Aline Roth

Vitamin D receptor is expressed within human carotid plaques and correlates with pro-inflammatory M1 macrophages

The role of Vitamin D system in cardiovascular diseases remains controversial. Here, we investigated whether intraplaque levels of vitamin D receptor (VDR) predicted major adverse cardiovascular events (MACEs) at 18month-follow-up and correlated with macrophage subsets in 164 patients undergoing endarterectomy for carotid stenosis. In human carotid plaque portions upstream and downstream the blood flow, VDR, lipid, collagen, as well as macrophage subsets were determined. Human primary monocytes were then differentiated in vitro to M1 and M2 macrophages and treated with 1,25(OH)2D3. Intraplaque VDR positively correlated with total and M1 macrophages. According to the result of ROC curve analysis, downstream portions of plaques having high VDR expression were characterized by increased M1 macrophages. Kaplan-Meier analysis showed that the risk of MACEs was greater in patients having low downstream VDR levels (8.2% vs. 1.3%; p=0.005). Cox proportional hazard regression analyses confirmed that MACE risk decreased with increasing downstream VDR (adjusted HR 0.78 [95% CI 0.62-0.98]; p=0.032). In vitro, VDR expression was prevalent in M1, but not M2. Incubation of M1 macrophages with 1,25(OH)2D3, increased VDR expression and suppressed toll-like receptor 4 expression. These results suggest that low intraplaque VDR expression predict MACEs in patients with carotid stenosis potentially involving M1 macrophages.

Anti-apolipoprotein A-1 auto-antibodies as active modulators of atherothrombosis

Humoral autoimmune-mediated inflammation plays a role in atherogenesis, and potentially in arterial thrombosis. Anti-apolipoprotein A-1 (apoA-1) IgG have been reported to represent emergent mediators of atherogenesis through Toll-like receptors (TLR) 2, 4 and CD14 signalling. We investigated the role of anti-apoA-1 IgG on tissue factor (TF) expression and activation, a key coagulation regulator underlying atherothrombosis. Atherothrombosis features were determined by immunohistochemical TF staining of human carotid biopsies derived from patients with severe carotid stenosis undergoing elective surgery (n=176), and on aortic roots of different genetic backgrounds mice (ApoE-/-; TLR2-/-ApoE-/- and TLR4-/-ApoE-/-) exposed to passive immunisation with anti-apoA-1 IgG. Human serum levels of anti-apoA-1 IgG were measured by ELISA. In vitro, on human-monocyte-derived-macrophages (HMDM) the anti-apoA-1 IgG increased TF expression and activity were analysed by FACS and chromogenic assays in presence of different pharmacological inhibitors. Human serum anti-apoA-1 IgG levels significantly correlated to intraplaque TF expression in carotid biopsies (r=0.31, p<0.001), which was predictive of clinically symptomatic lesions. On HMDM, anti-apoA-1 IgG induced a TLR2, 4 and CD14-dependent increase in TF expression and activity, involving NF-kappaB and a c-Jun N-terminal kinase-dependent AP-1 transcription factors. In ApoE-/- mice, anti-apoA-1 IgG passive immunisation significantly enhanced intraplaque TF expression when compared to control IgG. This effect was lost in both TLR2-/-ApoE-/- and TLR4-/-ApoE-/- mice. These results demonstrate that anti-apoA-1 IgG are associated with TF expression in human atherosclerotic plaques, induce TF expression in vitro and in vivo through TLR2 and 4 signalling, supporting a possible causal relationship between anti-apoA-1 IgG and atherothrombosis.

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.

Intraplaque Expression of C-Reactive Protein Predicts Cardiovascular Events in Patients with Severe Atherosclerotic Carotid Artery Stenosis

Serum c-reactive protein (CRP) was suggested for the assessment of intermediate cardiovascular (CV) risk. Here, systemic or intraplaque CRP levels were investigated as predictors of major adverse cardiovascular events (MACEs) in patients with severe carotid stenosis. CRP levels were assessed in the serum and within different portions (upstream and downstream) of carotid plaques of 217 patients undergoing endarterectomy. The association between CRP and intraplaque lipids, collagen, neutrophils, smooth muscle cells (SMC), and macrophage subsets was determined. No correlation between serum CRP and intraplaque biomarkers was observed. In upstream portions, CRP content was directly correlated with intraplaque neutrophils, total macrophages, and M1 macrophages and inversely correlated with SMC content. In downstream portions, intraplaque CRP correlated with M1 and M2 macrophages. According to the cut-off point (CRP > 2.9%) identified by ROC analysis in upstream portions, Kaplan-Meier analysis showed that patients with high CRP levels had a greater rate of MACEs. This risk of MACEs increased independently of age, male gender, serum CRP, and statin use. In conclusion, in patients with severe carotid artery stenosis, high CRP levels within upstream portions of carotid plaques directly and positively correlate with intraplaque inflammatory cells and predict MACEs at an 18-month follow-up period.

Treatment with the GPR55 antagonist CID16020046 increases neutrophil activation in mouse atherogenesis.

Endocannabinoids modulate atherogenesis by triggering different receptors. Recently, orphan G protein-coupled receptors (GPRs) were suggested to be activated by endocannabinoids, possibly regulating vasorelaxation. Here, we investigated whether GPR55 antagonism with CID16020046 would impact on atherosclerotic size and inflammation in two mouse models of early and more advanced atherogenesis. Eleven-week old ApoE-/- mice were fed either a normal diet ([ND] for 16 weeks) or a high-cholesterol diet ([HD] for 11 weeks), resulting in different degrees of hypercholesterolaemia and size of atherosclerosis. CID16020046 (0.5 mg/kg) or vehicle were intraperitoneally administrated five times per week in the last three weeks before euthanasia. Treatment with CID1602004 was well-tolerated, but failed to affect atherosclerotic plaque and necrotic core size, fibrous cap thickness, macrophage and smooth muscle cell content as well as Th cell polarisation. In ND mice, treatment with CID1602004 was associated with increased chemokine production, neutrophil and MMP-9 intraplaque content as well as reduced collagen as compared to vehicle-treated animals. In HD mice, CID1602004 increased intraplaque MMP-9 and abrogated collagen content without affecting neutrophils. In vitro, serum from CID1602004-treated ND mice increased mouse neutrophil chemotaxis towards CXCL2 as compared to serum from vehicle-treated animals. CID1602004 dose-dependently induced neutrophil degranulation that was reverted by co-incubation with the GPR55 agonist Abn-CBD. In supernatants from degranulation experiments, increased levels of the endocannabinoid and putative GPR55 ligand anandamide (AEA) were found, suggesting its possible autocrine control of neutrophil activity. These results indicate that GPR55 is critical for the negative control of neutrophil activation in different phases of atherogenesis.

Anti-ApoA-1 IgG serum levels predict worse poststroke outcomes

Autoantibodies to apolipoprotein A‐1 (anti‐ApoA‐1 IgG) were shown to predict major adverse cardiovascular events and promote atherogenesis. However, their potential relationship with clinical disability and ischaemic lesion volume after acute ischaemic stroke (AIS) remains unexplored.

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

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.

Alamandine abrogates neutrophil degranulation in atherosclerotic mice.

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.