Abstract Objectives Vascular calcification (VC) is a hallmark of atherosclerosis and commonly found in aging population, increasing the risk of myocardial infarction (MI), stroke and mortality. Our data shows that Wnt/β-catenin signaling pathway and its main inhibitor, sclerostin (Scl, Sost) could be central to this process. However, Scl inhibition with romosozumab has been associated with a higher incidence of major adverse cardiovascular events (MACE). This increases in MACE call into question the safety of romosozumab use, particularly in patients with cerebrovascular and cardiovascular history. However, whether Scl expression levels in atherosclerotic plaques are associated with clinical ischemic events remains unknown. Here we explored the association between Scl expression in human atherosclerosis plaques and the extend of VC, VSMCs phenotypic switch to osteoblast-like cells (OBL), and its correlation to clinical cerebrovascular events. Methods Carotid plaque specimens of symptomatic (ischemic stroke) and asymptomatic patients undergoing endarterectomy for severe carotid stenosis were co-stained with VSMCs specific marker (Myh11), OBL marker (RUNX2), microcalcification sensitive fluorescent imaging probes (OsteoSense) and Scl and used for quantification analyses. Results scRNA seq data showed that Sost is upregulated by VSMCs in the aortic region protected against VC, suggesting an inhibitory role of Sost on VSMCs differentiation into OBL. Carotid plaque specimens from asymptomatic patients exhibited a significantly higher percentage of VSMCs expressing Scl (Myh11+Scl+) in parallel with a greater Scl+ area versus symptomatic patients. A negative correlation was observed between the percentage of Myh11+Scl+ cells and the extent of microcalcification (OsteoSense) as well as between the percentage of total Scl+ cells and induction of VSMCs phenotype switch to osteoblast-like cells (quantified as RUNX positive area). Conclusion In atherosclerotic plaques of patients with ischemic stroke, total and VSMCs -specific Scl expression levels were reduced as compared to asymptomatic patients. Furthermore, reduction in Scl levels was positively associated with the extent of microcalcification, and VMSCs phenotypic switch to OBL. The presented findings suggest that sclerostin expression in human atherosclerotic plaques could contribute to plaque stabilization and be protective against ischemic stroke, explaining why Scl inhibition with romosozumab resulted in a higher incidence of MACE.Figure 1Figure 2

Aims: The heart rejuvenating effects of circulating growth differentiation factor 11 (GDF11), a transforming growth factor-beta superfamily member that shares 90% homology with myostatin (MSTN), remains controversial. Here, we aimed to probe the role of GDF11 in acute myocardial infarction (MI), a frequent cause of heart failure and premature death during ageing. Methods and results: In contrast to endogenous Mstn, myocardial Gdf11 declined during the course of ageing and was particularly reduced following ischaemia/reperfusion (I/R) injury, suggesting a therapeutic potential of GDF11 signalling in MI. Unexpectedly, boosting systemic Gdf11 by recombinant GDF11 delivery (0.1mg/kg body weight over 30 days) prior to myocardial I/R augmented myocardial infarct size in C57BL/6 mice irrespective of their age, predominantly by accelerating pro-apoptotic signalling. While intrinsic cardioprotective signalling pathways remained unaffected by high circulating GDF11, targeted transcriptomics and immunomapping studies focusing on GDF11-associated downstream targets revealed attenuated Nkx2-5 expression confined to CD105-expressing cells, with pro-apoptotic activity, as assessed by caspase-3 levels, being particularly pronounced in adjacent cells, suggesting an indirect effect. By harnessing a highly specific and validated liquid chromatography-tandem mass spectrometry-based assay, we show that in prospectively recruited patients with MI circulating GDF11 but not MSTN levels incline with age. Moreover, GDF11 levels were particularly elevated in those at high risk for adverse outcomes following the acute event, with circulating GDF11 emerging as an independent predictor of myocardial infarct size, as estimated by standardized peak creatine kinase-MB levels. Conclusion: Our data challenge the initially reported heart rejuvenating effects of circulating GDF11 and suggest that high levels of systemic GDF11 exacerbate myocardial injury in mice and humans alike. Persistently high GDF11 levels during ageing may contribute to the age-dependent loss of cardioprotective mechanisms and thus poor outcomes of elderly patients following acute MI.

Background: Atherosclerosis is a chronic inflammatory vascular disease and the main cause of death and morbidity. Emerging evidence suggests that ubiquitination plays an important role in the pathogenesis of atherosclerosis including control of vascular inflammation, vascular smooth muscle cell (VSMC) function and atherosclerotic plaque stability. Peli1 a type of E3 ubiquitin ligase has emerged as a critical regulator of innate and adaptive immunity, however, its role in atherosclerosis remains to be elucidated. Methods: Apoe−/− mice and Peli1-deficient Apoe−/− Peli1−/− mice were subject to high cholesterol diet. Post sacrifice, serum was collected, and atherosclerotic plaque size and parameters of atherosclerotic plaque stability were evaluated. Immunoprofiling and foam cell quantification were performed. Results: Peli1 deficiency does not affect atherosclerosis lesion burden and cholesterol levels, but promotes VSMCs foam cells formation, necrotic core expansion, collagen, and fibrous cap reduction. Apoe−/− Peli1−/− mice exhibit a storm of inflammatory cytokines, expansion of Th1, Th1, Th17, and Tfh cells, a decrease in regulatory T and B cells and induction of pro-atherogenic serum level of IgG2a and IgE. Conclusions: In the present study, we uncover a crucial role for Peli1 in atherosclerosis as an important regulator of inflammation and VSMCs phenotypic modulation and subsequently atherosclerotic plaque destabilization.

(1) Background: Monocytes and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome orchestrate lipid-driven amplification of vascular inflammation promoting the disruption of the fibrous cap. The components of the NLRP3 inflammasome are expressed in macrophages and foam cells within human carotid atherosclerotic plaques and VSMCs in hypertension. Whether monocytes and NLRP3 inflammasome activation are direct triggers of VSMC phenotypic switch and plaque disruption need to be investigated. (2) Methods: The direct effect of oxLDL-activated monocytes in VSMCs co-cultured system was demonstrated via flow cytometry, qPCR, ELISA, caspase 1, and pyroptosis assay. Aortic roots of VSMCs lineage tracing mice fed normal or high cholesterol diet and human atherosclerotic plaques were used for immunofluorescence quantification of NLRP3 inflammasome activation/VSMCs phenotypic switch. (3) Results: OxLDL-activated monocytes reduced α-SMA, SM22α, Oct-4, and upregulation of KLF-4 and macrophage markers MAC2, F4/80 and CD68 expression as well as caspase 1 activation, IL-1β secretion, and pyroptosis in VSMCs. Increased caspase 1 and IL-1β in phenotypically modified VSMCs was detected in the aortic roots of VSMCs lineage tracing mice fed high cholesterol diet and in human atherosclerotic plaques from carotid artery disease patients who experienced a stroke. (4) Conclusions: Taken together, these results provide evidence that monocyte promote VSMC phenotypic switch through VSMC NLRP3 inflammasome activation with a likely detrimental role in atherosclerotic plaque stability in human atherosclerosis.

Background and Aims : Non-Alcoholic Fatty Liver Disease (NAFLD) represents an increasing cause of liver disease worldwide. Cardiovascular disease is the leading cause of morbidity and mortality in these patients. Although NAFLD pathophysiology is not fully understood alterations in fat metabolism seem to play a role. Autoantibodies against apolipoprotein A-1 (AAA1) are a novel cardiovascular risk factor with a metabolic role and a function as a disruptor of the cholesterol pathway. This study aims at evaluating a possible role of anti-apoA-1 IgG in NAFLD.Methods: Sera from 137 NAFLD patients were tested for AAA1 prevalence. Protein expression was assessed in the human hepatic cell line HepaRG by western blot analysis. Bodipy and Oil Red O staining were used to evaluate lipid content in hepatocytes and liver sections from ApoE-/- mice respectively.Results: Elevated AAA1 seropositivity was found in patients with NAFLD (46%). In vitro, AAA1 induced lipid accumulation in hepatocytes (5.9 vs 2.5, p=0.0008) and this lipid overload was associated with a high SREBP1 but not SREBP2 expression. AAA1 caused a significant TLR2-mediated increase of the proinflammatory cytokines IL-6 (680 vs. 163 pg/mL, P=0.03), TNF-a (391 vs 266 pg/mL, P=0.04) and IL-8 (174.1 vs. 72.6 ng/mL, P=0.03) detected in hepatocyte supernatants. In vivo, AAA1 induced higher lipid accumulation in the livers of ApoE-/- mice (1.23 vs 0.53, p=0.03).Conclusions: Anti-apoA-1 IgG are frequent in NAFLD, cause a strong inflammatory response and promote lipid accumulation in human hepatocytes and mice. We hypothesize that AAA1 may be a potential contributor in the development of NAFLD. Background and Aims : Non-Alcoholic Fatty Liver Disease (NAFLD) represents an increasing cause of liver disease worldwide. Cardiovascular disease is the leading cause of morbidity and mortality in these patients. Although NAFLD pathophysiology is not fully understood alterations in fat metabolism seem to play a role. Autoantibodies against apolipoprotein A-1 (AAA1) are a novel cardiovascular risk factor with a metabolic role and a function as a disruptor of the cholesterol pathway. This study aims at evaluating a possible role of anti-apoA-1 IgG in NAFLD. Methods: Sera from 137 NAFLD patients were tested for AAA1 prevalence. Protein expression was assessed in the human hepatic cell line HepaRG by western blot analysis. Bodipy and Oil Red O staining were used to evaluate lipid content in hepatocytes and liver sections from ApoE-/- mice respectively. Results: Elevated AAA1 seropositivity was found in patients with NAFLD (46%). In vitro, AAA1 induced lipid accumulation in hepatocytes (5.9 vs 2.5, p=0.0008) and this lipid overload was associated with a high SREBP1 but not SREBP2 expression. AAA1 caused a significant TLR2-mediated increase of the proinflammatory cytokines IL-6 (680 vs. 163 pg/mL, P=0.03), TNF-a (391 vs 266 pg/mL, P=0.04) and IL-8 (174.1 vs. 72.6 ng/mL, P=0.03) detected in hepatocyte supernatants. In vivo, AAA1 induced higher lipid accumulation in the livers of ApoE-/- mice (1.23 vs 0.53, p=0.03). Conclusions: Anti-apoA-1 IgG are frequent in NAFLD, cause a strong inflammatory response and promote lipid accumulation in human hepatocytes and mice. We hypothesize that AAA1 may be a potential contributor in the development of NAFLD.

Background: Atherosclerosis is a chronic inflammatory disease where macrophages participate in the progression of the disease. However, the role of resident-like macrophages (res-like) in the atherosclerotic aorta is not completely understood. Methods: A single-cell RNA sequencing analysis of CD45+ leukocytes in the atherosclerotic aorta of apolipoprotein E–deficient (Apoe−/−) mice on a normal cholesterol diet (NCD) or a high cholesterol diet (HCD), respecting the side-to-specific predisposition to atherosclerosis, was performed. A population of res-like macrophages expressing hyaluronan receptor LYVE-1 was investigated via flow cytometry, co-culture experiments, and immunofluorescence in human atherosclerotic plaques from carotid artery disease patients (CAD). Results: We identified 12 principal leukocyte clusters with distinct atherosclerosis disease-relevant gene expression signatures. LYVE-1+ res-like macrophages, expressing a high level of CC motif chemokine ligand 24 (CCL24, eotaxin-2), expanded under hypercholesteremia in Apoe−/− mice and promoted VSMC phenotypic modulation to osteoblast/chondrocyte-like cells, ex vivo, in a CCL24-dependent manner. Moreover, the abundance of LYVE-1+CCL24+ macrophages and elevated systemic levels of CCL24 were associated with vascular calcification and CAD events. Conclusions: LYVE-1 res-like macrophages, via the secretion of CCL24, promote the transdifferentiation of VSMC to osteogenic-like cells with a possible role in vascular calcification and likely a detrimental role in atherosclerotic plaque destabilization.

Background Antiphospholipid antibody (aPL) syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies and thromboembolic or pregnancy complications. Although cryptic epitope R39-R43 belonging to beta-2-glycoprotein 1 (β2GP1) has been identified as the main antigenic determinant for aPLs, we have recently demonstrated that the epitope is a motif determined by the polarity, rather than by the sequence or charge of amino acids. Objective In the present study, we wanted to identify the association of residues needed to obtain the highest aPL affinity. Methods Based on the epitope R39-R43 and our identified motif, we generated a printed peptide microarray of 676 different peptides. These peptides have been then screened for their ability to interact with the plasmas from 11 well-characterized APS patients and confirmed by surface plasma resonance assay. Results and Conclusions We identified a peptide that selectively bound immunoglobulin G (IgG) derived from APS patients with 100 times more affinity than β2GP1, Domain I, or epitope R39-R43. This peptide is able to inhibit the activity of IgG derived from APS patients in vitro. We have also generated a monoclonal IgG antibody against this peptide. Using both peptide and monoclonal antibody, we have been able to develop a fully standardized indirect colorimetric immunoassay with highly sensitivity. The identification of the optimized peptide offers a new standardized and accurate tool for diagnostics of APS. Furthermore, having increased affinity for aPL, this peptide could represent a useful tool as prevention strategy for APS and an alternative to the use of anticoagulants.

Background and Aims: Atherosclerosis is a chronic inflammatory disease, in which lipids accumulate in arterial walls and lead to plaque formation. This process is largely driven by the immune system and monocytes/macrophages are the main immune cells present in atherosclerotic lesions. In this context, high levels of circulating LDL induce its engulfment and retention by macrophages, forming foam cells. The modulation of LDL absorbance is thus wildly used to manage atherosclerosis. The complex process permitting the regulation of LDL is however not fully understood.

Abstract Background Non-Alcoholic Fatty Liver Disease (NAFLD) represents an increasing cause of liver disease worldwide. Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in these patients. Although NAFLD pathophysiology is not fully understood alterations in fat metabolism seem to play a role. Autoantibodies against apolipoprotein A-1 (anti-apoA-1 IgG) are a novel cardiovascular risk factor to which have been recently attributed a metabolic role in addition to a well-established macrophage-mediated inflammatory effect and have a function as a disruptor of the cholesterol pathway. Purpose This study aims at evaluating a possible role of anti-apoA-1 IgG in NAFLD. Methods Serum from 137 NAFLD patients were tested for anti-apoA-1 IgG prevalence. In vitro, SREBP1, SREBP2 expressions were assessed in the human hepatic cell line HepaRG by western blot analysis and bodipy staining was used to evaluate the lipid droplet content. Mescoscale technology platform was used to measure TNF-α, IL-6 and IL-8 cytokines/chemokines in HepaRG supernatants. Oil Red O staining was used to detect lipid accumulation in liver sections from ApoE−/− mice. Results Elevated anti-apoA-1 IgG seropositivity was found in patients with NAFLD (46%). In vitro, anti-apoA-1 IgG and not control IgG induced lipid accumulation in hepatic cells (5.9 vs 2.5, P=0.0008) and this lipid overload was associated with a high SREBP1 but not SREBP2 expression. Furthermore, anti-apoA-1 IgG and not control antibodies caused a significant large increase of the proinflammatory cytokines IL-6 (680 vs. 163 pg/mL, P=0.03) and TNF-α (391 vs 266 pg/mL, P=0.04) as well as of the chemokine IL-8 (174.1 vs. 72.6 ng/mL, P=0.03) detected in the hepatic cell supernatants. In vivo, anti-apoA-1 IgG and not control IgG also induced higher lipid accumulation in the livers of ApoE−/− mice (1.23 vs 0.53, P=0.03). Conclusion Anti-apoA-1 IgG are frequent in NAFLD, cause a strong inflammatory response and promote lipid accumulation through SREBP1 activation in human hepatic cells. We hypothesize that anti-apoA1 IgG may be a potential contributor in the development of NAFLD. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Geneva University Hospital

(1) Background: Vascular smooth muscle cells (VSMCs) undergo a complex phenotypic switch in response to atherosclerosis environmental triggers, contributing to atherosclerosis disease progression. However, the complex heterogeneity of VSMCs and how VSMC dedifferentiation affects human carotid artery disease (CAD) risk has not been clearly established. (2) Method: A single-cell RNA sequencing analysis of CD45− cells derived from the atherosclerotic aorta of Apolipoprotein E-deficient (Apoe−/−) mice on a normal cholesterol diet (NCD) or a high cholesterol diet (HCD), respecting the site-specific predisposition to atherosclerosis was performed. Growth Differentiation Factor 10 (GDF10) role in VSMCs phenotypic switch was investigated via flow cytometry, immunofluorescence in human atherosclerotic plaques. (3) Results: scRNAseq analysis revealed the transcriptomic profile of seven clusters, five of which showed disease-relevant gene signature of VSMC macrophagic calcific phenotype, VSMC mesenchymal chondrogenic phenotype, VSMC inflammatory and fibro-phenotype and VSMC inflammatory phenotype. Osteoblast factor GDF10 involved in ossification and osteoblast differentiation emerged as a hallmark of VSMCs undergoing phenotypic switch. Under hypercholesteremia, GDF10 triggered VSMC osteogenic switch in vitro. The abundance of GDF10 expressing osteogenic-like VSMCs cells was linked to the occurrence of carotid artery disease (CAD) events. (4) Conclusions: Taken together, these results provide evidence about GDF10-mediated VSMC osteogenic switch, with a likely detrimental role in atherosclerotic plaque stability.

Background and Aims: Atherosclerosis is a chronic inflammatory disease characterized by the build-up of subendothelial deposition of cholesterol and the formation of leukocyte-rich plaques in the arteries. Peli1 ubiquitin ligase has an essential role in mediating activation of NF-κB via TLR3 and TLR4 shown to play a role in atherosclerosis. Peli1 has ligase activities, but whether it has a distinct function in atherosclerosis development remains unknown.

Background and Aims: Atherosclerosis is a lipid driven, chronic inflammatory disease. The monocytes expansion is causally linked to the enlargement of the atherosclerotic lesion, while NLRP3 inflammasome activation has a major role in atherogenesis progression. We aim to reveal the effect of monocytes/macrophages on NLRP3 inflammasome activation in vascular smooth cells (VSMCs) and their phenotypic switch/foam cells formation.

Abstract Objectives Autoantibodies against apolipoprotein A1 (anti‐apoA1 IgGs) and its C‐terminal region (cter apoA1) have emerged as an independent biomarker for cardiovascular disease. Cter apoA1 mimetic peptides were shown to reverse the deleterious anti‐apoA1 IgG effects in vitro. We evaluated the association of anti‐cter apoA1 IgGs with overall mortality in the general population and tested the ability of a cter apoA1 mimetic peptide to reverse the anti‐apoA1 IgG‐induced inflammatory response and mortality in vitro and in vivo, respectively. Methods Anti‐cter apoA1 IgGs were measured in serum samples of 6386 participants of the CoLaus study of which 5220 were followed for a median duration of 5.6 years. The primary outcome was overall mortality. The peptide inhibitory concentration 50% (IC50) was determined in vitro on HEK‐Blue‐4 and RAW cells. ApoE−/− mice were exposed to 16 weeks of anti‐apoA1IgG passive immunisation with and without peptide co‐incubation. Results Anti‐cter apoA1 IgGs were associated with higher interleukin 6 levels and independently predicted overall mortality; an increase of one standard deviation of anti‐cter apoA1 IgG level was associated with an 18% increase in mortality risk (hazard ratio: 1.18, 95% confidence interval: 1.04–1.33; P = 0.009). The cterApoA1 analogue reversed the antibody‐mediated inflammatory response with an IC50 of 1 µm in vitro but did not rescue the significant anti‐apoA1 IgG‐induced mortality rate in vivo (69% vs. 23%, LogRank P = 0.02). Conclusion Anti‐cter apoA1 IgG independently predicts overall mortality in the general population. Despite being effective in vitro, our cter apoA1 analogue did not reverse the anti‐apoA1 IgG‐induced mortality in mice. Our data suggest that these autoantibodies are not readily treatable through cognate peptide immunomodulation.

Background and Aims : B cells influence atherosclerotic plaque development with transforming growth factor-β (TGFβ) signaling as one of the main regulatory pathways in B cells. The present study aimed to investigate how TGFβ1 deficiency specifically in B cells affects atherosclerosis disease.Methods: Apoe-/- and Apoe-/- transgenic mice with B-cells specific TGFβ1 deficiency (BTGFβ1-/-) were fed high cholesterol diet for 11 weeks. At sacrifice, serum was collected, atherosclerotic plaques size and stability were evaluated, flow cytometry analyses of lymph nodes (LN), spleen, liquid peritoneal (LP) and aorta immune cells population were performed.Results: Apoe-/- BTGFβ1-/- mice exhibited a reduction in B1 and B2 cell subsets and a pronounced increase of plasma B cells in the spleen, LP and aorta versus Apoe-/- mice. Importantly, Apoe-/- BTGFβ1-/- mice showed lower plasma levels of cholesterol and LDL, while immunoglobulins IgA levels and IgG1-oxLDL-specific levels raised significantly compared to Apoe-/- mice. Importantly, Apoe-/- BTGFβ1-/-mice had smaller atherosclerotic plaque size and showed signs of plaques stability in comparison to Apoe-/- mice.Conclusions: This is the first study to demonstrate that B-cells specific TGFβ1 deficiency in Apoe-/- mice have a protective effect on atherosclerotic disease and regulates the levels of plasma levels of cholesterol. Background and Aims : B cells influence atherosclerotic plaque development with transforming growth factor-β (TGFβ) signaling as one of the main regulatory pathways in B cells. The present study aimed to investigate how TGFβ1 deficiency specifically in B cells affects atherosclerosis disease. Methods: Apoe-/- and Apoe-/- transgenic mice with B-cells specific TGFβ1 deficiency (BTGFβ1-/-) were fed high cholesterol diet for 11 weeks. At sacrifice, serum was collected, atherosclerotic plaques size and stability were evaluated, flow cytometry analyses of lymph nodes (LN), spleen, liquid peritoneal (LP) and aorta immune cells population were performed. Results: Apoe-/- BTGFβ1-/- mice exhibited a reduction in B1 and B2 cell subsets and a pronounced increase of plasma B cells in the spleen, LP and aorta versus Apoe-/- mice. Importantly, Apoe-/- BTGFβ1-/- mice showed lower plasma levels of cholesterol and LDL, while immunoglobulins IgA levels and IgG1-oxLDL-specific levels raised significantly compared to Apoe-/- mice. Importantly, Apoe-/- BTGFβ1-/-mice had smaller atherosclerotic plaque size and showed signs of plaques stability in comparison to Apoe-/- mice. Conclusions: This is the first study to demonstrate that B-cells specific TGFβ1 deficiency in Apoe-/- mice have a protective effect on atherosclerotic disease and regulates the levels of plasma levels of cholesterol.

Background and Aims: Atherosclerosis is an inflammatory disease with complex plaque diversity. We performed scRNAseq to characterize in-depth the phenotypic and transcriptional diversity of CD45- and CD45+ cells derived from athero-prone (arch/roots) and athero-resistant (thoracic) aorta in Apoe-/- mice.

AbstractAimsB cell functions in the process of atherogenesis have been investigated but several aspects remain to be clarified.Methods and resultsIn this study, we show that follicular regulatory helper T cells (TFR) control regulatory B cell (BREG) populations in Apoe−/− mice models on a high-cholesterol diet (HCD). Feeding mice with HCD resulted in up-regulation of TFR and BREG cell populations, causing the suppression of proatherogenic follicular helper T cell (TFH) response. TFH cell modulation is correlated with the growth of atherosclerotic plaque size in thoracoabdominal aortas and aortic root plaques, suggesting that TFR cells are atheroprotective. During adoptive transfer experiments, TFR cells transferred into HCD mice decreased TFH cell populations, atherosclerotic plaque size, while BREG cell population and lymphangiogenesis are significantly increased.ConclusionOur results demonstrate that, through different strategies, both TFR and TFH cells modulate anti- and pro-atherosclerotic immune processes in an Apoe−/− mice model since TFR cells are able to regulate both TFH and BREG cell populations as well as lymphangiogenesis and lipoprotein metabolism.

Anti-apolipoprotein A-1 (anti-apoA-1 IgG) and anti-double stranded DNA (anti-dsDNA IgG) autoantibodies have been described as mediators of atherogenesis in mice and humans. In the present study, we aim to investigate the association between atherosclerotic parameters, autoantibodies and plaque vulnerability in the context of systemic lupus erythematosus (SLE). We therefore bred a lupus prone-mouse model (Nba2.Yaa mice) with Apoe−/− mice resulting in Apoe−/−Nba2.Yaa mice spontaneously producing anti-apoA-1 IgG antibodies. Although Apoe−/−Nba2.Yaa and Apoe−/− mice subject to a high cholesterol diet displayed similar atherosclerosis lesions size in aortic roots and abdominal aorta, the levels of macrophage and neutrophil infiltration, collagen, MMP-8 and MMP-9 and pro-MMP-9 expression in Apoe−/−Nba2.Yaa mice indicated features of atherosclerotic plaque vulnerability. Even though Apoe−/−Nba2.Yaa mice and Apoe−/− mice had similar lipid levels, Apoe−/−Nba2.Yaa mice showed higher anti-apoA-1 and anti-dsDNA IgG levels. Apoe−/−Nba2.Yaa mice displayed a reduction of the size of the kidney, splenomegaly and lymph nodes (LN) hypertrophy. In addition, anti-apoA-1 and anti-dsDNA IgG increased also in relation with mRNA levels of GATA3, IL-4, Bcl-6 and CD20 in the spleen and aortic arch of Apoe−/−Nba2.Yaa mice. Our data show that although atherosclerosis-lupus-prone Apoe−/−Nba2.Yaa mice did not exhibit exacerbated atherosclerotic lesion size, they did show features of atherosclerotic plaque destabilization in correlation with the increase of pro-atherogenic autoantibodies.

Background: Neutrophils accumulate in atherosclerotic plaques. Neutrophil extracellular traps (NET) were recently identified in experimental atherosclerosis and in complex human lesions. However, not much is known about the NET marker citrullinated histone-3 (H3Cit) expression and functionality in human carotid plaques. Moreover, the association between the proatherosclerotic autoantibody anti-apolipoprotein A-1 (anti-ApoA-1 IgG) and NET has never been investigated. Methods: Atherosclerotic plaques have been obtained from 36 patients with severe carotid stenosis that underwent carotid endarterectomy for severe carotid stenosis. Samples were sectioned into upstream and downstream regions from the same artery segment. Plaque composition and expression of NET markers neutrophil elastase (NE) and H3Cit were quantified by immunohistochemistry. H3Cit expression and function was evaluated by immunofluorescence and confocal analysis in a subset of patients. Results: Pathological features of vulnerable phenotypes were exacerbated in plaques developed at downstream regions, including higher accumulation of neutrophils and enhanced expression of NE and H3Cit, as compared to plaques from upstream regions. The H3Cit signal was also more intense in downstream regions, with significant extracellular distribution in spaces outside of neutrophils. The percentage of H3Cit colocalization with CD66b (neutrophils) was markedly lower in downstream portions of carotid plaques, confirming the extrusion of NET in this region. In agreement, the maximum distance of the H3Cit signal from neutrophils, extrapolated from vortex distance calculation in all possible directions, was also higher in downstream plaques. The serum anti-ApoA-1index positively correlated with the expression of H3Cit in downstream segments of plaques. Expression of the H3Cit signal outside of neutrophils and H3Cit maximal distance from CD66b-positive cells increased in plaques from serum positive anti-ApoA-1 patients compared with serum negative patients. Conclusion: NET elements are differentially expressed in upstream versus downstream regions of human carotid plaques and may be influenced by circulating levels of anti-ApoA-1 IgG. These findings could warrant the investigation of NET elements as potential markers of vulnerability.

Cardiotrophin-1 (CT-1) is associated with cardiovascular (CV) diseases. We investigated the effect of CT-1 deficiency in the development and progression of atherosclerosis in double knockout Apoe −/− ct-1 −/− mice. Apoe −/− C57Bl/6 or Apoe −/− ct-1 −/− C57Bl/6 mice were fed a normal chow diet (NCD) or a high-cholesterol diet (HCD). After sacrifice, serum triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-C), free fatty acids and systemic paracrine factors were measured. Intraplaque lipid and collagen content were quantified in the aortic sections. Immune cell populations in spleen, lymph nodes and aorta were analysis by flow cytometry. Apoe −/− ct-1 −/− mice in accelerated atherosclerosis exhibited a reduction of total cholesterol, LDL-C, atherosclerotic plaques size in the aortic root and in the abdominal aorta and improved plaque stability in comparison to Apoe −/− mice. CT-1 deficiency in Apoe −/− mice on (HCD) promoted atheroprotective immune cell responses, as demonstrated by a rise in plasma anti-inflammatory immune cell populations (regulatory T cells, Tregs; regulatory B cells, Bregs and B1a cells) and atheroprotective IgM antibodies. CT-1 deficiency in advanced atherosclerosis mediated regulation of paracrine factors, such as interleukin (IL)-3, IL-6, IL-9, IL-15, IL-27, CXCL5, MCP-3, MIP-1α and MIP-1β. In a model of advanced atherosclerosis, CT-1 deficiency induced anti-inflammatory and atheroprotective effects which resulted in abrogation of atheroprogression.

Bonaventura, Aldoa,b; Carbone, Federicoa,c; Liberale, Lucaa,d; Mach, Françoise; Roth, Alinee; Burger, Fabiennee; Pende, Aldoa,c; Vecchié, Alessandraa,b; Bertolotto, Mariaa; Spinella, Giovannif; Pane, Biancaf; Palombo, Domenicof; Dallegri, Francoa,c; Montecucco, Fabrizioc,g Author Information