap via beneficial EndoMT to myofibroblast transitions that we have shown normally occur when SMC investment into fibrous cap of lesions is impaired. C_LIO_LIKnock out (KO) of Klf4 in SMC, which results in smaller but more stable atherosclerotic lesions, was associated with reduced expression of pro-senescence markers, but preserved expression of the anti-senescence marker, telomerase reverse transcriptase although it is unclear if the latter is causal or an effect. C_LI

AimsNucleotide-binding oligomerization domain-containing protein (NOD)1 is an intracellular pattern recognition receptor that initiates immune responses upon ligation of molecules such as bacterial peptidoglycan containing a D-glutamyl-meso-diaminopimelic acid (iE-DAP) moiety. NOD1 ligation has been shown to promote vascular inflammation and atherosclerosis. In this study, we investigate the functional role of NOD1 in atherosclerotic plaques and characterize the vascular cells responsible for NOD1 expression and function. Methods and resultsNOD1 was mainly expressed in a subtype of vascular smooth muscle cells (SMC) in human atherosclerotic lesions. In ex vivo cultures, human endarterectomy specimens reacted to NOD1 ligand by activation of mitogen-activated protein kinase (MAPK) pathways, leading to cytokine expression. Levels of NOD1 mRNA were higher in carotid endarterectomy specimens obtained from symptomatic patients compared to asymptomatic ones. NOD1high SMC were also found in arteries of atherosclerosis-prone Ldlr-/- mice. Challenging these mice with a NOD1 agonist resulted in transmural vascular inflammation, severe arterial damage, accelerated atherogenesis throughout the aorta, and evidence of occlusive coronary artery disease. In rats, mechanic injury to carotid arteries promoted NOD1high SMC expansion and neointima formation. In vitro, neointima derived NOD1high SMCs responded to NOD1 ligand exposure by enhanced migration, increased iNOS+ cells and amplified CCL5 production. ConclusionOur findings show that NOD1 promotes vascular inflammation, vascular injury responses and atherosclerosis by acting on a NOD1high subtype of SMC.

BackgroundAtherosclerosis (ATH) is a chronic systemic inflammatory disease affecting the vessel wall, wherein regulating non-coding RNAs play a crucial role. We previously demonstrated that miR-125b is upregulated in ATH and is a main regulator of cholesterol metabolism in macrophages. Herein we hypothesized that inhibiting miR-125b may attenuate ATH. Methods and resultsIn the ApoE-/- mice model fed with a high fat diet for 14 weeks, we inhibited miR-125b using an antagomiR over a 4-week period. We observed a significant reduction in plaque size, accompanied by diminished infiltration of F4/80 macrophages and attenuation of NF-{kappa}B+ activation within plaques. We explored the mechanism using a Vas-on-Chip adhesion assay using Human Aortic Endothelial Cells (HAoEC) stimulated with TNF. We observed an impairment in the trafficking of miR-125b transfected THP-1 monocytes, accompanied by the downregulation of the CD11b/CD18 integrin and the CCR7 receptor. Furthermore, we demonstrated a direct regulation of the CCR7 receptor by miR-125b using a reporter plasmid construct (p_CCR7.WT) containing the 3UTR region of CCR7 gene fused with a luciferase coding sequence. In addition, miR-125b transfected monocytes inhibited CCR7 cell migration induced by the CCL21 ligand but did not affect migration induced by others ligands such as MCP1. Finally, we confirmed the downregulation of CCR7 in coronary plaques in both ApoE-/- mice and patients with coronary artery disease. ConclusionsInhibiting miR-125b offers a novel therapeutic approach for ameliorating ATH that results in a reduction of macrophage content and plaque lesion size. This improvement occurs through the enhancement of monocyte trafficking via CCR7 that facilitates the exit of foam cells from the plaque. CLINICAL PERSPECTIVEO_ST_ABSWhat is New?C_ST_ABS- We found evidences of a new therapeutic approach for atherosclerosis, in which miR-125b inhibition reduces macrophage content and plaque size. - We described the molecular mechanism underlying miR-125b, which involves regulating of monocyte trafficking to plaques and the downregulation of the chemokine receptor CCR7. CCR7 plays a crucial role in facilitating the egress of macrophages and foam cells from plaques, and its downregulation contribute to progression of ATH. - The results have been validated in a cohort of patients with coronary artery disease, where CCR7 expression was reduced in plaques. What are the clinical implications?- We highlight the pivotal role of monocyte trafficking in the inflammatory mechanism of atherosclerosis. Managing miR-125b/CCR7 signaling may improve the resolution of ATH promoting the exit of foam cells from plaque. - Inhibition of miR-125b in plaque macrophages represents a novel and promising therapeutic approach for cardiovascular disease.

Background and aimsThe accumulation of erythrocyte membranes within an atherosclerotic plaque may contribute to the deposition of free cholesterol and thereby the enlargement of the necrotic core. Erythrocyte membranes can be visualized and quantified in the plaque by immunostaining for the erythrocyte marker glycophorin C. Consequently, we hypothesized, that the accumulation of erythrocytes quantified by glycophorin C is a marker for plaque vulnerability and may therefore reflect intraplaque hemorrhage (IPH), vulnerability of plaques and predict pre-procedural neurological symptoms. MethodsWe employed the CellProfiler-integrated slideToolKit workflow to visualize and quantify glycophorin C, defined as the total plaque area that is positive for glycophorin C, in single slides of culprit lesions obtained from the Athero-Express Biobank of 1,819 consecutive asymptomatic and symptomatic patients who underwent carotid endarterectomy. Our assessment included the evaluation of various parameters such as lipid core, calcifications, collagen content, SMC content, and macrophage burden. These parameters were evaluated using a semi-quantitative scoring method, and the resulting data was dichotomized as predefined criteria into categories of no/minor or moderate/heavy staining. In addition, the presence or absence of IPH was also scored. ResultsThe prevalence of IPH and pre-procedural neurological symptoms were 62.4% and 87.1%, respectively. The amount of glycophorin staining was significantly higher in samples from men compared to samples of women (median 7.15 (IQR:3.37, 13.41) versus median 4.06 (IQR:1.98, 8.32), p<0.001). Glycophorin C was associated with IPH adjusted for clinical confounders (OR 1.90; 95% CI 1.63, 2.21; p=<0.001). Glycophorin C was significantly associated with ipsilateral pre-procedural neurological symptoms (OR:1.27, 95%CI:1.06-1.41, p=0.005). Sex-stratified analysis, showed that this was also the case for men (OR 1.37; 95%CI 1.12, 1.69; p=0.003), but not for women (OR 1.15; 95%CI 0.77, 1.73; p=0.27). Glycophorin C was associated with classical features of a vulnerable plaque, such as a larger lipid core, a higher macrophage burden, less calcifications, a lower collagen and SMC content. There were marked sex differences, in men, glycophorin C was associated with calcifications and collagen while these associations were not found in women. ConclusionsThe accumulation of erythrocytes in atherosclerotic plaque quantified and visualized by glycophorin C was independently associated with the presence of IPH, preprocedural symptoms in men, and with a more vulnerable plaque composition in both men and women. These results strengthen the notion that the accumulation of erythrocytes quantified by glycophorin C can be used as a marker for plaque vulnerability.

BackgroundSex and plaque histology are intertwined, with fibrous atherosclerotic plaques being more prevalent in women and pointing to general smooth muscle cell plasticity and estrogen signaling. Plaque erosion, a significant contributor to acute coronary syndromes (ACSs), is linked to fibrous plaques and is more prevalent in women as compared to men. We hypothesize that the molecular drivers of histologically determined fibrous plaques differ between men and women. MethodsHuman end-stage atherosclerotic plaques were isolated from consecutive patients who underwent carotid endarterectomy and were included in the Athero-Express biobank. Fibrous plaques from both female and male patients were histologically assessed and further processed to obtain protein, bulk RNA, single-cell RNA and DNA methylation data. We leveraged sex-differential expression and deconvolution analyses to uncover sex-biased molecular mechanisms and cellular dynamics which were experimentally validated using an EndMT in vitro model. ResultsOut of 1,889 atherosclerotic plaques (1,309 male and 580 female), fibrous lesions were observed in 50% of female (n=290) and 31% of male patients (n=416). Compared to patients with atheromatous plaques (n=494), women with fibrous plaques exhibited a higher prevalence of smoking (41% vs. 33%), while men with fibrous plaques presented more often with diabetes (29% vs. 20%). Transcriptional and proteomic phenotyping highlighted more immune-dependent and inflammatory processes in male fibrous plaques. Genes and proteins with higher abundance in female fibrous plaques pointed to endothelial-to-mesenchymal transition (EndMT) and extracellular matrix remodelling. Using single-cell RNA sequencing, we identified a dominant role of endothelial and smooth muscle cells in female plaques, and more macrophages in males. Finally, at the cellular level, we show that sex - specific, smoking-mediated promoter methylation changes may explain these differences. ConclusionsPatients with end-stage fibrous atherosclerotic plaques have a distinct clinical profile, with men more often having diabetes and women more often smoking. This clinical profile associates with sex differences that point to different cellular and molecular compositions of fibrous plaques. These mechanisms might be candidate pathways to understand plaque erosion from a molecular point of view and may provide promising targets for atherosclerosis therapies, as they account for the sex-specific differences in plaque phenotype. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/24314739v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@f3f474org.highwire.dtl.DTLVardef@3606d4org.highwire.dtl.DTLVardef@fa8801org.highwire.dtl.DTLVardef@1f719f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundLipoprotein(a) (Lp(a)) is a causal risk-factor for atherosclerotic cardiovascular disease including acute ischemic stroke (AIS). The underlying pathomechanisms mediating this risk are less well understood, especially in AIS caused by large artery atherosclerosis (LAA). In this observational cohort study we evaluated the association of Lp(a) with markers of LAA, namely carotid intima media thickness (cIMT) and the presence of extra- or intracranial vessel narrowing plaques. MethodsAmong participants of the BIOSIGNAL cohort study we determined Lp(a) levels within 24h after symptom onset in 1161 AIS patients from the single center of Zurich. cIMT was determined using a semi-automated computerized edge tracking software, internal carotid artery (ICA) stenosis was graded according to the North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria, intracranial ultrasound was performed by transcranial color-coded duplex (TCCD). ResultsIn 1161 participants higher Lp(a) levels were not associated with an increased cIMT in univariable or multivariable regression models containing known cardiovascular risk-factors. Higher Lp(a) levels were not associated with the presence of neither extracranial high-grade ICA-stenosis nor significant intracranial stenosis assessed by neurovascular ultrasound. ConclusionIn AIS patients higher Lp(a) levels were not associated with clinical markers of arteriosclerotic burden despite its association with LAA-stroke etiology and an increased risk for stroke recurrence. RegistrationRegistration-URL: http://www.clinicaltrials.gov; Unique identifier: NCT-02274727

BackgroundLipoprotein(a) [Lp(a)] has been independently associated with increased cardiovascular risk. We examined the effect of monoclonal antibody proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) on plasma Lp(a) levels across multiple clinical trials. MethodsStudies were retrieved comparing the effect of PCSK9i vs. placebo on Lp(a) levels. The primary outcome was percent change in Lp(a) levels. Secondary outcomes included percent change in additional cholesterol markers. Factors associated with the treatment effect were determined by meta-regression analysis. Subgroup analyses were done to explore potential treatment effect differences based on comparator, PCSK9i type, treatment duration, and presence of familial hypercholesterolemia (FH). Results47 studies with 67,057 patients were analyzed. PCSK9i reduced Lp(a) levels on average of -27% (95% CI: -29.8 to -24.1, p<0.001). Concurrent reduction in LDL-C, non-HDL-C, total cholesterol, triglycerides ApoB, ApoA-1, and increased HDL-C were also observed with PCSK9i use. Factors associated with the treatment effect included mean percent change in LDL-C (p=0.02, tau2=177.1, R2=0.00) and Apo-B (p<0.00, tau2=114.20, R2=1.42). Subgroup analyses revealed consistent treatment effect amongst comparators (vs. placebo: -27.69% (95% CI: - 30.85 to -24.54, p<0.00), vs. ezetimibe: -24.0% (95% CI: -29.95% to -18.01, p<0.00), type of PCSK9i, evolocumab: -29.35% (95% CI: -33.56 to -25.14, p<0.00) vs. alirocumab: -24.50% (95% CI: -27.96 to -21.04, p<0.00), and presence of FH: -25.63% (95% CI: -31.96% to -19.30, p<0.00 vs. no FH: -27.22% (95% CI: -30.34. to -24.09, p<0.00). Varying treatment effects were noted in the duration of treatment (12 weeks or shorter: -32.43% (95% CI: -36.63 to -28.23 vs. >12 weeks: -22.31% (95% CI: -25.13 to -19.49, p<0.00), p interaction <0.01. ConclusionPCSK9 inhibitors reduce Lp(a) levels by an average of 27%. Mean percent change in LDL-C and Apo-B were associated with treatment effect. PCSK9i also significantly reduced other atherogenic lipoproteins. Across multiple clinical trials, PCSK9i has a consistent effect of significantly lowering Lp(a) levels.

BackgroundThromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions are the leading cause of death in the world. The most common and effective means to reduce these major adverse cardiovascular events (MACE), including myocardial infarction (MI) and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, little is known regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. MethodsSmooth muscle cell (SMC)-lineage tracing Apoe-/- mice were fed a Western diet (WD) for 18 weeks and then switched to a low-fat chow diet for 12 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery (BCA) lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of SMC, and other lesion cells by SMC-lineage tracing combined with scRNA-seq, CyTOF, and immunostaining plus high resolution confocal microscopic z-stack analysis. In addition, to determine if treatment with a potent inhibitor of inflammation could augment the benefits of chow diet-induced reductions in LDL-cholesterol, SMC-lineage tracing Apoe-/- mice were fed a WD for 18 weeks and then chow diet for 12 weeks prior to treating them with an IL-1{beta} or control antibody (Ab) for 8-weeks. ResultsLipid-lowering by switching Apoe-/- mice from a WD to a chow diet reduced LDL-cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1{beta} Ab treatment resulted in multiple detrimental changes including increased plaque burden, BCA lesion size, as well as increased cholesterol crystal accumulation, intra-plaque hemorrhage, necrotic core area, and senescence as compared to IgG control Ab treated mice. Furthermore, IL-1{beta} Ab treatment upregulated neutrophil degranulation pathways but down-regulated SMC extracellular matrix pathways likely important for the protective fibrous cap. ConclusionsTaken together, IL-1{beta} appears to be required for chow diet-induced reductions in plaque burden and increases in multiple indices of plaque stability. Clinical PerspectiveAlthough one must be cautious in extrapolating results of mouse studies to humans, the current and our previous studies (Gomez et al. 2018 Nature Medicine) suggest that efforts to identify anti-inflammatory therapies for treating patients with advanced atherosclerosis should consider the possibility that inhibiting a given cytokine may have a mixture of beneficial and detrimental effects that vary between individuals. What is New?In a mouse model of advanced atherosclerosis followed by diet-induced reductions in cholesterol, IL-1{beta} inhibition unexpectedly had multiple detrimental effects including increasing overall plaque burden and decreasing various indices of plaque stability, as well as markedly increasing the number of senescent cells, and cholesterol crystal accumulation in lesions as compared to IgG control Ab treated mice. What are the clinical Implications?Results suggest that some anti-inflammatory therapies may have limited efficacy for treating patients with advanced atherosclerosis because such therapies inhibit not only detrimental pro-inflammatory responses, but also evolutionarily conserved beneficial inflammatory processes, which play a critical role in resistance to pathogenic microorganisms, in tissue repair following injury, and resolution of inflammation. We propose that the latter includes clearance of senescent cells and cholesterol from advanced atherosclerotic lesions induced by dietary-induced lipid lowering.

BackgroundPreviously we reported a time dependent change in atherosclerotic carotid plaque characteristics, with decreasing destabilising characteristics between 2002 -2011. This observation was considered confirmative with a parallel improved adherence to medication and decrease in overall cardiovascular mortality in Western Europe. In the present study, we investigated if these time dependent changes in plaque characteristics still occurred over the last decade. MethodsUsing the Athero Express biobank, atherosclerotic plaques of 1,277 consecutive carotid endarterectomy patients included between 2010 and 2021 were analysed to examine time dependent changes in histological plaque characteristics in intervals of 2 years. These results were compared with our previous time dependent plaque composition data between 2002-2011. ResultsIn contrast to the period 2002-2011, the period 2010-2021 showed a significant increase in vulnerable plaque characteristics: large lipid cores, intraplaque hemorrhage, macrophages, and calcification. When adjusted for confounders related to these plaque characteristics, such as time to operation and pre-operative type of symptoms, the adjusted odds ratios per 2 years increase in time were 1,177 (95% confidence interval, 1,070-1,293; p<0,001) for calcification, 1,352 (95% confidence interval, 1,229-1,487; p<0,000) for intraplaque hemorrhage, 1,277 (95% confidence interval, 1,159-1,407; p<0,001) for plaques consisting of >40% of fat and 1,388 (95% confidence interval, 1,262-1,528; p<0,001) for macrophages. Use of statins increased in both the 2002-2011 and 2010-2021 period. ConclusionOur study did not support a further increase in plaque stabilizing features in carotid atherosclerotic plaques between 2010-2021. In contrast, plaques even showed features of destabilisation in the present cohort.

AimsCarotid atherosclerosis is a leading cause of ischemic stroke, driven by plaque inflammation. While current carotid imaging focuses on luminal stenosis, quantitative perivascular adipose tissue attenuation (PVAT) on computed tomography angiography has emerged as a potential non-invasive biomarker for arterial inflammation and plaque instability in coronary arteries. This systematic review and meta-analysis consider the utility of PVAT for imaging and risk-stratifying inflamed and vulnerable carotid atherosclerotic plaques. Methods and ResultsFollowing PRISMA guidelines, a systematic literature search identified 11 studies suitable for meta-analysis, incorporating 2316 patients. We compared continuous PVAT measures between symptomatic and asymptomatic carotid arteries using mean differences and 95% confidence intervals, employing a random-effects meta-analysis due to substantial heterogeneity (I{superscript 2} = 92%). The pooled mean difference (MD) was 12.63 HU (95% CI: 8.39-16.88), indicating significantly higher PVAT in symptomatic arteries. While funnel plot asymmetry was visually observed, Eggers test (intercept = 7.29, p = 0.044) confirmed statistically significant publication bias, highlighting the need to apply caution when interpreting the results given the high heterogeneity. Notably, the leave-one-out sensitivity analysis showed no significant change in the overall p-value or substantial shifts in statistical heterogeneity. ConclusionThis meta-analysis supports PVAT as a promising non-invasive imaging marker for inflammation and vulnerable plaques in symptomatic carotid atherosclerosis. The findings, while subject to heterogeneity and requiring further validation against conflicting individual study results, suggest a possible role of carotid PVAT for risk stratification. Future directions may PVAT use for monitoring treatment response in clinical trials.