Increased circulating levels of MIP-1α and CD14 are associated with the presence of severe stenosis and hypoechoic plaques in patients with carotid atherosclerosis.

OBJECTIVE: Carotid atherosclerosis, a major cause of ischemic cerebrovascular events, is characterized by a pro-inflammatory and pro-oxidant vascular microenvironment. The current risk score models based on traditional risk factors for cardiovascular risk assessment have some limitations. The identification of novel blood biomarkers could be useful to improve patient management. The aim of the study was to evaluate the association of selected inflammation- and oxidative stress-related markers with the presence of severe stenosis and/or vulnerable plaques. METHODS: Circulating levels of soluble CD40 ligand, interleukin-10, macrophage inflammatory protein (MIP)-1α, endoglin, CD163, CD14, E-selectin, tumor necrosis factor-α, monocyte chemoattractant protein-1, C-Reactive protein, CD40 L + T lymphocytes, total antioxidant capacity, glutathione reductase activity, and protein carbonyl content were determined in patients with carotid atherosclerosis. RESULTS: Multiparametric analysis showed significantly higher levels of MIP-1α in patients with stenosis ≥70% than in patients with stenosis <70%, and significantly higher levels of CD14 in patients with hypoechoic (vulnerable) lesions compared to those with hyperechoic (stable) ones. The area under the curve obtained by the receiver operating characteristic curve analysis was 0.7253 for MIP-1α and 0.6908 for CD14. CONCLUSIONS: Our data suggest that circulating MIP-1α and CD14 levels are associated with the presence of advanced stenosis and of vulnerable carotid plaques.

Hemorheological profiles and chronic inflammation markers in transfusion-dependent and non-transfusion- dependent thalassemia.

The rheological properties of blood play an important role in regulating blood flow in micro and macro circulation. In thalassemia syndromes red blood cells exhibit altered hemodynamic properties that facilitate microcirculatory diseases: increased aggregation and reduced deformability, as well as a marked increase in adherence to the vascular endothelial cells. A personalized approach to treating thalassemia patients (transfusions, iron chelation, and splenectomy), has increased patients' life expectancy, however they generally present many complications and several studies have demonstrated the presence of high incidence of thromboembolic events. In this study the hemorheological profiles of thalassemia patients have been characterized to point out new indices of vascular impairment in thalassemia. Plasma viscosity, blood viscosities at low and high shear rates (η1 and η200, respectively), erythrocyte aggregation index (η1/η200), and the erythrocyte viscoelastic profile (elastic modulus G', and viscous modulus G") have been studied in transfusion-dependent and non-transfusion-dependent thalassemia patients. Moreover, the levels of inflammation biomarkers in thalassemia have been evaluated to investigate a relationship between the biomarkers, the disease severity and the rheological parameters. The biomarkers studied are the main components of the immune and endothelial systems or are related to vascular inflammation: cytokines (IL-2, IL-6, IL-10, IL-17A, TNF-alpha), chemokines (IL-8, MIP-1alpha), adipocytokines (leptin and adiponectin), growth factors (VEGF, angiopoietin-1), adhesion molecules (ICAM-1, VCAM-1, E-selectin, L-selectin), and a monocyte/macrophage activation marker (CD163). This study shows that transfusion-dependent thalassemia patients, both major and intermedia, have blood viscosities comparable to those of healthy subjects. Non-transfusion-dependent thalassemia intermedia patients show high blood viscosities at low shear rates (η1), corresponding to the flow conditions of the microcirculation, an increase in erythrocyte aggregation, and high values of the elastic G' and viscous G" modules that reflect a reduced erythrocyte deformability and an increase in blood viscosity. Levels of cytokines, chemokines and adhesion molecules are different in transfusion- and non-transfusion dependent patients and positive correlations between η1 or η1/η200 and the cytokines IL-6 and IL-10 have been observed. The evaluation of the hemorheological profiles in thalassemia can provide new indicators of vascular impairment and disease severity in thalassemia in order to prevent the onset of thromboembolic events.

Lupeol Counteracts the Proinflammatory Signalling Triggered in Macrophages by 7-Keto-Cholesterol: New Perspectives in the Therapy of Atherosclerosis.

Macrophage activation and polarization play a central role in atherosclerotic plaque fate. The M1/M2 activation phenotypes represent two profiles of the macrophage polarization state. During atherosclerosis regression or stabilization, macrophages switch from M1 proinflammatory phenotype to M2 anti-inflammatory reparative one. Here, we investigated whether the natural compound lupeol, a pentacyclic triterpene, induces phenotypical and functional changes in human M1 macrophages and counteracts the proinflammatory signalling triggered by 7-keto-cholesterol (7KC), a major product of oxidative stress-mediated cholesterol oxidation. Flow cytometric and immunochemical analysis showed that the treatment with lupeol of M1 monocyte-derived macrophages M (IFN-γ/LPS) specifically stimulated these cells to upregulate the expression of the anti-inflammatory cytokines interleukin- (IL-)10 and TGF-ß, and of the scavenger receptor CD36, whereas downregulated the proinflammatory cytokine IL-12 and the M1 activation marker HLA-DR. Pretreatment of macrophages with lupeol prevented the release of IL-12, IL-1ß, and the upregulation of HLA-DR expression triggered by 7KC and increased the IL-10 production and CD36 expression. This treatment also prevented the impairment of endocytosis triggered by 7KC and prevented 7KC-induced foam cell formation by reducing the lipid droplet accumulation in M1-polarized THP-1 macrophages, whereas showed an additive effect in reactive oxygen species (ROS) production. Western blotting analysis of autophagy markers LC3-I/II and p62-SQSTM1 in M1-polarized THP-1 macrophages demonstrated that lupeol activated autophagy as indicated by increased LC3-II levels, and by marked inhibition of p62. These findings indicate that lupeol has a cytoprotective effect on 7KC-proinflammatory signalling by efficiently switching the macrophage polarization toward an anti-inflammatory phenotype, probably through the activation of the autophagy pathway by increasing ROS production, the reduction of cellular lipid accumulation, and an overall reduction of proinflammatory phenotype. Thus, our data demonstrating an anti-inflammatory and immunomodulatory activity of lupeol in human M1 macrophages suggest its usefulness as an adjunctive drug in the therapy of atherosclerosis.

Phenotypical and functional abnormalities of circulating neutrophils in patients with ß-thalassemia.

ß-Thalassemia is an inherited single gene disorder related to reduced synthesis of the ß-globin chain of hemoglobin. Patients with ß-thalassemia present variable clinical severity ranging from asymptomatic trait to severe transfusion-dependent anemia and multiple organs complications. Moreover, multiple immune abnormalities are a major concern in ß-thalassemia patients. Aberrant neutrophil effector function plays a pivotal role in infection susceptibility in these patients. In severe and persistent inflammation, immature neutrophils are released from the bone marrow and are functionally different compared with mature ones. Despite some abnormalities reported for thalassemia patient's immune system, few data exist on the characterization of human neutrophils in ß-thalassemia. The aim of this study was to investigate the phenotype and function of circulating neutrophil subsets in patients with ß-thalassemia major and with ß-thalassemia intermedia divided in transfusion-dependent and non-transfusion-dependent. By the use of immunochemical and cytofluorimetric analyses, we observed that patients' CD16+ neutrophils exhibit abnormalities in their phenotype and functions and the abnormalities vary according to the clinical form of the disease and to the neutrophil subset (CD16bright and CD16dim). Abnormalities include altered surface expression of the innate immune receptor CD45, Toll-like receptor 4, and CD32, reduced ability to produce an oxidative burst, and elevated levels of membrane lipid peroxidation, especially in patients with a more severe form of the disease. Overall, our results indicating the occurrence of an immuno-senescent phenotype on circulating neutrophils from thalassemia patients suggest the usefulness of neutrophil feature assessment as a tool for better clinical management of ß-thalassemia.

Post-translational modifications of proteins in antiphospholipid antibody syndrome.

Antiphospholipid antibody syndrome (APS) is a systemic autoimmune disease characterized by arterial and venous thrombosis and/or pregnancy morbidity. The incidence is around five new cases/100,000 persons per year and the prevalence is around 40-50 cases/100,000. The prevalence is higher (about 30%) among patients with systemic lupus erythematosus. APS is associated with circulating antiphospholipid antibodies (aPLs), a heterogeneous group of autoantibodies directed against negatively charged molecules and a combination of protein-complexed phospholipids. The predominant protein antigen in this disorder is beta 2-glycoprotein I (ß2GPI). Despite the discovery of "new" antigenic targets and development of "new" methodological approaches, the laboratory diagnosis of APS is still an evolving field and studies to identify further antigenic target(s) as potential diagnostic markers and risk predictors are in progress. In particular, recent studies were aimed at analyzing the pathogenic role of post-translational modifications (PTMs) of proteins induced by inflammation and/or oxidative stress as modulators of protein structure and function and possibly as a source of antigenic epitopes. The present review is focused on PTMs of self-proteins responsible for autoimmune reactions in patients with APS. At present, the known PTMs in APS involve ß2GPI. In particular, the PTM of ß2GPI via thiol-exchange reactions is a highly specific phenomenon that makes the protein more antigenic. Other PTMs, including sialylation and acetylation, may affect ß2GPI antigenicity. Moreover, the addition or loss of carbohydrate chains affects ß2GPI immunoreactivity since carbohydrates are determining factors for ß2GPI conformation. In addition to ß2GPI, PTMs of other self proteins such as vimentin and annexins may play a role in the immune response during APS. The study of PTMs is useful to clarify the role of modified proteins in the pathogenesis of APS as well as to design more efficient diagnostic/prognostic tools and more targeted therapeutic approaches.

Oxidative Stress Induces HSP90 Upregulation on the Surface of Primary Human Endothelial Cells: Role of the Antioxidant 7,8-Dihydroxy-4-methylcoumarin in Preventing HSP90 Exposure to the Immune System.

We have previously demonstrated that human heat shock protein 90 (HSP90), an intracellular self protein, is the target of cellular and humoral autoimmune responses in patients with carotid atherosclerosis. In this study, we evaluated in vitro whether oxidative stress, a feature of atherosclerotic plaque, alters HSP90 expression in endothelial cells, thus inducing surface localization of this molecule and whether the antioxidant compound 7,8-dihydroxy-4-methylcoumarin (7,8-DHMC) is able to prevent oxidative stress-induced alterations of HSP90 localization. By the use of flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay, and semiquantitative reverse-transcription polymerase chain reaction, we demonstrated that exposure of human umbilical vein endothelial cells (HUVEC) to the prooxidant compound H2O2 upregulated HSP90 surface expression and reduced its secretion without altering HSP90 gene expression and intracytoplasmic protein levels. Pretreatment of HUVEC with 7,8-DHMC prevented H2O2-induced alterations of HSP90 cellular distribution and secretion. Our results suggest that the strong oxidative conditions of atherosclerotic plaques promote the upregulation of HSP90 surface expression on endothelial cells, thus rendering the protein a possible target of autoimmune reactions. The antioxidant 7,8-DHMC, by preventing oxidative-stress-triggered HSP90 surface upregulation, may be useful to counteract possible autoreactive reactions to HSP90.

The Nutraceutical Dehydrozingerone and Its Dimer Counteract Inflammation- and Oxidative Stress-Induced Dysfunction of In Vitro Cultured Human Endothelial Cells: A Novel Perspective for the Prevention and Therapy of Atherosclerosis.

Atherosclerosis is characterized by endothelial dysfunction, mainly induced by inflammation and oxidative stress. Increased reactive oxygen species (ROS) production together with increased adhesion molecules and thrombogenic tissue factor (TF) expression on endothelial cells has a key role in proatherogenic mechanisms. Therefore downmodulation of these molecules could be useful for reducing the severity of inflammation and atherosclerosis progression. Dehydrozingerone (DHZ) is a nutraceutical compound with anti-inflammatory and antioxidant activities. In this study we evaluated the ability of DHZ and its symmetric dimer to modulate hydrogen peroxide- (H2O2-) induced ROS production in human umbilical vein endothelial cells (HUVEC). We also evaluated intercellular adhesion molecule- (ICAM-) 1, vascular cell adhesion molecule- (VCAM-) 1, and TF expression in HUVEC activated by tumor necrosis factor- (TNF-) α. HUVEC pretreatment with DHZ and DHZ dimer reduced H2O2-induced ROS production and inhibited adhesion molecule expression and secretion. Of note, only DHZ dimer was able to reduce TF expression. DHZ effects were in part mediated by the inhibition of the nuclear factor- (NF-) κB activation. Overall, our findings demonstrate that the DHZ dimer exerts a potent anti-inflammatory, antioxidant, and antithrombotic activity on endothelial cells and suggest potential usefulness of this compound to contrast the pathogenic mechanisms involved in atherosclerosis progression.

Crosstalk between red blood cells and the immune system and its impact on atherosclerosis.

Atherosclerosis is a chronic multifactorial disease of the arterial wall characterized by inflammation, oxidative stress, and immune system activation. Evidence exists on a pathogenic role of oxidized red blood cells (RBCs) accumulated in the lesion after intraplaque hemorrhage. This review reports current knowledge on the impact of oxidative stress in RBC modifications with the surface appearance of senescent signals characterized by reduced expression of CD47 and glycophorin A and higher externalization of phosphatidylserine. The review summarizes findings indicating that oxidized, senescent, or stored RBCs, due to surface antigen modification and release of prooxidant and proinflammatory molecules, exert an impaired modulatory activity on innate and adaptive immune cells and how this activity contributes to atherosclerotic disease. In particular RBCs from patients with atherosclerosis, unlike those from healthy subjects, fail to control lipopolysaccharide-induced DC maturation and T lymphocyte apoptosis. Stored RBCs, accompanied by shedding of extracellular vesicles, stimulate peripheral blood mononuclear cells to release proinflammatory cytokines, augment mitogen-driven T cell proliferation, and polarize macrophages toward the proinflammatory M1 activation pathway. Collectively, literature data suggest that the crosstalk between RBCs with immune cells represents a novel mechanism by which oxidative stress can contribute to atherosclerotic disease progression and may be exploited for therapeutic interventions.

Pleiotropic effects of statins in atherosclerotic disease: focus on the antioxidant activity of atorvastatin.

Cardiovascular diseases, which are the leading cause of mortality in the Western World, are closely associated with atherosclerosis development. Atherosclerosis is a chronic multifactorial disease of the arterial wall characterized by endothelial dysfunction, inflammation and oxidative stress. Oxidative stress is an alteration of the balance between pro-oxidant and antioxidant mechanisms which promotes vascular complications and represents a valid therapeutic target to prevent or treat cardiovascular diseases. Statins are enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that have been included in the therapeutic regimen of cardiovascular diseases due to their lipid-lowering activity. Experimental and clinical data demonstrated that the antiatherogenic effects of these drugs are also related to other pleiotropic activities, particularly to their anti-inflammatory, anti-thrombotic and antioxidant effects. This review summarizes experimental and clinical studies demonstrating the impact of statins on atherosclerotic disease with a focus on the antioxidant activity of atorvastatin. Atorvastatin is a synthetic statin characterized by a high efficacy, in part due to its longer half-life compared to other molecules of the same group. It also exerts high antioxidant effects, independent from its hypolipidemic activity, beneficial for the prevention and therapy of atherosclerosis.

Resveratrol counteracts inflammation in human M1 and M2 macrophages upon challenge with 7-oxo-cholesterol: potential therapeutic implications in atherosclerosis.

MACROPHAGES CONSIST OF TWO MAIN SUBSETS: the proinflammatory M1 subset and the anti-inflammatory M2 one. 7-oxo-cholesterol, the most abundant cholesterol autoxidation product within atherosclerotic plaque, is able to skew the M1/M2 balance towards a proinflammatory profile. In the present study, we explored the ability of the polyphenolic compound resveratrol to counteract the 7-oxo-cholesterol-triggered proinflammatory signaling in macrophages. Resveratrol-pretreated human monocyte-derived M1 and M2 macrophages were challenged with 7-oxo-cholesterol and analyzed for phenotype and endocytic ability by flow cytometry, for metalloproteinase- (MMP-) 2 and MMP-9 by gelatin zymography, and for cytokine, chemokine, and growth factor secretome by a multiplex immunoassay. We also investigated the NF-κB signaling pathway. In the M1 subset, resveratrol prevented the downregulation of CD16 and the upregulation of MMP-2 in response to 7-oxo-cholesterol, whereas in M2 macrophages it prevented the upregulation of CD14, MMP-2, and MMP-9 and the downregulation of endocytosis. Resveratrol prevented the upregulation of several proinflammatory and proangiogenic molecules in both subsets. We identified modulation of NF-κB as a potential mechanism implicated in 7-oxo-cholesterol and resveratrol effects. Our results strengthen previous findings on the immunomodulatory ability of resveratrol and highlight its role as potential therapeutic or preventive compound, to counteract the proatherogenic oxysterol signaling within atherosclerotic plaque.

Neuropeptide Y induces potent migration of human immature dendritic cells and promotes a Th2 polarization.

Neuropeptide Y (NPY), a major autonomic nervous system and stress mediator, is emerging as an important regulator of inflammation, implicated in autoimmunity, asthma, atherosclerosis, and cancer. Yet the role of NPY in regulating phenotype and functions of dendritic cells (DCs), the professional antigen-presenting cells, remains undefined. Here we investigated whether NPY could induce DCs to migrate, mature, and polarize naive T lymphocytes. We found that NPY induced a dose-dependent migration of human monocyte-derived immature DCs through the engagement of NPY Y1 receptor and the activation of ERK and p38 mitogen-activated protein kinases. NPY promoted DC adhesion to endothelial cells and transendothelial migration. It failed to induce phenotypic DC maturation, whereas it conferred a T helper 2 (Th2) polarizing profile to DCs through the up-regulation of interleukin (IL)-6 and IL-10 production. Thus, during an immune/inflammatory response NPY may exert proinflammatory effects through the recruitment of immature DCs, but it may exert antiinflammatory effects by promoting a Th2 polarization. Locally, at inflammatory sites, cell recruitment could be amplified in conditions of intense acute, chronic, or cold stress. Thus, altered or amplified signaling through the NPY-NPY-Y1 receptor-DC axis may have implications for the development of inflammatory conditions.-Buttari, B., Profumo, E., Domenici, G., Tagliani, A., Ippoliti, F., Bonini, S., Businaro, R., Elenkov, I., Riganò, R. Neuropeptide Y induces potent migration of human immature dendritic cells and promotes a Th2 polarization.

Subclinical atherosclerosis in systemic lupus erythematosus and antiphospholipid syndrome: focus on ß2GPI-specific T cell response.

OBJECTIVE: Systemic Lupus Erythematosus (SLE) and antiphospholipid syndrome (APS) are associated with a high prevalence of atherosclerosis. ß2 glycoprotein I (ß2GPI) represents a link between autoimmunity and endothelial dysfunction. Recently, ß2GPI reactive T cells have been identified; however, their role in atherosclerosis is still under investigation. We evaluated early atherosclerosis in patients with SLE and APS and investigated T cell reactivity to ß2GPI and its relationship with atherosclerotic process. APPROACH AND RESULTS: Fifty SLE, 18 patients with primary APS (PAPS), and 25 healthy controls were enrolled. Demographic and clinical data, including traditional cardiovascular risk factors, were recorded. Monocyte ß2GPI and Tissue Factor (TF) expression and peripheral blood mononuclear cell response to ß2GPI stimulation were evaluated. Doppler ultrasound was performed to investigate flow-mediated dilatation (FMD) and carotid intima-media thickness (IMT). We detected an increase in mean IMT and a decrease in FMD in patients with SLE versus controls (P<0.05 and P=0.0001, respectively) and a decrease in FMD in patients with PAPS versus controls (P<0.05). Monocyte ß2GPI and TF expression was higher in patients with SLE and PAPS than in controls (P=0.006 and P=0.001, respectively); no correlation of monocyte ß2GPI and TF with IMT or FMD was detected. ß2GPI induced peripheral blood mononuclear cell proliferation in 32% of patients with SLE, 25% of patients with PAPS yet in none of the controls. Proliferative response to ß2GPI correlated with a history of arterial thrombosis, thrombocytopenia, and IMT >0.9 mm. CONCLUSIONS: A significant percentage of patients with SLE and PAPS show a ß2GPI-specific T cell reactivity, which is associated with subclinical atherosclerosis.

Actin is a target of T-cell reactivity in patients with advanced carotid atherosclerotic plaques.

Atherosclerosis is a chronic inflammatory disease of the arterial wall associated with autoimmune reactions. In a previous study, we observed the presence of actin-specific antibodies in sera from patients with carotid atherosclerosis. To extend our previous results we evaluated the possible role of actin as antigenic target of cell-mediated immune reactions in carotid atherosclerosis. Peripheral blood mononuclear cells (PBMC) from 17 patients and 16 healthy subjects were tested by cell proliferation assay and by ELISA for cytokine production. Actin induced a proliferative response in 47% of patients' PBMC samples, with SI ranging from 2.6 to 21.1, and in none of the healthy subjects' samples (patients versus healthy subjects, P = 0.02). The presence of diabetes in patients was significantly associated with proliferative response to actin (P = 0.04). IFN- γ and TNF- α concentrations were higher in PBMC from patients than in those from healthy subjects and in PBMC proliferating to actin than in nonproliferating ones. Our data demonstrate for the first time a role of actin as a target autoantigen of cellular immune reactions in patients with carotid atherosclerosis. The preferential proinflammatory Th1 activation suggests that actin could contribute to endothelial dysfunction, tissue damage, and systemic inflammation in carotid atherosclerosis.

Resveratrol prevents dendritic cell maturation in response to advanced glycation end products.

Advanced glycation end products (AGEs), generated through nonenzymatic glycosylation of proteins, lipids, and nucleic acids, accumulate in the body by age thus being considered as biomarkers of senescence. Senescence is characterized by a breakdown of immunological self-tolerance, resulting in increased reactivity to self-antigens. Previous findings suggest that AGE and its receptor RAGE may be involved in the pathogenesis of autoimmune reactions through dendritic cell (DC) activation. The aim of this study was to investigate whether resveratrol, a polyphenolic antioxidant compound with tolerogenic effects on DCs, was able to counteract the mechanisms triggered by AGE/RAGE interaction on DCs. By immunochemical and cytofluorimetric assays, we demonstrated that in vitro pretreatment of human monocyte-derived DCs with resveratrol prevents DC activation in response to glucose-treated albumin (AGE-albumin). We found that resveratrol exerts an inhibitory effect on DC surface maturation marker and RAGE up-regulation in response to AGE-albumin. It also inhibited proinflammatory cytokine expression, allostimulatory ability upregulation, mitogen-activated protein (MAP) kinases, and NF-κB activation in AGE-albumin-stimulated DCs. We suggest that resveratrol, by dismantling AGE/RAGE signaling on DCs may prevent or reduce increased reactivity to self-molecules in aging.

Oxidized haemoglobin-driven endothelial dysfunction and immune cell activation: novel therapeutic targets for atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by endothelial dysfunction, and in which innate and adaptive immune responses have a crucial role. Autoimmune reactions against several self molecules and modified self molecules have been identified in patients with atherosclerotic disease. Oxidative stress, increasingly reported in these patients is the major event causing protein structural modifications, thus inducing the appearance of neo/cryptic epitopes. Following intraplaque haemorrhage large amounts of cell-free haemoglobin (Hb) accumulate within atheroma, due to its impaired clearance by the haptoglobin-CD163 scavenging system. The pro-oxidative intraplaque microenvironment may induce Hb structural changes, thus generating neo/cryptic autoantigenic epitopes and rendering the oxidized self molecule as a dangerous signal for both immune and endothelial cells. In this review, we will present the most relevant information on Hb as a candidate self antigen involved in the pathogenesis of atherosclerotic disease and on its ability to trigger signals that drive endothelial dysfunction and immune cell activation. On these grounds, we will also discuss how these new paradigms may lead to novel therapeutic targets for cardiovascular diseases.

7-Oxo-cholesterol potentiates pro-inflammatory signaling in human M1 and M2 macrophages.

Macrophages, the major cellular components of atherosclerotic plaques, consist of two main subsets: the pro-inflammatory, M1 or classically activated macrophages, and the anti-inflammatory, M2 or alternatively activated macrophages. The molecular and cellular mechanisms that orchestrate the macrophage polarization and activation that may play a role in plaque progression and stability are poorly understood. Recent studies suggest that oxysterols, oxidative stress-mediated cholesterol oxidation products that are abundant in atherosclerotic lesions, may affect macrophage biology. We investigated whether 7-oxo-cholesterol (7oxo-C) affected polarized human M1 and M2 macrophage phenotypes and functions. Monocyte-derived M1 and M2 macrophages were challenged with 7oxo-C and their phenotype analyzed using flow cytometric analysis, and their function via secretome profiling, the presence of endocytosis and matrix metalloproteinase-9 (MMP-9) release. 7oxo-C increased the expression of HLA-DR in M1 macrophages, and CD14 on M2 macrophages. The oxysterol also reduced CD16 expression on M1 macrophages, while reducing their endocytotic capability and increasing MMP-9 secretion in M2 macrophages. Secretome profiling from cultured cell supernatants showed that 7oxo-C stimulated the production of key pro-atherogenic mediators involved in pro-inflammatory, pro-invasive and pro-angiogenic mechanisms both in M1 and M2 cells. Hypoxic conditions potentiated the effects of 7oxo-C on M1 and M2 cells. The ability of 7oxo-C to polarize macrophages toward a pro-inflammatory state represents a potentially novel mechanism by which oxidative stress can contribute to atherosclerotic lesion progression.

Lack of haptoglobin results in unbalanced VEGFα/angiopoietin-1 expression, intramural hemorrhage and impaired wound healing after myocardial infarction.

Decreased haptoglobin (Hp) functionality due to allelic variations is associated with worsened outcome in patients after myocardial infarction (MI). However, mechanisms through which haptoglobin deficiency impairs cardiac repair remain to be elucidated. In the present study, we identified novel molecular alterations mediated by Hp involved in early and late cardiac repair responses after left coronary artery ligation in Hp(-/-) and wild-type (WT) mice. We observed a higher mortality rate in Hp(-/-) mice despite similar infarct size between groups. Deaths were commonly caused by cardiac rupture in Hp(-/-) animals. Histological analysis of 3 and 7days old non-ruptured infarcted hearts revealed more frequent and more severe intramural hemorrhage and increased leukocyte infiltration in Hp(-/-) mice. Analyses of non-ruptured hearts revealed increased oxidative stress, reduced PAI-1 activity and enhanced VEGFα transcription in Hp(-/-) mice. In line with these observations, we found increased microvascular permeability in Hp(-/-) hearts 3days after infarction. In vitro, haptoglobin prevented hemoglobin-induced oxidative stress and restored VEGF/Ang-1 balance in endothelial cell cultures. During long-term follow-up of the surviving animals, we observed altered matrix turnover, impaired scar formation and worsened cardiac function and geometry in Hp(-/-)mice. In conclusion, haptoglobin deficiency severely deteriorates tissue repair and cardiac performance after experimental MI. Haptoglobin plays a crucial role in both short- and long-term cardiac repair responses by reducing oxidative stress, maintaining microvascular integrity, myocardial architecture and proper scar formation.

Cellular and molecular players in the atherosclerotic plaque progression.

Atherosclerosis initiation and progression is controlled by inflammatory molecular and cellular mediators. Cells of innate immunity, stimulated by various endogenous molecules that have undergone a transformation following an oxidative stress or nonenzymatic glycation processes, activate cells of the adaptive immunity, found at the borders of atheromas. In this way, an immune response against endogenous modified antigens takes place and gives rise to chronic low-level inflammation leading to the slow development of complex atherosclerotic plaques. These lesions will occasionally ulcerate, thus ending with fatal clinical events. Plaque macrophages represent the majority of leukocytes in the atherosclerotic lesions, and their secretory activity, including proinflammatory cytokines and matrix-degrading proteases, may be related to the fragilization of the fibrous cap and then to the rupture of the plaque. A considerable amount of work is currently focused on the identification of locally released proinflammatory factors that influence the evolution of the plaque to an unstable phenotype. A better understanding of these molecular processes may contribute to new treatment strategies. Mediators released by the immune system and associated with the development of carotid atherosclerosis are discussed.

Biomarkers of subclinical atherosclerosis in patients with autoimmune disorders.

Atherosclerosis is accelerated in rheumatoid arthritis (RA) and psoriatic arthritis (PsA). We investigated a possible association of oxidized low-density lipoproteins (ox-LDLs), nitric oxide (NO), 3-nitrotyrosine, vitamin A, vitamin E, and ß-carotene serum levels with subclinical atherosclerosis in RA and PsA. By the use of ELISA, we observed higher ox-LDL levels in patients with intima-media thickness (IMT) > 1 than in patients with IMT ≤ 1 and a negative correlation between NO levels and IMT values. By the use of high-performance liquid chromatography, we determined higher levels of vitamin A in patients with PsA and IMT ≤ 1 than in controls and lower levels of ß-carotene in patients with RA and PsA than in controls. ß-carotene concentrations were negatively correlated to the duration of disease in RA. Our study confirms that ox-LDLs and NO may be markers of accelerated atherosclerosis in RA and PsA whereas vitamins seem to be associated only to the presence of the autoimmune disorders.