Circulating GDF11 exacerbates myocardial injury in mice and associates with increased infarct size in humans.

AIMS: The heart rejuvenating effects of circulating growth differentiation factor 11 (GDF11), a transforming growth factor-ß 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 (0.1 mg/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.

Epitope of antiphospholipid antibodies retrieved from peptide microarray based on R39-R43 of ß2-glycoprotein I.

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 (ß2GP1) 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 ß2GP1, 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.

The E3 Ubiquitin Ligase Peli1 Deficiency Promotes Atherosclerosis Progression.

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.

Single-Cell RNA-Seq Reveals a Crosstalk between Hyaluronan Receptor LYVE-1-Expressing Macrophages and Vascular Smooth Muscle Cells.

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.

Single-Cell Analysis Uncovers Osteoblast Factor Growth Differentiation Factor 10 as Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation Associated with Plaque Rupture in Human Carotid Artery Disease.

(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.

NLRP3 Inflammasome Activation Controls Vascular Smooth Muscle Cells Phenotypic Switch in Atherosclerosis.

(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-1ß secretion, and pyroptosis in VSMCs. Increased caspase 1 and IL-1ß 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.

Follicular regulatory helper T cells control the response of regulatory B cells to a high-cholesterol diet.

AIMS: B cell functions in the process of atherogenesis have been investigated but several aspects remain to be clarified. METHODS AND RESULTS: In 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. CONCLUSION: Our 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.

Prognostic and therapeutic considerations of antibodies against c-ter apolipoprotein A-1 in the general population.

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.

Atherosclerotic plaque vulnerability is increased in mouse model of lupus.

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.

Anti-Apolipoprotein A-1 IgG Influences Neutrophil Extracellular Trap Content at Distinct Regions of Human Carotid Plaques.

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 Deficiency Abrogates Atherosclerosis Progression.

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-1ß. In a model of advanced atherosclerosis, CT-1 deficiency induced anti-inflammatory and atheroprotective effects which resulted in abrogation of atheroprogression.

Cardiomyocyte-Specific JunD Overexpression Increases Infarct Size following Ischemia/Reperfusion Cardiac Injury by Downregulating Sirt3.

Ischemia/reperfusion (I/R) injury in acute myocardial infarction activates several deleterious molecular mechanisms. The transcription factor JunD regulates pathways involved in oxidative stress as well as in cellular proliferation, differentiation, and death. The present study investigated the potential role of JunD as a modulator of myocardial injury pathways in a mouse model of cardiac I/R injury. Infarct size, systemic and local inflammation, and production of reactive oxygen species, as well as cytosolic and mitochondrial apoptotic pathways were investigated in adult males after myocardial I/R. In wild-type (WT) mice, 30 minutes after ischemia and up to 24 hours following reperfusion, cardiac JunD messenger ribonucleic acid expression was reduced while JunB increased. Cardiac-specific JunD overexpressing mice (JunDTg/0 ) displayed larger infarcts compared with WT. However, postischemic inflammatory or oxidative responses did not differ. JunD overexpression reduced Sirt3 transcription by binding to its promoter, thus leading to mitochondrial dysfunction, myocardial cell death, and increased infarct size. On the other hand, JunD silencing reduced, while Sirt3 silencing increased infarct size. In human myocardial autopsy specimens, JunD-positive areas within the infarcted left ventricle staining corresponded to undetectable Sirt3 areas in consecutive sections of the same heart. Cardiac-specific JunD overexpression increases myocardial infarct size following I/R. These effects are mediated via Sirt3 transcriptional repression, mitochondrial swelling, and increased apoptosis, suggesting that JunD is a key regulator of myocardial I/R injury. The present data set the stage for further investigation of the potential role of Sirt3 activation as a novel target for the treatment of acute myocardial infarction.

Serum PCSK9 levels predict the occurrence of acute coronary syndromes in patients with severe carotid artery stenosis.

BACKGROUND: The pharmacological inhibition of Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) has shown to dramatically impact on low-density lipoprotein-cholesterol (LDL-C) levels and associated cardiovascular (CV) diseases. However, the potential use of PCSK9 serum levels as a CV risk biomarker remains to be clarified. METHODS: 189 patients with severe carotid artery atherosclerosis undergoing carotid endarterectomy (CEA) and whose clinical records and serum sample aliquots for PCSK9 level measurement were available both directly before CEA and at 24-month follow-up were included in the present pilot study. The study endpoint was to determine whether PCSK9 serum levels prior to CEA would predict the occurrence of acute coronary syndromes (ACS) at 24-month follow-up. RESULTS: PCSK9 serum levels were significantly accurate in predicting ACS at 24-month follow-up, as assessed by ROC curve analysis (AUC: 0.719 [95% CI 0.649-0.781]). According to the cut-off point indicated by Youden's index, PCSK9 values >431.3 ng/mL were correlated with a higher risk of ACS occurrence (Log Rank test, p = 0.0003). At Cox regression analysis, the predictive ability of high serum PCSK9 was confirmed also after adjustment for age, gender, baseline statin treatment and active smoking, dyslipidemia, and chronic coronary artery disease (HR 17.04 [95% CI 3.34-86.81]; p = 0.001). CONCLUSIONS: High serum PCSK9 levels predict ACS occurrence at 24-month follow-up after CEA in patients with severe carotid artery stenosis. Larger clinical studies are needed to evaluate whether PCSK9 serum levels could be used towards predicting the risk of ACS in patients with advanced carotid atherosclerosis.

Serum levels of osteopontin predict major adverse cardiovascular events in patients with severe carotid artery stenosis.

BACKGROUND: Inflammatory mediators in the blood stream and within plaques are key determinants in atherogenesis. Here, we investigated serum osteopontin (OPN) as a potential predictor of poor outcome in patients with severe carotid atherosclerosis. METHODS: Carotid plaques and serum were collected from patients asymptomatic (n=185) or symptomatic (n=40) for ischemic stroke. Plaques were stained for lipids, smooth muscle cells, neutrophils, M1 and M2 macrophage subsets and matrix metallopropteinase-9 (MMP-9). Serum levels of OPN and interleukin-6 (IL-6) were determined by colorimetric enzyme-linked immunosorbent assays. RESULTS: Symptomatic patients showed a two-fold increase in serum OPN levels. In both symptomatic and asymptomatic patients, OPN levels positively correlated with intraplaque count of neutrophils, total macrophages, and MMP-9 content. In asymptomatic patients, OPN levels also positively correlated with lipids and M1 macrophage subsets. Receiver operating characteristic curve analysis identified serum OPN concentration of 70ng/ml as the best cut-off value to predict major adverse cardiovascular events (MACEs). Patients with high OPN levels had more vulnerable plaque phenotype and reduced levels of HDL-cholesterol and IL-6 as compared to low OPN levels. Kaplan-Meier curve confirmed that patients with OPN levels >70ng/ml had more MACEs at a 24-month follow-up. In the multivariate survival analysis, OPN levels >70ng/ml predicted MACEs, independently of age, gender, and symptomatic status. CONCLUSION: High circulating OPN levels were strongly correlated with vulnerability parameters within plaques and predict MACEs in patients with severe carotid artery stenosis. Although confirmation is needed from larger trials, OPN could be a promising clinical tool to assess atherosclerotic outcomes.

Serum adiponectin levels predict acute coronary syndrome (ACS) in patients with severe carotid stenosis.

As endocrine organ, adipose tissue may modulate inflammatory response by releasing a wide range of mediators, known as adipocytokines. Due to the complex balance between pro- and anti-inflammatory activity their pathophysiological and prognostic role in cardiovascular (CV) diseases still remains debated. Here, we consider the potential associations of circulating adipocytokines adiponectin, leptin and their ratio (LAR), with metabolic and inflammatory profiles in 217 patients with severe carotid stenosis. A prospective analysis investigating their predictive role toward acute coronary syndromes (ACS) was also drawn over a 12-month follow-up period. Serum leptin was positively associated with fasting insulinemia and HOMA-IR, but not with lipid profile and inflammation. Conversely, adiponectin was negatively associated with glucose, insulin, HOMA-IR, triglycerides and both systemic and intraplaque inflammatory markers whereas a positive association with high-density lipoprotein cholesterol (HDL-c) was observed. Accordingly, a significant association with metabolic profile was reported for LAR. According to the cut-off point identified by ROC curve, adiponectin values≤2.56µg/mL were correlated with a higher risk of ACS occurrence at 12months' follow-up (p-value for Log Rank test=0.0003). At Cox regression analysis the predictive ability of low serum adiponectin was confirmed also after adjustment for age, male gender and diabetes. In conclusion, adiponectin may be considered a biomarker of metabolic compensation, inversely associated with chronic low-grade inflammation. Circulating adiponectin is also associated with lower risk of adverse CV events in patients with severe carotid stenosis.

Apelin-13 treatment enhances the stability of atherosclerotic plaques.

BACKGROUND: Apelin is an endogenous peptidergic system which modulates cardiovascular function. Recent studies pointed out a fundamental contribution of apelin on atherosclerosis development; however, such reports revealed contradictory data, and to date, it is difficult to accurately define a beneficial or deleterious role. To better understand apelin function on atherosclerosis, we aimed to investigate apelin-13 treatment effects on atherosclerotic plaques composition. DESIGN: Apolipoprotein E gene-deleted mice were fed on Western-type diet for 11 weeks. Atherosclerotic plaque formation was induced in the carotid artery by a shear stress modifier device, which exposes the same vessel to distinct patterns of shear stress enabling the formation of plaques with different composition. Mice were treated with apelin-13 (2 mg kg-1 day-1 ) or vehicle for the last 3 weeks. RESULTS: Apelin-13 treatment did not alter the lipid content of low shear stress- and oscillatory shear stress-induced plaques in the carotid. However, apelin-13 greatly ameliorated plaque stability by increasing intraplaque collagen content and reducing MMP-9 expression. Furthermore, apelin-13 decreased the infiltration of inflammatory cells (neutrophil and macrophage) and intraplaque reactive oxygen species content. Interestingly, apelin-13 treatment reduced total cholesterol, LDL levels and free fatty acid serum levels, while HDL, triglycerides serum levels were not significantly changed. CONCLUSIONS: Apelin-13 treatment for 3 weeks did not alter the lesion size, but it significantly enhanced the stable phenotype of atherosclerotic plaques and improved serum lipid profile. These results indicate that activation of apelin system decreases plaque vulnerability.

Serum lipoprotein (a) predicts acute coronary syndromes in patients with severe carotid stenosis.

BACKGROUND: Different cut-off values of serum lipoprotein (a) [Lp (a)] were recently identified to better stratify cardiovascular risk categories. Both pathophysiological and prognostic values of Lp (a) remain unclear. MATERIALS AND METHODS: Here, the prognostic value of Lp (a) and its correlation with intraplaque features were assessed in patients with severe carotid artery stenosis undergoing endarterectomy (n = 180). The cut-off value of 10 mg/dL for serum Lp (a) was selected to predict 24-month follow-up acute coronary syndrome (ACS). In addition, the association between serum Lp (a) and intraplaque lipids, collagen, inflammatory and vascular cells was assessed. Serum Lp (a) levels were measured by nephelometric assay. RESULTS: Patients with high Lp (a) had similar comorbidities, medications and laboratory parameters as compared to low Lp (a) levels. At 24-month follow-up, patients with high Lp (a) had more ACS as compared to low levels. Histological parameters within plaques were comparable in the study groups. No significant correlation between Lp (a) serum levels and intraplaque parameters was found, except for a weak positive association with smooth muscle cells in upstream plaque portions. When adjusted for gender, the presence of dyslipidaemia and chronic coronary artery disease, Lp (a) ≥10 mg/dL remained predictive for ACS. CONCLUSIONS: Lp (a) determination could be a useful tool to predict ACS in patients with severe carotid stenosis.

Alamandine abrogates neutrophil degranulation in atherosclerotic mice.

BACKGROUND: Neutrophil-mediated inflammation was recently identified as an active contributor to athero-progression. Therapeutic strategies inhibiting neutrophil degranulation or recruitment were hypothesized to positively impact on plaque vulnerability. In this study, we investigated whether treatment with the recently discovered agonist of the Mas-related G-coupled receptor type D (MrgD) alamandine would impact on neutrophil degranulation in vivo and in vitro. MATERIALS AND METHODS: Fifteen-week-old ApoE-/- mice were fed with a Western-type diet for an additional 11 weeks. After the first 2 weeks of diet, mice were surgically implanted with a carotid 'cast' device that alters the blood shear stress and induces different carotid plaque phenotypes. During the last 4 weeks before euthanasia, mice were randomly assigned to subcutaneously receive vehicle (NaCl 0·15 M) or alamandine (24 µg/kg/h) by micropump. For in vitro experiments, neutrophils were obtained after thioglycollate intraperitoneal injection in ApoE-/- mice. RESULTS: Treatment with alamandine was well-tolerated, but failed to affect lipid, macrophage, neutrophil or collagen content within carotid and aortic root plaques. Also, treatment with alamandine did not affect Th-cell polarization in lymphoid organs. Conversely, alamandine administration was associated with a reduction in serum levels of neutrophil granule enzymes, such as MMP-9 and MPO as well as MMP-9 content within aortic root plaques. In vitro, preincubation with alamandine dose-dependently abrogated PMA-induced neutrophil degranulation of MMP-9 and MPO. CONCLUSION: These results suggest that treatment with the MrgD agonist alamandine led to a reduced release of neutrophil granule products, potentially interfering with pro-atherosclerotic neutrophil activation.