Bianca Pane

Anti-Apolipoprotein A-1 auto-antibodies are active mediators of atherosclerotic plaque vulnerability

AIMS Anti-Apolipoprotein A-1 auto-antibodies (anti-ApoA-1 IgG) represent an emerging prognostic cardiovascular marker in patients with myocardial infarction or autoimmune diseases associated with high cardiovascular risk. The potential relationship between anti-ApoA-1 IgG and plaque vulnerability remains elusive. Thus, we aimed to investigate the role of anti-ApoA-1 IgG in plaque vulnerability. METHODS AND RESULTS Potential relationship between anti-ApoA-1 IgG and features of cardiovascular vulnerability was explored both in vivo and in vitro. In vivo, we investigated anti-ApoA-1 IgG in patients with severe carotid stenosis (n = 102) and in ApoE-/- mice infused with polyclonal anti-ApoA-1 IgG. In vitro, anti-ApoA-1 IgG effects were assessed on human primary macrophages, monocytes, and neutrophils. Intraplaque collagen was decreased, while neutrophil and matrix metalloprotease (MMP)-9 content were increased in anti-ApoA-1 IgG-positive patients and anti-ApoA-1 IgG-treated mice when compared with corresponding controls. In mouse aortic roots (but not in abdominal aortas), treatment with anti-ApoA-1 IgG was associated with increased lesion size when compared with controls. In humans, serum anti-ApoA-1 IgG levels positively correlated with intraplaque macrophage, neutrophil, and MMP-9 content, and inversely with collagen. In vitro, anti-ApoA-1 IgG increased macrophage release of CCL2, CXCL8, and MMP-9, as well as neutrophil migration towards TNF-α or CXCL8. CONCLUSION These results suggest that anti-ApoA-1 IgG might be associated with increased atherosclerotic plaque vulnerability in humans and mice.

Systemic and Intraplaque Mediators of Inflammation Are Increased in Patients Symptomatic for Ischemic Stroke

Background and Purpose— The concept of “vulnerable plaque” has been extended to the more recent definition of the “cardiovascular vulnerable patient,” in which “intraplaque” and “systemic” factors contribute to the cumulative risk of acute cardiovascular events. Thus, we investigated the possible role of systemic and intraplaque inflammation in patients asymptomatic versus symptomatic for ischemic stroke. Methods— Regions upstream and downstream the blood flow were isolated from internal carotid plaques of patients asymptomatic (n=63) or symptomatic (n=18) for ischemic stroke. Specimens were analyzed for lipid, collagen, macrophage, lymphocyte, neutrophil, mast cell and smooth muscle cell content, and chemokine and cytokine mRNA expression. Chemokine receptors and adhesion molecules were assessed on circulating leukocytes by flow cytometry. Systemic inflammatory markers and biochemical parameters were measured on total blood, plasma, and serum. Results— Tumor necrosis factor-&agr; and CCL5 serum levels as well as intercellular adhesion molecule-1 expression on circulating neutrophils were increased in symptomatic as compared with asymptomatic patients. Collagen content and smooth muscle cell infiltration were decreased in symptomatic plaques. In upstream regions of symptomatic plaques, lipid content and lymphocyte infiltration were increased. In downstream regions of symptomatic plaques, macrophage, neutrophil, and mast cell infiltration were increased. Intraplaque collagen content was positively correlated with smooth muscle cell infiltration and inversely correlated with macrophages, neutrophils, or serum tumor necrosis factor-&agr;. Collagen reduction in downstream regions and serum tumor necrosis factor-&agr; were independently associated with the likelihood of being symptomatic. Conclusions— Inflammatory mediators are increased in ischemic stroke. Despite statistically significant, the correlation between tumor necrosis factor-&agr; serum level and intraplaque vulnerability was weak and probably of limited biological importance.

The activation of the cannabinoid receptor type 2 reduces neutrophilic protease-mediated vulnerability in atherosclerotic plaques

AIMS The activation of cannabinoid receptor type 2 (CB(2))-mediated pathways might represent a promising anti-atherosclerotic treatment. Here, we investigated the expression of the endocannabinoid system in human carotid plaques and the impact of CB(2) pharmacological activation on markers of plaque vulnerability in vivo and in vitro. METHODS AND RESULTS The study was conducted using all available residual human carotid tissues (upstream and downstream the blood flow) from our cohort of patients symptomatic (n = 13) or asymptomatic (n = 27) for ischaemic stroke. Intraplaque levels of 2-arachidonoylglycerol, anandamide N-arachidonoylethanolamine, N-palmitoylethanolamine, N-oleoylethanolamine, and their degrading enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) were not different in human plaque portions. In the majority of human samples, CB(1) (both mRNA and protein levels) was undetectable. In downstream symptomatic plaques, CB(2) protein expression was reduced when compared with asymptomatic patients. In these portions, CB(2) levels were inversely correlated (r = -0.4008, P = 0.0170) with matrix metalloprotease (MMP)-9 content and positively (r = 0.3997, P = 0.0174) with collagen. In mouse plaques, CB(2) co-localized with neutrophils and MMP-9. Treatment with the selective CB(2) agonist JWH-133 was associated with the reduction in MMP-9 content in aortic root and carotid plaques. In vitro, pre-incubation with JWH-133 reduced tumour necrosis factor (TNF)-α-mediated release of MMP-9. This effect was associated with the reduction in TNF-α-induced ERK1/2 phosphorylation in human neutrophils. CONCLUSION Cannabinoid receptor type 2 receptor is down-regulated in unstable human carotid plaques. Since CB(2) activation prevents neutrophil release of MMP-9 in vivo and in vitro, this treatment strategy might selectively reduce carotid vulnerability in humans.

Resveratrol counteracts systemic and local inflammation involved in early abdominal aortic aneurysm development

BACKGROUND Monocyte activation, macrophage infiltration, vascular oxidative stress and matrix proteolysis are inflammatory key steps contributing to abdominal aortic aneurysm (AAA) development. A phenotypical and functional heterogeneity is recognizable in monocytes by the differential expression of surface molecules: CD62L- subset corresponds to activated monocytes, while CD143/ACE surface expression increases during their differentiation into macrophages. In this work, Resveratrol, which is an antioxidant polyphenol with vasoprotective properties, has been evaluated for its potential to limit aneurysm development and monocyte-dependent inflammatory response in a model of elastase-induced AAA. METHODS Male Sprague-Dawley rats received Resveratrol (10 mg/kg/die) (Rsv group, n=15) or vehicle (ethanol) alone (Et-OH group, n=15) continuously from 7 d before until 14 d after the AAA induction with elastase; five littermates were used as untreated control group (Ctr group, n=5). At the end of treatment, CD143 and CD62L monocyte expression was analyzed by flow cytometry, serum antioxidant capacity was evaluated using the TRAP method and circulating TNFα, and MMP-9 were measured with ELISA and gel zymography, respectively. Aortas were subjected to histology and immunohistochemistry for morphological analysis, macrophage infiltration, and MMP-9, TNFα, and VEGF expression. RESULTS Resveratrol counteracted the CD62L-monocyte subset expansion, CD143 monocyte expression, and circulating levels of MMP-9 activity and TNFα associated to AAA induction. Similarly, treatment with Resveratrol significantly attenuated AAA expansion, vessel wall macrophage infiltration and MMP-9, VEGF, and TNFα expression, compared with AAA from Et-OH group. CONCLUSIONS Resveratrol limited the monocyte-dependent inflammatory response, macrophage differentiation and aortic lumen enlargement in elastase-induced AAA. These data suggest that Resveratrol might be tested in selected patients with small AAA to modulate the early systemic and local inflammatory response associated to AAA progression.

Treatment with Angiotensin-(1-7) reduces inflammation in carotid atherosclerotic plaques

Angiotensin (Ang)-(1-7), acting through the receptor Mas, has atheroprotective effects; however, its role on plaque vulnerability has been poorly studied. Here, we investigated the expression of the renin-angiotensin system (RAS) components in stable and unstable human carotid plaques. In addition, we evaluated the effects of the chronic treatment with an oral formulation of Ang-(1-7) in a mouse model of shear stress-determined carotid atherosclerotic plaque. Upstream and downstream regions of internal carotid plaques were obtained from a recently published cohort of patients asymptomatic or symptomatic for ischaemic stroke. Angiotensinogen and renin genes were strongly expressed in the entire cohort, indicating an intense intraplaque modulation of the RAS. Intraplaque expression of the Mas receptor mRNA was increased in the downstream portion of asymptomatic patients as compared to corresponding region in symptomatic patients. Conversely, AT1 receptor gene expression was not modified between asymptomatic and symptomatic patients. Treatment with Ang-(1-7) in ApoE-/- mice was associated with increased intraplaque collagen content in the aortic root and low shear stress-induced carotid plaques, and a decreased MMP-9 content and neutrophil and macrophage infiltration. These beneficial effects were not observed in the oscillatory shear stress-induced plaque. In vitro incubation with Ang-(1-7) did not affect ICAM-1 expression and apoptosis on cultured endothelial cells. In conclusion, Mas receptor is up regulated in the downstream portions of human stable carotid plaques as compared to unstable lesions. Treatment with the oral formulation of Ang-(1-7) enhances a more stable phenotype in atherosclerotic plaques, depending on the local pattern of shear stress forces.

Effects of polyphenol extract from olive pomace on anoxia-induced endothelial dysfunction.

Anoxia modulates the expression of molecules associated with endothelial dysfunction and vascular diseases. Polyphenols have potent antioxidant properties due to their ability to modulate genes involved in oxidative tissue damage. In this study, we investigated the effect of polyphenol extract from olive pomace (PEOP) and its main constituents, Tyrosol and Oleuropein, on endothelial cells subjected to anoxia by evaluating the expression of molecules critical for endothelial function, proliferation and migration, and the signaling pathway involved. EAhy926 human endothelial cells were exposed to anoxic stress in the presence or absence of PEOP. Anoxia increased the nitric oxide (NO) level and the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNFα). These effects were prevented by PEOP treatment in a dose-dependent manner. Moreover, PEOP prevented the proliferation and migration associated with anoxia in EAhy926 cells, down-regulated the levels of matrix metalloproteinase (MMP)-2, MMP-9 and membrane type-1 MMP (MT1-MMP) and increased tissue MMP inhibitor-1 (TIMP-1) expression. Purified Oleuropein or Tyrosol restored to a basal level the anoxia-induced expression of MMP-9 and partially of MMP-2. The expression of TNFα was reduced by both polyphenols in a dose-dependent manner, but more efficiently by Tyrosol. Conversely, Oleuropein and Tyrosol had no significant effects on iNOS, COX-2 and TIMP-1 expression when used at the concentration found in PEOP. PEOP induced a time-dependent phosphorylation of p38 MAPK and ERK1/2 and inhibited anoxia-induced NF-κB activation. PEOP treatment restores the endothelial functions that are impaired by anoxia by regulating the expression of genes involved in proteolysis, angiogenesis and inflammation more efficiently than the single purified components. Therefore, the combined use of polyphenols, as in PEOP, could represent a powerful tool for the treatment and chemoprevention of endothelial dysfunction-associated vascular diseases.

Serum levels of anti-apolipoprotein A-1 auto-antibodies and myeloperoxidase as predictors of major adverse cardiovascular events after carotid endarterectomy

We aimed at challenging the prognostic accuracies of myeloperoxidase (MPO) and antibodies anti-apolipoprotein A-1 (anti-apoA-1 IgG), alone or in combination, for major adverse cardiovascular events (MACE) prediction, one year after carotid endarterectomy (CEA). In this prospective single centre study, 178 patients undergoing elective CEA were included. Serum anti-apoA-1 IgG and MPO were assessed by enzyme-linked immunosorbent assay prior to the surgery. Post-hoc determination of the MPO cut-off was performed by receiver operating characteristics (ROC) analyses. MACE was defined by the occurrence of fatal or non-fatal acute coronary syndromes or stroke during one year follow-up. Prognostic accuracy of anti-apoA-1 IgG was assessed by ROC curve analyses, survival analyses and reclassification statistics. During follow-up, 5% (9/178) of patients presented a MACE, and 29% (52/178) were positive for anti-apoA-1 IgG. Patients with MACE had higher median MPO and anti-apoA-1 IgG levels at admission (p=0.01), but no difference for the 10-year global Framingham risk score (FRS) was observed (p=0.22). ROC analyses indicated that both MPO and anti-apoA-1 IgG were significant predictors of subsequent MACE (area under the curve [AUC]: 0.75, 95% confidence interval [95%CI]: 0.61-0.89, p=0.01; and 0.74, 95%CI: 0.59-90; p=0.01), but combining anti-apoA-1 IgG positivity and MPO>857 ng/ml displayed the best predictive accuracy (AUC: 0.78, 95%CI: 0.65-0.91; p=0.007). It was associated with a poorer MACE-free survival (98.2% vs. 57.1%; p<0.001, LogRank), with a positive likelihood ratio of 13.67, and provided incremental predictive ability over FRS. In conclusion, combining the assessment of anti-apoA-1 IgG and MPO appears as a promising risk stratification tool in patients with severe carotid stenosis.

A positron emission tomography/computed tomography (PET/CT) evaluation of asymptomatic abdominal aortic aneurysms: another point of view.

BACKGROUND To assess the prevalence of increased (18)F-fluorodeoxyglucose (FDG) uptake in aneurysmal walls, adopting a case-control approach in a population of asymptomatic patients with abdominal aortic aneurysm (AAA). METHODS This study included 40 males (mean age: 74 years, range: 59-93 years), consecutive, white Caucasian patients, with asymptomatic infrarenal AAA. The mean diameter of AAA was 4.9 cm (range: 4.8-5.4 cm), detected by computed tomography (CT) scan. Control Subjects: 44 age-matched controls subjects (mean age: 71 years, range: 59-85 years, 24 males, 20 females) who were selected according to a case-control criterion among a population of patients without any clinical evidence of atherosclerotic disease. Patients and controls underwent simultaneous FDG-positron emission tomography (PET) and CT imaging from the skull base to the femoral neck by using an integrated PET/CT scanner. PET/CT studies were analysed both visually and quantitatively. For quantitative analysis, circular CT-based regions of interest (ROIs) were drawn on the AAA, on all the aortic segments, and on the large vessel included in the study (carotid, subclavian, and iliac arteries). FDG uptake was quantified by calculating the mean and maximum standardized uptake values (SUVs) within each ROI and normalizing for the blood-pool SUV to obtain the final target-to-background ratio. Arterial calcium load was graded according to a semiquantitative five-point scale based on calcification of the arterial ring. RESULTS Metabolic activity in the aneurysmal aortic segment was even lower with respect to both the adjacent--nonaneurysmal--samples of patient group and the corresponding arterial segments of control subjects (P < 0.001 and P < 0.01, respectively). In visual analysis, no patients showed an increased focal uptake of degree adequate to identify the aneurysmal arterial wall. AAA patients showed significantly higher values of total calcium load (ACL) than controls in ascending aorta and subclavian and iliac arteries (P < 0.01), and only in AAA patient group, a significant correlation was present between values of ACL in both iliac arteries and abdominal aorta on one side and wall metabolic activity in the same arteries on the other (P < 0.05). CONCLUSIONS In conclusion, our results suggest that FDG hot spot, as well an increased diffuse uptake of FDG, in PET/CT studies is an extremely rare finding in patients with AAA of diameter close to surgical indications.

An economic evaluation of an abdominal aortic aneurysm screening program in Italy

OBJECTIVES Abdominal aortic aneurysm (AAA) is defined as a localized dilatation of an aortic vessel. Though predominantly asymptomatic, it is a chronic degenerative condition associated with life-threatening risk of rupture. The early diagnosis of AAA, ie, before it ruptures, is therefore important; a simple, effective diagnostic method is ultrasound examination. To assess the benefit of screening in Italy, we developed a cost-effective Markov model comparing screening vs nonscreening scenarios. METHODS A 13-health-states Markov model was developed to compare two cohorts of 65- to 75-year-old men: the first group undergoing screening for AAA by means of ultrasound (US), the second following the current practice of incidental detection. The following health states were distinguished: no AAA, unknown small AAA (3-3.9 cm), followed-up small AAA (1 year), unknown medium-sized AAA (4-4.9 cm), followed-up medium-sized AAA (6 months), unknown large AAA (>5 cm), elective repair, emergency repair, postelective-repair AAA, postemergency-repair AAA, rejected large AAA, and death. Transitions between health states were simulated by using 6-month cycles. Transition probabilities were derived from a literature review of relevant randomized controlled trial and from a screening program that is currently ongoing at San Martino Hospital in Genoa, Italy. The Italian National Health Service (NHS) perspective was adopted and incremental cost per life-year saved was calculated with a lifetime horizon; costs and health benefits were discounted at an annual rate of 3% from year 2 onward. Uncertainty surrounding the model inputs was tested by means of univariate, multivariate, and probabilistic sensitivity analyses. RESULTS Considering an attendance rate of 62%, the individual cost per invited subject was €60 (US

Statin Treatment Is Associated with Reduction in Serum Levels of Receptor Activator of NF-κB Ligand and Neutrophil Activation in Patients with Severe Carotid Stenosis

83.2); 0.011 additional quality adjusted life years (QALY) were gained per patient in the screened cohort, corresponding to an incremental cost-effectiveness ratio (ICER) of €5673/QALY (US