Atherogenesis and atherosclerosis in primary antiphospholipid syndrome

ABSTRACT: In the late seventies the term “Haematological Stress Syndrome” defined some haematological abnormalities appearing in the course of acute and chronic disorders, such as raised plasma levels of fibrinogen (FNG) and factor VIII, reduced fibrinolytic activity and hyperviscosity. In the early nineties the “Membrane stress syndrome hypothesis” proposed the unification of the concepts of haematological stress syndrome with those of oxidation, inflammation and immune activation to explain the pathogenesis of the antiphospholipid syndrome (APS) Antiphospholipid antibodies, coagulation, fibrinolysis and thrombosis. This chapter investigated the occurrence of the “Haematological Stress Syndrome” and thrombosis in 144 participants positive for aPL detected by clotting and immune tests. Among the clotting assays for the detection of lupus anticoagulant, dilute Russell's viper venom time better correlated with a history of venous thrombosis than activated partial thromboplastin time (p<0.0002 vs p<0.009) and was the only test correlated with a history of arterial thrombosis (p<0.01). By regression analysis, serum levels of IgG anticardiolipin antibodies (aCL) associated with the number of venous occlusions (p<0.001). With regards to FNG and von Willebrand factor (vWF), the former rose by 36% (95% CI; 21%, 53%) and the latter by 50% (95% CI; 29%, 75%) at the first venous occlusion and remained unchanged after subsequent occlusions. At variance FNG rose by 45% (95% CI; 31%, 60%) per arterial occlusion and vWF by 27% (95% CI; 10%, 47%) per arterial occlusion throughout. The coagulation/fibrinolytic balance was cross-sectionally evaluated on 18 thrombotic PAPS patients, 18 subjects with persistence of idiopathic aPL and in healthy controls. Markers of thrombin generation prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT) and of fibrin turnover D-Dimer (D-D) were higher in thrombotic (p=0.006)and non-thrombotic subjects (p=0.0001) than in controls as were those of D-D (p<0.0001 and p=0.003 respectively). TAT levels did not differ. Gender analysed data revealed blunted tPA release (hence a negative venous occlusion test) in thrombotic females but neither in thrombotic males (p=0.01) nor in asymptomatic subjects of either sex. Also, in both patient groups females had higher mean PAI than males (p<0.0002) and control females (p<0.02). The activity of factor XIII (FXIIIa) was evaluated was evaluated in 29 patients with PAPS, 14 persistent carriers of aPL without thrombosis, 24 thrombotic patients with inherited thrombophilia, 28 healthy controls and 32 patients with mitral and aortic valve prosthesis as controls for FXIII only. FXIIIa was highest in PAPS (p=0.001), particularly in patients with multiple (n=12) than single occlusion (p=0.02) and in correlation with PAI (p=0.003) and FNG (p=0.005). Moreover FXIIIa was strongly associated with IgG aCL and IgG anti-2GPI (p=0.005 for both) in the PAPS group and to a lesser degree in the aPL group (FXIIIa with IgG aCL, p=0.02, with IgG anti-2GPI, p=0.04). Altogether these results indicate: 1) a differential relationship of aPL, vWF and FNG with venous and arterial thrombosis; 2) heightened thrombin generation, accelerated fibrin turnover and fibrinolysis abnormalities also in asymptomatic carriers of aPLs; 3) enhanced FXIIIa that may contribute to atherothrombosis via increased fibrin/fibrinogen cross-linking. Lipid profile, lipid peroxidation and anti-lipoprotein antibodies in thrombotic primary antiphospholipid syndrome. Given the atherogenic lipid profile of SLE, the same possibility was explored in PAPS by comparing high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (CHO), apolipoprotein AI (ApoAI), apolipoprotein B (ApoB), triglycerides (TG), anti-lipoprotein antibodies, beta-2-glycoprotein I complexed to oxidized low-density lipoprotein (oxLDL-2GPI) and C-reactive protein (CRP) in 34 thrombotic PAPS patients compared to 36 thrombotic patients with inherited thrombophilia (IT), to 18 subjects persistently positive for antiphospholipid antibodies (aPL) with no underlying autoimmune or non-autoimmune disorders and to 28 healthy controls. Average concentrations of HDL (p<0.0001), LDL (p<0.0001), CHO (p=0.0002), ApoAI (p=0.002) were lower in PAPS whereas average TRY was higher (p=0.01) than other groups. Moreover PAPS showed higher IgG anti-HDL (p=0.01) and IgG anti-ApoAI (p<0.0001) as well as greater average oxLDL-2GPI (p=0.001) and CRP (p=0.003). Within PAPS, IgG anti-HDL correlated negatively to HDL (p=0.004) and was an independent predictor of oxLDL-2GPI (p=0.009). HDL and ApoAI correlated negatively with CRP (p=0.001 and p=0.007, respectively). IgG anti-HDL may hamper the antioxidant and anti-inflammatory effect of HDL favouring low-grade inflammation and enhanced oxidation in thrombotic PAPS. Indeed plasma 8-epi-prostaglandin F2α (a very specific marker of lipid peroxidation) was significantly higher in 10 patients with PAPS than 10 age and sex matched healthy subjects (p=0.0002) and strongly related to the titre of plasma IgG aCL (r=0.89, p=0.0004). Hence oxidative stress, a major player in atherogenesis, also characterises PAPS. Nitric oxide and nitrative stress in thrombotic primary antiphosholipid syndrome. Oxidative stress goes hand in hand with nitrative stress and to address the latter plasma nitrotyrosine (NT, marker of nitrative stress), nitrite (NO2-) and nitrate (NO3-) were measured in 46 thrombotic PAPS patients, 21 asymptomatic but persistent carriers of antiphospholipid antibodies (PCaPL), 38 patients with inherited thrombophilia (IT), 33 patients with systemic lupus erythematosus (SLE) and 29 healthy controls (CTR). Average crude NT was higher in PAPS and SLE (p=0.01) whereas average plasma NO2- was lower in PAPS and average NO3- highest in SLE (p<0.0001). In PAPS, IgG aCL titer and number of vascular occlusions negatively predicted NO2-, (p=0.03 and p=0.001, respectively) whereas arterial occlusions and smoking positively predicted NO3- (p=0.05 and p=0.005). Moreover CRP (an inflammatory marker) positively predicted NT (p=0.004). Nitric oxide metabolites relates to type and number of vascular occlusions and to aPL titers, whereas nitrative stress relates to low grade marker) positively predicted NT (p=0.004). Nitric oxide metabolites relates to type and number of vascular occlusions and to aPL titers, whereas nitrative stress relates to low grade inflammation and both phenomena may have implications for thrombosis and atherosclerosis in PAPS Inflammation and immune activation in thrombotic primary antiphospholipid syndrome. To investigate inflammation and immune activation in thrombotic PAPS high-sensitivity CRP (hs-CRP), serum amyloid A (SAA), oxLDL-2GPI, CRP bound to oxLDL-2GPI (CRP-oxLDL-2GPI) (as inflammatory markers) neopterin (NPT) and soluble CD14 (sCD14) (as immune activation markers) were measured by ELISA in 41 PAPS patients, in 44 patients with inherited thrombophilia (IT) and 39 controls (CTR). Compared to other groups, PAPS presented with higher plasma concentrations of inflammatory, hs-CRP (p=0.0004), SAA (p<0.01), CRP-oxLDL-2GPI (p=0.0004) and immune activation markers, NPT (p<0.0001) and sCD14 (p=0.007). By regression analysis SAA independently predicted thrombosis number (p=0.003) and NPT independently predicted thrombosis type (arterial, p=0.03) and number (p=0.04). These data confirm that low-grade inflammation and immune activation occur and relate to vascular features of PAPS. Antiphosholipid antibodies, haemostatic variables and atherosclerosis in thrombotic primary antiphospholipid syndrome To evaluate whether IgG aCL titre, haemostatic variables and the lipid profile bore any relationship to the intima media thickness (IMT) of carotid arteries high-resolution sonography was applied to the common carotid (CC), carotid bifurcation (CB) and internal carotid (IC) of 42 aPL subjects, 29 with primary thrombotic antiphospholipid syndrome and 13 with persistence of aPL in the absence of any underlying disorder. The following were measured: plasma FNG, vWF, PAI, homocysteine (HC), CHO, TG, HDL, LDL, platelet numbers and aCL of IgG and IgM isotype. By multiple regression analysis, IgG aCL titre independently predicted IMT at all carotid segments examined (p always <0.005). Plasma FNG and HC independently predicted IMT at the CB (p=0.001 and p<0.0001, respectively) and IC (p=0.03 and p<0.0001, respectively). These data strongly support an atherogenic role for IgG aCL in patients with aPL in addition to traditional risk factors. The atherosclerosis hypothesis was investigated in an age and sex-matched case-double-control study including 49 thrombotic PAPS patients (18 M, 31 F, mean age 37 ± 11), 49 thrombotic patients for IT and 49 healthy subjects. Average IMT was always greater in PAPS than control patients (CC: p=0.004, CB: p=0.013, IC: p=0.001). By dividing participants into age tertiles the IMT was greater in the second (CC: p=0.003, CB: p=0.023, IC: p=0.003) and third tertiles (CC: p=0.03, CB: p=0.004, IC: p=0.007). Conclusion: Coagulation activation, fibrinolysis depression, hightened fibrin turnover, oxidative and nitrative stress in parallel with low grade inflammation and immune activation characterise thrombotic PAPS: all these are early atherogenic processes and contribute to the demonstrated premature atherosclerosis that should be considered a clinical feature of PAPS.

Humoral response towards high density lipoprotein : a new mechanism for atherogenesis

RESUMO:Aterosclerose é uma das principais causas de morbilidade e mortalidade no mundo ocidental. É responsável, direta ou indiretamente, pela maior percentagem de gastos com a saúde na maioria dos países europeus. A “teoria lipídica” da aterosclerose, que se baseia na dislipidemia como causa primária para a doença vascular tem algumas implicações práticas importantes: permite a definição de linhas de orientação e protocolos simples e ainda estabelece alvos terapêuticos que podem ser atingidos na maior parte dos casos com a atual intervenção farmacológica. A associação da aterosclerose com o sistema imunológico (a “teoria imunológica”), forneceu por sua vez novas formas de explorar os mecanismos envolvidos e abriu novas perspetivas para um conhecimento mais completo da doença. No entanto, levanta dificuldades evidentes no que diz respeito às possibilidades terapêuticas. De todos os intervenientes no processo aterosclerótico (bioquímicos, imunológicos e anatómicos), as lipoproteínas de elevada densidade (HDL) são atualmente reconhecidas como um dos fatores mais importantes na aterogénese. Isto é baseado no reconhecimento das múltiplas propriedades anti-aterogénicas das HDL como por exemplo: a anti-oxidante, a anti-inflamatória e a antitrombótica, bem como o seu importante papel na melhoraria da função endotelial. Atualmente, é consensual que as funções anti-aterogénicas das HDL vão além do seu papel no transporte reverso do colesterol (RCT) e a importância das HDL no processo aterosclerótico baseia-se não apenas no seu papel protetor impedindo a formação da placa de ateroma, mas também na estabilização destas, prevenindo a sua ruptura e, consequentemente o evento trombótico. Como fundamentais no processo aterosclerótico estão reconhecidos dois principais conjuntos de eventos: um caracterizado por alterações no metabolismo das lipoproteínas que resultam em lipoproteínas pró-inflamatórias e pró-oxidantes que interagem com os componentes celulares da parede arterial e que conduzem à formação da placa de ateroma; o outro evento é a resposta imunológica desencadeada contra um novo conjunto de antigénios que por sua vez leva à produção de citoquinas pró-inflamatórias. Dada a complexidade da HDL e das suas múltiplas funções estas lipoproteínas tornaram-se um potencial alvo para a resposta auto-imune, e cujas consequências podem explicar algumas das associações identificados em estudos clínicos e epidemiológicos. Contudo esta interação entre o sistema imunológico e HDL nunca foi exaustivamente estudada. Portanto, pomos a hipótese de que em condições oxidativas e pró-inflamatórias, um aumento do antigénio (HDL) conduz a um consequente acréscimo na produção de anticorpos anti-HDL (aHDL) responsáveis pela alteração quantitativa e / ou qualitativa das HDL. O conceito de que estes anticorpos podem contribuir tanto para a evolução a longo prazo do processo aterosclerótico, como para o desencadeamento de eventos clínicos pode também explicar a heterogeneidade encontrada em cada doente e nos grandes estudos clínicos, no que diz respeito aos fatores de risco e outcomes clínicos. Para além disso, a confirmação desta hipótese pode permitir explicar porque é que as intervenções terapêuticas atualmente em desenvolvimento para aumentar os níveis de HDL, não conseguem mostrar a tão esperada redução do risco vascular. O objetivo geral desta tese foi identificar e caracterizar a resposta humoral contra os componentes da HDL, e avaliar possíveis mecanismos que possam contribuir para a modificação das propriedades anti-aterogénicas das HDL. Para alcançar este objetivo investigou-se: 1) A presença de anticorpos aHDL em doentes com lúpus eritematoso sistémico (SLE) e em doentes com manifestações clínicas de aterosclerose, como os doentes com doença arterial coronária (CAD), acidente vascular cerebral isquémico (IS) e diabetes tipo 2; 2) Os principais alvos antigénicos dentro do complexo das HDL e a associação entre os títulos de anticorpos aHDL e diferentes características clínicas destas doenças; 3) As modificações das funções normais associadas às HDL, em particular da função anti-oxidante e anti-inflamatória; 4) A atividade biológica dos anticorpos aHDL isolados do soro de doentes através de um conjunto de experiências in vitro de inibição da atividade da paraoxonase 1 (PON1) e da expressão de moléculas de adesão em culturas de células endoteliais. Para tal foi necessário estabelecer um método de isolamento dos anticorpos. Os anticorpos aHDL isolados do soro de doentes foram utilizados de forma a identificar as potenciais alterações dos sistemas celulares utilizados; 5) O efeito de fármacos usados no tratamento das dislipidemias, em particular o ácido nicotínico e as estatinas, na variação dos títulos de anticorpos aHDL através de ensaios clínicos randomizados, controlados com placebo e em dupla ocultação. Os métodos utilizados neste trabalho incluíram: técnicas imunológicas (como por exemplo, enzyme-linked immunoabsorbent assay - ELISA, ensaio imunoturbidimetrico e cromatografia de imuno-afinidade) técnicas bioquímicas (tais como a quantificação de atividade enzimática por espectrofotometria e por luminescência), experiências com cultura de células e citometria de fluxo. Os nossos resultados mostram que: 1) A presença de anticorpos aHDL, e mais especificamente anticorpos contra alguns do seus principais componentes como a apolipoproteína A-I (ApoA-I, principal apolipoproteína presente nas HDL) e a PON1 (o enzima que mais contribui para a propriedade anti-oxidante das HDL), quer em doentes com doenças auto-imunes, como o SLE, quer em doentes com manifestações clínicas de aterosclerose, como CAD, IS e diabetes tipo 2. Os doentes apresentaram títulos de anticorpos IgG aHDL, aApoA-I e aPON1 significativamente mais elevados do que controlos saudáveis com a mesma idade e sexo. 2) A correlação positiva estatisticamente significativa entre os títulos de aHDL e aApoA-I e aPON1 sugere que estes sejam dois dos principais alvos antigénicos dentro do complexo das HDL. Os anticorpos encontrados nestes doentes estão associados com a diminuição da atividade da PON1 e a uma redução da capacidade anti-oxidante total (TAC) do soro, um aumento dos biomarcadores de disfunção endotelial (como por exemplo dos metabolitos do óxido nítrico - NO2- e NO3-, as moléculas de adesão vascular e intracelular - VCAM-1 e ICAM-1 e os níveis de 3-nitrotirosina). Nos doentes com SLE os títulos destes estão associados a um aumento do dano cardiovascular e à atividade global da doença avaliados pelas escalas SLICC/ACR DI e BILAG score, respetivamente. Enquanto que nos doentes com diabetes tipo 2 estes anticorpos estão associados com um aumento dos níveis de glicemia em jejum (FGP) e hemoglobina glicada (HbA1c). 3) Após se ter estabelecido um método de isolamento dos anticorpos que permite isolar quantidades significativas de anticorpos do soro de doentes sem perder a sua especificidade, foi identificada a capacidade dos anticorpos isolados do soro de doentes inibirem de uma forma dependente da concentração a atividade da PON1 até um máximo de 70% no caso dos doentes com SLE e ente 7-52% no caso dos anticorpos isolados de doentes com CAD e IS. 4) O efeito anti-inflamatório das HDL na inibição da produção de VCAM-1 induzida por citoquinas (como o TNF-) foi revertido em mais de 80% pelos anticorpos aHDL isolados do soro de doentes. 5) A angiogenesis induzida por HDL através do aumento do fator de crescimento do endotélio vascular (VEGF) foi anulada em 65% pelos anticorpos aHDL isolados do soro de doentes. 6) Os atuais agentes farmacológicos disponíveis para aumentar as concentrações de HDL-C estão associados a um aumento dos títulos de anticorpos.-------- ABSTRACTAtherosclerosis is the major cause of morbidity and mortality in the western world. It is also responsible, directly or indirectly, for the highest percentage of health costs in most European countries. Despite the use of new technologies for the diagnosis of vascular disease and regardless of the major advances in treatment, the atherosclerosis-related clinical burden is still raising. The “lipid theory” of atherogenesis, which identifies dyslipidemia as the primary cause of this vascular disease has some important practical implications: it allows the definition of simple guidelines and establishes therapeutic targets which can be generally met with current pharmacologic intervention. The association between atherosclerosis an the immune system (the immune concept) has in turn provided new ways of exploring the mechanisms involved in this condition and has opened new perspectives in the understanding of the disease. However, it raises obvious difficulties when it comes to treatment options. Of all the players (biochemical, immunological and anatomical) involved in this matter, high-density lipoproteins (HDL) are currently recognised as one of the most important factors in atherogenesis. This is based on the recognition of HDL's multiple anti-atherogenic properties: anti-oxidant, anti-inflammatory and antithrombotic, as well as its capacity to improve endothelial function. Nowadays, it is widely recognized that the anti-atherogenic functions of HDL go beyond reverse cholesterol transport (RCT), and the importance of HDL is based not just on its ability to reduce atheroma formation but also on its ability to stabilise plaques, therefore preventing their rupture and ultimately thrombosis. Two main set of events have been recognised as fundamental in atherogenesis: one, characterized by lipoprotein metabolism alterations, resulting in pro-inflammatory and pro-oxidative lipoproteins, which interact with the normal cellular elements of the arterial wall leading to atheroma formation; the other, the immune cellular response towards new sets of antigens which lead to the production of pro-inflammatory cytokines. Given to HDL complexity and multiple functions this lipoprotein has became a potential target for an auto-immune response, the consequences of which may explain some of the association identified in epidemiological and clinical studies, though the interaction between the immune system and HDL has never been thoroughly addressed. Therefore, we hypothesized that under oxidative and pro-inflammatory conditions, the increase in the antigen (HDL) would lead to a consequent increase in the production of anti-HDL (aHDL) antibodies be responsible for quantitative and/or qualitative changes of HDL. The concept that these antibodies may contribute either to the long-term evolution of atherosclerosis or to the triggering of clinical events may also explain the heterogeneity found in individual patients and in large cohorts regarding risk factors and clinical outcomes. Moreover this may be a major breakthrough in understanding why therapeutic interventions that increase HDL levels, failed to show the anticipated reduction in vascular risk. The overall aims of this thesis were to identified and characterize the humoral response towards HDL components and to evaluate the possible mechanisms that may contribute to the modifications of the anti-atherogenic properties of HDL. To achieve this objective we investigated: 1) the presence of aHDL antibodies in patients with systemic lupus erythematosus (SLE) and in patients with atherosclerosis-related clinical events, such as coronary artery disease (CAD), ischemic stroke (IS) and type 2 diabetes; 2) the association between the titres of aHDL antibodies and different clinical features of these diseases; 3) the modifications of the anti-atherogenic properties of HDL; 4) the biologic effect of aHDL antibodies isolated from serum of patients on the anti-oxidant and anti-inflammatory properties of HDL; 5) the effect of different pharmacologic treatments for dyslipidemia on the prevalence and activity of aHDL antibodies. The methodologies used in this work included immunologic-related techniques (e.g. enzyme-linked immunoabsorbent assay – ELISA, immunoturbidimetric immunoassay and immunoaffinity chromatography), biochemical techniques (enzymatic assays with quantification by spectrophotometry and luminescence methods), cell culture experiments and flow cytometry. Our results indicate that: 1) The titres of IgG aHDL, anti-apolipoprotein A-I (aApoA-I) and anti-paraoxonase 1 (aPON1) antibodies were higher in patients with SLE, CAD, IS and type 2 diabetes when compared with age and sex matched healthy controls. 2) The antibodies found in these patients were associated with decreased PON1 activity, (the enzyme responsible for most of the anti-oxidant effect of HDL), reduced total anti-oxidant capacity (TAC) of serum and increased biomarkers of endothelial dysfunction (nitric oxide metabolites, adhesion molecules, nitrotyrosine). In patients with SLE the antibody titres were associated with an increase in disease-related cardiovascular damage and activity whereas in patients with type 2 diabetes they were directly related with the fasting glucose plasma (FGP) levels and the glycosylated haemoglobin (HbA1c). 3) The antibodies isolated from serum of our patients, directly inhibited HDL-associated PON1 activity in a dose dependent way ranging from 7 to 52%. 4) The anti-inflammatory effect of HDL, measured by the percentage of inhibition of the cytokine-induced production of vascular adhesion molecules (VCAM-1), was reduced in more than 80% by aHDL antibodies isolated from our patients. 5) The HDL-induced angiogenesis by increasing vascular endothelial growth factor (VEGF) levels was abrogated in 65% by the antibodies isolated from serum of patients. 6) The current available pharmacologic agents for increasing HDL-C concentrations were associated with an increase in the titres of IgG aApoA-I antibodies. This increase was higher in the extended release niacin when compared to statins probably due to their dampening effect on oxidative stress. In conclusion, aHDL antibodies are present in different pathologic conditions. aHDL antibodies represent a family of self-reacting immunoglobulins, of which ApoA-I and PON1 might be the most relevant targets. These antibodies are biologically active, interfering with the HDL anti-oxidant and anti-inflammatory properties and, consequently, with the atherosclerotic process. The pathogenic potential of these antibodies may lead to the identification of a new biomarker for vascular disease, whilst presenting itself as a novel target for a different treatment approach which may redefine the treatment strategies and clinical trials design for HDL interventions in the future.

Influence of CD4+/CD25+ regulatory T cells on atherogenesis in patients with end-stage kidney disease.

Atherosclerosis, which is influenced by both traditional and nontraditional cardiovascular risk factors and has been characterized as an inflammatory process, is considered to be the main cause of the elevated cardiovascular risk associated with chronic kidney disease. The inflammatory component of atherosclerosis can be separated into an innate immune response involving monocytes and macrophages that respond to the excessive uptake of lipoproteins and an adaptive immune response that involves antigen-specific T cells. Concurrent with the influx of immune cells to the site of atherosclerotic lesion, the role of the adaptive immune response gradually increases. One of those cells are represented by the CD4+/CD25+ Tregs, which play indispensable roles in the maintenance of natural self-tolerance and negative control of pathological, as well as physiological, immune responses. Altered self-antigens such as oxidized LDLs may induce the development of CD4+/CD25+ Tregs with atheroprotective properties. However, atherosclerosis may be promoted by an imbalance between regulatory and pathogenic immunity that may be represented by the low expression of the forkhead box transcription factor (Foxp3) in CD4+/CD25+ Tregs. Such a defect may break immunologic tolerance and alter both specific and bystander immune suppression, leading to exacerbation of plaque development. Patients with end-stage kidney disease (ESKD) display a cellular immune dysfunction and accelerated atherosclerosis. Uremic solutes that accumulate during ESKD may be involved in these processes. In patients with ESKD and especially in those that are chronically hemodialyzed, oxidative stress induced by oxidized LDLs may increase CD4+/CD25+ Treg sensitivity to Fas-mediated apoptosis as a consequence of specific dysregulation of IL-2 expression. This review will focus on the current state of knowledge regarding the influence of CD4+/CD25+ Tregs on atherogenesis in patients with ESKD, and the potential effect of statins on the circulating number and the functional properties of these cells.

Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis.

BACKGROUND: Macrophage-mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg-II) in macrophage function remains elusive. This study characterizes the role of Arg-II in macrophage inflammatory responses and its impact on obesity-linked type II diabetes mellitus and atherosclerosis. METHODS AND RESULTS: In human monocytes, silencing Arg-II decreases the monocytes' adhesion to endothelial cells and their production of proinflammatory mediators stimulated by oxidized low-density lipoprotein or lipopolysaccharides, as evaluated by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Macrophages differentiated from bone marrow cells of Arg-II-deficient (Arg-II(-/-)) mice express lower levels of lipopolysaccharide-induced proinflammatory mediators than do macrophages of wild-type mice. Importantly, reintroducing Arg-II cDNA into Arg-II(-/-) macrophages restores the inflammatory responses, with concomitant enhancement of mitochondrial reactive oxygen species. Scavenging of reactive oxygen species by N-acetylcysteine prevents the Arg-II-mediated inflammatory responses. Moreover, high-fat diet-induced infiltration of macrophages in various organs and expression of proinflammatory cytokines in adipose tissue are blunted in Arg-II(-/-) mice. Accordingly, Arg-II(-/-) mice reveal lower fasting blood glucose and improved glucose tolerance and insulin sensitivity. Furthermore, apolipoprotein E (ApoE)-deficient mice with Arg-II deficiency (ApoE(-/-)Arg-II(-/-)) display reduced lesion size with characteristics of stable plaques, such as decreased macrophage inflammation and necrotic core. In vivo adoptive transfer experiments reveal that fewer donor ApoE(-/-)Arg-II(-/-) than ApoE(-/-)Arg-II(+/+) monocytes infiltrate into the plaque of ApoE(-/-)Arg-II(+/+) mice. Conversely, recipient ApoE(-/-)Arg-II(-/-) mice accumulate fewer donor monocytes than do recipient ApoE(-/-)Arg-II(+/+) animals. CONCLUSIONS: Arg-II promotes macrophage proinflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis. Targeting Arg-II represents a potential therapeutic strategy in type II diabetes mellitus and atherosclerosis. (J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.).

Lipoproteins and mitogen-activated protein kinase signaling: a role in atherogenesis?

PURPOSE OF REVIEW: Lipoproteins play a critical role in the development of atherosclerosis, which might result partly from their capacity to induce specific intracellular signaling pathways. The goal of this review is to summarize the signaling properties of lipoproteins, in particular, their capacity to induce activation of mitogen-activated protein kinase pathways and the resulting modulation of cellular responses in blood vessel cells. RECENT FINDINGS: Lipoproteins activate the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways in all blood vessel cell types. This may require lipoprotein docking to scavenger receptor B1, allowing transfer of cholesterol and sphingosine-1-phosphate to plasma membranes. Subsequent propagation of the signals probably requires the stimulation of G protein-coupled receptors, followed by the transactivation of receptor tyrosine kinases. Lipoprotein-induced extracellular signal-regulated kinase activity favors cell proliferation, whereas lipoprotein-induced p38 mitogen-activated protein kinase activity leads to cell hyperplasia and promotes cell migration. Some signaling pathways and cellular effects induced by lipoproteins have been observed in atherosclerotic plaques and therefore represent potential targets for the development of anti-atherosclerotic drugs. SUMMARY: The main blood vessel cell types have the capacity to activate protein kinase pathways in the presence of lipoproteins. This induces cell proliferation, hyperplasia and migration, known to be dysregulated in atherosclerotic lesions.

Effect of Lactobacillus delbrueckii on cholesterol metabolism in germ-free mice and on atherogenesis in apolipoprotein E knock-out mice

Elevated blood cholesterol is an important risk factor associated with atherosclerosis and coronary heart disease. Several studies have reported a decrease in serum cholesterol during the consumption of large doses of fermented dairy products or lactobacillus strains. The proposed mechanism for this effect is the removal or assimilation of intestinal cholesterol by the bacteria, reducing cholesterol absorption. Although this effect was demonstrated in vitro, its relevance in vivo is still controversial. Furthermore, few studies have investigated the role of lactobacilli in atherogenesis. The aim of the present study was to determine the effect of Lactobacillus delbrueckii on cholesterol metabolism in germ-free mice and the possible hypocholesterolemic and antiatherogenic action of these bacteria using atherosclerosis-prone apolipoprotein E (apo E) knock-out (KO) mice. For this purpose, Swiss/NIH germ-free mice were monoassociated with L. delbrueckii and fed a hypercholesterolemic diet for four weeks. In addition, apo E KO mice were fed a normal chow diet and treated with L. delbrueckii for 6 weeks. There was a reduction in cholesterol excretion in germ-free mice, which was not associated with changes in blood or liver cholesterol concentration. In apo E KO mice, no effect of L. delbrueckii was detected in blood, liver or fecal cholesterol. The atherosclerotic lesion in the aorta was also similar in mice receiving or not these bacteria. In conclusion, these results suggest that, although L. delbrueckii treatment was able to reduce cholesterol excretion in germ-free mice, no hypocholesterolemic or antiatherogenic effect was observed in apo E KO mice.

Treatment With Methotrexate Inhibits Atherogenesis in Cholesterol-Fed Rabbits

A decrease in the number of cardiovascular events in patients with rheumatoid arthritis or psoriasis treated with methotrexate (MTX) has been observed in the literature. The aim of this study was to test whether MTX could promote anti-inflammatory effects and reduce the atherosclerotic lesions in rabbits with atherosclerosis induced by cholesterol feeding. Twenty male New Zealand rabbits were fed a 1% cholesterol diet for 60 days. Starting from day 30 of cholesterol feeding, 10 animals were treated with 4 weekly intravenous injections of MTX (4 mg/kg) and 10 with 4 weekly saline solution injections for 30 days. MTX reduced the size of the lesion areas of cholesterol-fed animals by 75% and intima-media ratio 2- fold. The drug inhibited macrophage migration into the intima by 50% and the presence of apoptotic cells by 84% but did not inhibit the intimal proliferation of smooth muscle cells. MTX treatment also diminished the positive staining area of metalloproteinase 9 in the intima, which is probably beneficial. In the tumor necrosis factor-alpha-treated human umbilical vein endothelial cell line, incubation with MTX led to downregulation of 5 pro-inflammatory genes, TNF-alpha, VAP-1, IL-1 beta, CXCL2, and TLR2, and upregulation of the antiinflammatory TGF-beta 1 gene, thus showing endothelium-protective properties. In conclusion, MTX showed direct in vivo anti-atherosclerotic action and may have potential in the treatment of this disorder.

Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis

Macrophage-mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg-II) in macrophage function remains elusive. This study characterizes the role of Arg-II in macrophage inflammatory responses and its impact on obesity-linked type II diabetes mellitus and atherosclerosis.

Acute systemic inflammation up-regulates secretory sphingomyelinase in vivo: A possible link between inflammatory cytokines and atherogenesis

Inflammation plays a critical role in atherogenesis, yet the mediators linking inflammation to specific atherogenic processes remain to be elucidated. One such mediator may be secretory sphingomyelinase (S-SMase), a product of the acid sphingomyelinase gene. The secretion of S-SMase by cultured endothelial cells is induced by inflammatory cytokines, and in vivo data have implicated S-SMase in subendothelial lipoprotein aggregation, macrophage foam cell formation, and possibly other atherogenic processes. Thus, the goal of this study was to seek evidence for S-SMase regulation in vivo during a physiologically relevant inflammatory response. First, wild-type mice were injected with saline or lipopolysaccharide (LPS) as a model of acute systemic inflammation. Serum S-SMase activity 3 h postinjection was increased 2- to 2.5-fold by LPS (P < 0.01). To determine the role of IL-1 in the LPS response, we used IL-1 converting enzyme knockout mice, which exhibit deficient IL-1 bioactivity. The level of serum S-SMase activity in LPS-injected IL-1 converting enzyme knockout mice was ≈35% less than that in identically treated wild-type mice (P < 0.01). In LPS-injected IL-1-receptor antagonist knockout mice, which have an enhanced response to IL-1, serum S-SMase activity was increased 1.8-fold compared with LPS-injected wild-type mice (P < 0.01). Finally, when wild-type mice were injected directly with IL-1β, tumor necrosis factor α, or both, serum S-SMase activity increased 1.6-, 2.3-, and 2.9-fold, respectively (P < 0.01). These data show regulation of S-SMase activity in vivo and they raise the possibility that local stimulation of S-SMase may contribute to the effects of inflammatory cytokines in atherosclerosis.

Acceleration of atherogenesis by COX-1-dependent prostanoid formation in low density lipoprotein receptor knockout mice

The cyclooxygenase (COX) product, prostacyclin (PGI2), inhibits platelet activation and vascular smooth-muscle cell migration and proliferation. Biochemically selective inhibition of COX-2 reduces PGI2 biosynthesis substantially in humans. Because deletion of the PGI2 receptor accelerates atherogenesis in the fat-fed low density lipoprotein receptor knockout mouse, we wished to determine whether selective inhibition of COX-2 would accelerate atherogenesis in this model. To address this hypothesis, we used dosing with nimesulide, which inhibited COX-2 ex vivo, depressed urinary 2,3 dinor 6-keto PGF1α by approximately 60% but had no effect on thromboxane formation by platelets, which only express COX-1. By contrast, the isoform nonspecific inhibitor, indomethacin, suppressed platelet function and thromboxane formation ex vivo and in vivo, coincident with effects on PGI2 biosynthesis indistinguishable from nimesulide. Indomethacin reduced the extent of atherosclerosis by 55 ± 4%, whereas nimesulide failed to increase the rate of atherogenesis. Despite their divergent effects on atherogenesis, both drugs depressed two indices of systemic inflammation, soluble intracellular adhesion molecule-1, and monocyte chemoattractant protein-1 to a similar but incomplete degree. Neither drug altered serum lipids and the marked increase in vascular expression of COX-2 during atherogenesis. Accelerated progression of atherosclerosis is unlikely during chronic intake of specific COX-2 inhibitors. Furthermore, evidence that COX-1-derived prostanoids contribute to atherogenesis suggests that controlled evaluation of the effects of nonsteroidal anti-inflammatory drugs and/or aspirin on plaque progression in humans is timely.

Testosterone inhibits early atherogenesis by conversion to estradiol: Critical role of aromatase

The effects of testosterone on early atherogenesis and the role of aromatase, an enzyme that converts testosterone to estrogens, were assessed in low density lipoprotein receptor-deficient male mice fed a Western diet. Castration of male mice increased the extent of fatty streak lesion formation in the aortic origin compared with testes-intact animals. Administration of anastrazole, a selective aromatase inhibitor, to testes-intact males increased lesion formation to the same extent as that observed with orchidectomized animals. Testosterone supplementation of orchidectomized animals reduced lesion formation when compared with orchidectomized animals receiving the placebo. This attenuating effect of testosterone was not observed when the animals were treated simultaneously with the aromatase inhibitor. The beneficial effects of testosterone on early atherogenesis were not explained by changes in lipid levels. Estradiol administration to orchidectomized males attenuated lesion formation to the same extent as testosterone administration. Aromatase was expressed in the aorta of these animals as assessed by reverse transcription–PCR and immunohistochemistry. These results indicate that testosterone attenuates early atherogenesis most likely by being converted to estrogens by the enzyme aromatase expressed in the vessel wall.

Multiple roles of angiopoietins in atherogenesis

Purpose of review: The roles of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) during vascular development have been extensively investigated, as has been their role in controlling the responsiveness of the endothelium to exogenous cytokines. However, very little is known about the role of these vascular morphogenic molecules in the pathogenesis of atherosclerosis. Here, we summarize the recent research into angiopoietins in atherosclerosis. Recent findings: Angiopoietin-2 is a context-dependent agonist that protects against the development of arteriosclerosis in rat cardiac allograft. A recent study showed, contrary to expectations, that a single systemic administration of adenoviral Ang-2 to apoE-/- mice, fed a Western diet, reduced atherosclerotic lesion size and LDL oxidation in a nitric oxide synthase dependent manner. In contrast, overexpression of Ang-1 fails to protect from rat cardiac allograft due to smooth muscle cell activation. The potential proatherogenic effect of Ang-1 is further supported by the induction of chemotaxis of monocytes by Ang-1 in a manner that is independent of Tie-2 and integrin binding. These studies highlight the need for extensive research to better understand the role of angiopoietins in the cardiovascular setting. Summary: Ang-2 inhibits atherosclerosis by limiting LDL oxidation via stimulation of nitric oxide production. In contrast, Ang-1 can promote monocyte and neutrophil migration. The angiopoietin–Tie-2 system provides an important new target for modulating vascular function.

Lipid Metabolism in Subclinical Hypothyroidism: Plasma Kinetics of Triglyceride-Rich Lipoproteins and Lipid Transfers to High-Density Lipoprotein Before and After Levothyroxine Treatment

Background: Subclinical hypothyroidism (SCH) has been associated with atherosclerosis, but the abnormalities in plasma lipids that can contribute to atherogenesis are not prominent. The aim of this study was to test the hypothesis that patients with normocholesterolemic, normotriglyceridemic SCH display abnormalities in plasma lipid metabolism not detected in routine laboratory tests including abnormalities in the intravascular metabolism of triglyceride-rich lipoproteins, lipid transfers to high-density lipoprotein (HDL), and paraoxonase 1 activity. The impact of levothyroxine (LT4) treatment and euthyroidism in these parameters was also tested. Methods: The study included 12 SCH women and 10 matched controls. Plasma kinetics of an artificial triglyceride-rich emulsion labeled with radioactive triglycerides and cholesteryl esters as well as in vitro transfer of four lipids from an artificial donor nanoemulsion to HDL were determined at baseline in both groups and after 4 months of euthyroidism in the SCH group. Results: Fractional clearance rates of triglycerides (SCH 0.035 +/- 0.016 min(-1), controls 0.029 +/- 0.013 min(-1), p=0.336) and cholesteryl esters (SCH 0.009 +/- 0.007 min(-1), controls 0.009 +/- 0.009 min(-1), p=0.906) were equal in SCH and controls and were unchanged by LT4 treatment and euthyroidism in patients with SCH, suggesting that lipolysis and remnant removal of triglyceride-rich lipoproteins were normal. Transfer of triglycerides to HDL (SCH 3.6 +/- 0.48%, controls 4.7 +/- 0.63%, p=0.001) and phospholipids (SCH 16.2 +/- 3.58%, controls 21.2 +/- 3.32%, p=0.004) was reduced when compared with controls. After LT4 treatment, transfers increased and achieved normal values. Transfer of free and esterified cholesterol to HDL, HDL particle size, and paraoxonase 1 activity were similar to controls and were unchanged by treatment. Conclusions: Although intravascular metabolism of triglyceride-rich lipoproteins was normal, patients with SCH showed abnormalities in HDL metabolism that were reversed by LT4 treatment and achievement of euthyroidism.