Therapeutic targeting of chronic kidney disease-associated DAMPs differentially contributing to vascular pathology.

Chronic Kidney Disease (CKD) is associated with markedly increased cardiovascular (CV) morbidity and mortality. Chronic inflammation, a hallmark of both CKD and CV diseases (CVD), is believed to drive this association. Pro-inflammatory endogenous TLR agonists, Damage-Associated Molecular Patterns (DAMPs), have been found elevated in CKD patients' plasma and suggested to promote CVD, however, confirmation of their involvement, the underlying mechanism(s), the extent to which individual DAMPs contribute to vascular pathology in CKD and the evaluation of potential therapeutic strategies, have remained largely undescribed. A multi-TLR inhibitor, soluble TLR2, abrogated chronic vascular inflammatory responses and the increased aortic atherosclerosis-associated gene expression observed in nephropathic mice, without compromising infection clearance. Mechanistically, we confirmed elevation of 4 TLR DAMPs in CKD patients' plasma, namely Hsp70, Hyaluronic acid, HMGB-1 and Calprotectin, which displayed different abilities to promote key cellular responses associated with vascular inflammation and progression of atherosclerosis in a TLR-dependent manner. These included loss of trans-endothelial resistance, enhanced monocyte migration, increased cytokine production, and foam cell formation by macrophages, the latter via cholesterol efflux inhibition. Calprotectin and Hsp70 most consistently affected these functions. Calprotectin was further elevated in CVD-diagnosed CKD patients and strongly correlated with the predictor of CV events CRP. In nephropathic mice, Calprotectin blockade robustly reduced vascular chronic inflammatory responses and pro-atherosclerotic gene expression in the blood and aorta. Taken together, these findings demonstrated the critical extent to which the DAMP-TLR pathway contributes to vascular inflammatory and atherogenic responses in CKD, revealed the mechanistic contribution of specific DAMPs and described two alternatives therapeutic approaches to reduce chronic vascular inflammation and lower CV pathology in CKD.

(+)-Catechin Attenuates Multiple Atherosclerosis-Associated Processes In Vitro, Modulates Disease-Associated Risk Factors in C57BL/6J Mice and Reduces Atherogenesis in LDL Receptor Deficient Mice by Inhibiting Inflammation and Increasing Markers of Plaque Stability.

SCOPE: A prospective study of 34492 participants shows an inverse association between (+)-catechin intake and coronary heart disease. The effects of (+)-catechin on atherosclerosis and associated risk factors are poorly understood and are investigated. METHODS AND RESULTS: (+)-Catechin attenuates reactive oxygen species production in human macrophages, endothelial cells and vascular smooth muscle cells, chemokine-driven monocytic migration, and proliferation of human macrophages and their expression of several pro-atherogenic genes. (+)-Catechin also improves oxidized LDL-mediated mitochondrial membrane depolarization in endothelial cells and attenuates growth factor-induced smooth muscle cell migration. In C57BL/6J mice fed high fat diet (HFD) for 3 weeks, (+)-catechin attenuates plasma levels of triacylglycerol and interleukin (IL)-1ß and IL-2, produces anti-atherogenic changes in liver gene expression, and reduces levels of white blood cells, myeloid-derived suppressor cells, Lin- Sca+ c-Kit+ cells, and common lymphoid progenitor cells within the bone marrow. In LDL receptor deficient mice fed HFD for 12 weeks, (+)-catechin attenuates atherosclerotic plaque burden and inflammation with reduced macrophage content and increased markers of plaque stability; smooth muscle cell and collagen content. CONCLUSION: This study provides novel, detailed insights into the cardio-protective actions of (+)-catechin together with underlying molecular mechanisms and supports further assessments of its beneficial effects in human trials.

The Role of Punicalagin and Its Metabolites in Atherosclerosis and Risk Factors Associated with the Disease.

Atherosclerotic cardiovascular disease (ACVD) is the leading cause of death worldwide. Although current therapies, such as statins, have led to a marked reduction in morbidity and mortality from ACVD, they are associated with considerable residual risk for the disease together with various adverse side effects. Natural compounds are generally well-tolerated; a major recent goal has been to harness their full potential in the prevention and treatment of ACVD, either alone or together with existing pharmacotherapies. Punicalagin (PC) is the main polyphenol present in pomegranates and pomegranate juice and demonstrates many beneficial actions, including anti-inflammatory, antioxidant, and anti-atherogenic properties. The objective of this review is to inform on our current understanding of the pathogenesis of ACVD and the potential mechanisms underlying the beneficial actions of PC and its metabolites in the disease, including the attenuation of dyslipidemia, oxidative stress, endothelial cell dysfunction, foam cell formation, and inflammation mediated by cytokines and immune cells together with the regulation of proliferation and migration of vascular smooth muscle cells. Some of the anti-inflammatory and antioxidant properties of PC and its metabolites are due to their strong radical-scavenging activities. PC and its metabolites also inhibit the risk factors of atherosclerosis, including hyperlipidemia, diabetes mellitus, inflammation, hypertension, obesity, and non-alcoholic fatty liver disease. Despite the promising findings that have emerged from numerous in vitro, in vivo, and clinical studies, deeper mechanistic insights and large clinical trials are required to harness the full potential of PC and its metabolites in the prevention and treatment of ACVD.

Anti-Atherogenic Actions of the Lab4b Consortium of Probiotics In Vitro.

Probiotic bacteria have many protective effects against inflammatory disorders, though the mechanisms underlying their actions are poorly understood. The Lab4b consortium of probiotics contains four strains of lactic acid bacteria and bifidobacteria that are reflective of the gut of newborn babies and infants. The effect of Lab4b on atherosclerosis, an inflammatory disorder of the vasculature, has not yet been determined and was investigated on key processes associated with this disease in human monocytes/macrophages and vascular smooth muscle cells in vitro. The Lab4b conditioned medium (CM) attenuated chemokine-driven monocytic migration, monocyte/macrophage proliferation, uptake of modified LDL and macropinocytosis in macrophages together with the proliferation and platelet-derived growth factor-induced migration of vascular smooth muscle cells. The Lab4b CM also induced phagocytosis in macrophages and cholesterol efflux from macrophage-derived foam cells. The effect of Lab4b CM on macrophage foam cell formation was associated with a decrease in the expression of several key genes implicated in the uptake of modified LDL and induced expression of those involved in cholesterol efflux. These studies reveal, for the first time, several anti-atherogenic actions of Lab4b and strongly implicate further studies in mouse models of the disease in vivo and in clinical trials.

The Beneficial Effects of Pine Nuts and Its Major Fatty Acid, Pinolenic Acid, on Inflammation and Metabolic Perturbations in Inflammatory Disorders.

Inflammatory disorders such as atherosclerosis, diabetes and rheumatoid arthritis are regulated by cytokines and other inflammatory mediators. Current treatments for these conditions are associated with significant side effects and do not completely suppress inflammation. The benefits of diet, especially the role of specific components, are poorly understood. Polyunsaturated fatty acids (PUFAs) have several beneficial health effects. The majority of studies on PUFAs have been on omega-3 fatty acids. This review will focus on a less studied fatty acid, pinolenic acid (PNLA) from pine nuts, which typically constitutes up to 20% of its total fatty acids. PNLA is emerging as a dietary PUFA and a promising supplement in the prevention of inflammatory disorders or as an alternative therapy. Some studies have shown the health implications of pine nuts oil (PNO) and PNLA in weight reduction, lipid-lowering and anti-diabetic actions as well as in suppression of cell invasiveness and motility in cancer. However, few reviews have specifically focused on the biological and anti-inflammatory effects of PNLA. Furthermore, in recent bioinformatic studies on human samples, the expression of many mRNAs and microRNAs was regulated by PNLA indicating potential transcriptional and post-transcriptional regulation of inflammatory and metabolic processes. The aim of this review is to summarize, highlight, and evaluate research findings on PNO and PNLA in relation to potential anti-inflammatory benefits and beneficial metabolic changes. In this context, the focus of the review is on the potential actions of PNLA on inflammation along with modulation of lipid metabolism and oxidative stress based on data from both in vitro and in vivo experiments, and human findings, including gene expression analysis.

Pinolenic acid exhibits anti-inflammatory and anti-atherogenic effects in peripheral blood-derived monocytes from patients with rheumatoid arthritis.

Pinolenic acid (PNLA), an omega-6 polyunsaturated fatty acid from pine nuts, has anti-inflammatory and anti-atherogenic effects. We aimed to investigate the direct anti-inflammatory effect and anti-atherogenic effects of PNLA on activated purified CD14 monocytes from peripheral blood of patients with rheumatoid arthritis (RA) in vitro. Flow cytometry was used to assess the proportions of CD14 monocytes expressing TNF-α, IL-6, IL-1ß, and IL-8 in purified monocytes from patients with RA after lipopolysaccharide (LPS) stimulation with/without PNLA pre-treatment. The whole genomic transcriptome (WGT) profile of PNLA-treated, and LPS-activated monocytes from patients with active RA was investigated by RNA-sequencing. PNLA reduced percentage of monocytes expressing cytokines: TNF-α by 23% (p = 0.048), IL-6 by 25% (p = 0.011), IL-1ß by 23% (p = 0.050), IL-8 by 20% (p = 0.066). Pathway analysis identified upstream activation of peroxisome proliferator-activated receptors (PPARs), sirtuin3, and let7 miRNA, and KLF15, which are anti-inflammatory and antioxidative. In contrast, DAP3, LIF and STAT3, which are involved in TNF-α, and IL-6 signal transduction, were inhibited. Canonical Pathway analysis showed that PNLA inhibited oxidative phosphorylation (p = 9.14E-09) and mitochondrial dysfunction (p = 4.18E-08), while the sirtuin (SIRTs) signalling pathway was activated (p = 8.89E-06) which interfere with the pathophysiological process of atherosclerosis. Many miRNAs were modulated by PNLA suggesting potential post-transcriptional regulation of metabolic and immune response that has not been described previously. Multiple miRNAs target pyruvate dehydrogenase kinase-4 (PDK4), single-immunoglobulin interleukin-1 receptor molecule (SIGIRR), mitochondrially encoded ATP synthase membrane subunit 6 (MT-ATP6) and acetyl-CoA acyltranferase2 (ACAA2); genes implicated in regulation of lipid and cell metabolism, inflammation, and mitochondrial dysfunction. PNLA has potential anti-atherogenic and immune-metabolic effects on monocytes that are pathogenic in RA and atherosclerosis. Dietary PNLA supplementation regulates key miRNAs that are involved in metabolic, mitochondrial, and inflammatory pathways.

Key Roles of Inflammation in Atherosclerosis: Mediators Involved in Orchestrating the Inflammatory Response and Its Resolution in the Disease Along with Therapeutic Avenues Targeting Inflammation.

Inflammation is a critical driver of all stages of atherosclerosis, from lesion development to plaque rupture. Cytokines are mediators of the immune response and in atherosclerosis, the balance of anti- and pro-inflammatory cytokines is tipped in favor of the latter, resulting in persistent and unresolved inflammation. Although reducing plasma cholesterol levels mainly via the use of statins has positively impacted patient outcomes and reduced mortality rates, the presence of significant residual inflammation and cardiovascular risk posttherapy emphasizes the prevailing risk of primary and secondary events driven by inflammation independently of hyperlipidemia. Given the dominant role of inflammation in driving pathogenesis, alternative therapeutic avenues beyond targeting lowering of plasma lipids are required. This chapter will discuss the role of inflammation and pro-inflammatory cytokines in driving atherogenesis and disease progression, the therapeutic potential of targeting cytokines for atherosclerosis and promising avenues in this area.

Atherosclerosis: Pathogenesis and Key Cellular Processes, Current and Emerging Therapies, Key Challenges, and Future Research Directions.

Atherosclerosis is the principal cause of cardiovascular disease that continues to be a substantial drain on healthcare systems, being responsible for about 31% of all global deaths. Atherogenesis is influenced by a range of factors, including oxidative stress, inflammation, hypertension, and hyperlipidemia, and is ultimately driven by the accumulation of low-density lipoprotein cholesterol within the arterial wall of medium and large arteries. Lipoprotein accumulation stimulates the infiltration of immune cells (such as monocytes/macrophages and T-lymphocytes), some of which take up the lipoprotein, leading to the formation of lipid-laden foam cells. Foam cell death results in increased accumulation of dead cells, cellular debris and extracellular cholesterol, forming a lipid-rich necrotic core. Vascular smooth muscle cells from the arterial media also migrate into the intima layer and proliferate, taking up the available lipids to become foam cells and producing extracellular matrix proteins such as collagen and elastin. Plaque progression is characterized by the formation of a fibrous cap composed of extracellular matrix proteins and smooth muscle cells, which acts to stabilize the atherosclerotic plaque. Degradation, thinning, and subsequent rupture of the fibrous cap leads to lumen-occlusive atherothrombosis, most commonly resulting in heart attack or stroke. This chapter describes the pathogenesis of atherosclerosis, current and emerging therapies, key challenges, and future directions of research.

Survey of In Vitro Model Systems for Investigation of Key Cellular Processes Associated with Atherosclerosis.

Atherosclerosis progression is associated with a complex array of cellular processes in the arterial wall, including endothelial cell activation/dysfunction, chemokine-driven recruitment of immune cells, differentiation of monocytes to macrophages and their subsequent transformation into lipid laden foam cells, activation of inflammasome and pro-inflammatory signaling, and migration of smooth muscle cells from the media to the intima. The use of in vitro model systems has considerably advanced our understanding of these atherosclerosis-associated processes and they are also often used in drug discovery and other screening platforms. This chapter will describe key in vitro model systems employed frequently in atherosclerosis research.

Survey of Approaches for Investigation of Atherosclerosis In Vivo.

Although in vitro model systems are useful for investigation of atherosclerosis-associated processes, they represent simplification of complex events that occur in vivo, which involve interactions between many different cell types together with their environment. The use of animal model systems is important for more in-depth insights of the molecular mechanisms underlying atherosclerosis and for identifying potential targets for agents that can prevent plaque formation and even reverse existing disease. This chapter will provide a survey of such animal models and associated techniques that are routinely used for research of atherosclerosis in vivo.

Monitoring Modified Lipoprotein Uptake and Macropinocytosis Associated with Macrophage Foam Cell Formation.

Macrophage foam cell formation plays a crucial role in the initiation and progression of atherosclerosis. Macrophages uptake native and modified low density lipoprotein (LDL) through either receptor-dependent or receptor-independent mechanisms to transform into lipid laden foam cells. Foam cells are involved in the formation of fatty streak that is seen during the early stages of atherosclerosis development and therefore represents a promising therapeutic target. Normal or modified lipoproteins labeled with fluorescent dyes such as 1,1'-dioctadecyl-3-3-3',3'-tetramethylindocarbocyanine perchlorate (Dil) are often used to monitor their internalization during foam cell formation. In addition, the fluorescent dye Lucifer Yellow (LY) is widely used as a marker for macropinocytosis activity. In this chapter, we describe established methods for monitoring modified lipoprotein uptake and macropinocytosis during macrophage foam cell formation.

Investigation of Mitochondrial Bioenergetic Profile and Dysfunction in Atherosclerosis.

Mitochondrial function and activity are key indicators of overall cell health and mitochondrial dysfunction is closely associated with disruptions in normal cellular function. Altered mitochondrial function and cellular metabolism has been implicated in processes involved in ageing and associated pathologies. In atherosclerosis, compromised mitochondrial respiration can promote plaque instability and other processes that encourage pathogenesis and dysfunction. For example, increasing respiration promotes vascular smooth muscle cell (VSMC) proliferation and attenuates macrophage and VSMC apoptosis. Use of Agilent Seahorse technology to study mitochondrial bioenergetics has largely replaced previous outdated methods which provided limited insight into mitochondrial function and were associated with various issues. This chapter describes the use of Seahorse Agilent technology (Mito Stress Test) to study key parameters of mitochondrial respiration on cultured cells relevant to atherosclerosis.

Probing Inflammasome Activation in Atherosclerosis.

Atherosclerosis is driven by chronic inflammation in all stages of the disease. Inflammation is fueled by elevated levels of pro-inflammatory cytokines. Interleukins (IL) are cytokines of particular importance in atherosclerosis, due to their key involvement in various pro-atherogenic processes, including infiltration of immune cells to the lesion, stimulation of the production of other pro-inflammatory mediators by other sources, and generation of lipid laden foam cells, all of which contribute to plaque development and progression. Various stimuli that are abundant in atherosclerotic plaques, including oxidized low-density lipoprotein, cholesterol crystals and reactive oxygen species can trigger inflammasome activation. Importantly, activation of the nucleotide oligomerization domain leucine-rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome activates the caspase-1 protease and results in the generation and release of potent pro-inflammatory cytokines, IL-1ß and IL-18. Both cytokines are influential in driving chronic inflammation and atherogenesis. This chapter describes the use of enzyme-linked immunosorbent assay (ELISA) and Western blot to quantify these cytokines in cell supernatant and lysate respectively, after stimulating inflammasome activation in cultured cells.

Evaluation of Plaque Burden and Lipid Content in Atherosclerotic Plaques.

Atherosclerosis is a chronic inflammatory disease characterized by the formation of lipid-rich, fibrous plaques within the arterial wall of medium and large arteries. Plaques prone to rupture are typically rich in lipids and pro-inflammatory markers. Cells within the plaque can take up lipids via different mechanisms leading to the formation and accumulation of lipid-rich foam cells, a key hallmark of the disease. Evaluation of plaque burden and lipid content is hence important to determine disease progression and severity. This chapter describes the most commonly used staining methods that enable visualization and analysis of mouse atherosclerotic plaques. These methods include en face preparation of mouse aorta, and staining sections of arteries using hematoxylin and eosin, Oil Red O, and Masson's Trichrome.

Monitoring Cellularity and Expression of Key Markers in Atherosclerotic Plaques.

Atherosclerotic plaques are highly diverse and heterogeneous structures, even within the same individual, and can vary depending on its anatomical location within the vascular bed. Early in the disease and throughout its progression, immune cells infiltrate the lesion, contributing to the plaque phenotype via different mechanisms. Detailed characterization of constituent cell populations within plaques is hence required for more accurate assessment of disease severity and inflammatory burden. A wide range of fluorophore-conjugated antibodies targeted to key cell types implicated in all stages of the disease are commercially available, enabling visualization of the dynamic cellular landscape present within lesions. This chapter describes the use of immunofluorescence staining of atherosclerotic plaque sections to study plaque cellularity and expression of key markers.

Anti-inflammatory and immunoregulatory effects of pinolenic acid in rheumatoid arthritis.

OBJECTIVES: In pre-clinical studies, pinolenic acid (PNLA), an omega-6-polyunsaturated fatty acid from pine nuts, has shown anti-inflammatory effects. We aimed to investigate the effect of PNLA in human cell lines and peripheral blood mononuclear cells (PBMCs) from RA patients and healthy controls (HCs). METHODS: A modified Boyden chamber was used to assess chemokine-induced migration of THP-1 monocytes. Macropinocytosis was assessed using Lucifer yellow and oxidized low-density lipoprotein (oxLDL) uptake using DiI-labelled oxLDL in THP-1 macrophages and human monocyte-derived macrophages (HMDMs). IL-6, TNF-α and prostaglandin E2 (PGE2) release by lipopolysaccharide (LPS)-stimulated PBMCs from RA patients and HCs was measured by ELISA. The transcriptomic profile of PNLA-treated, LPS-activated PBMCs was investigated by RNA-sequencing. RESULTS: PNLA reduced THP-1 cell migration by 55% (P < 0.001). Macropinocytosis and DiI-oxLDL uptake were reduced by 50% (P < 0.001) and 40% (P < 0.01), respectively, in THP-1 macrophages and 40% (P < 0.01) and 25% (P < 0.05), respectively, in HMDMs. PNLA reduced IL-6 and TNF-α release from LPS-stimulated PBMCs from RA patients by 60% (P < 0.001) and from HCs by 50% and 35%, respectively (P < 0.01). PNLA also reduced PGE2 levels in such PBMCs from RA patients and HCs (P < 0.0001). Differentially expressed genes whose expression was upregulated included pyruvate dehydrogenase kinase-4, plasminogen activator inhibitor-1, fructose bisphosphatase1 and N-Myc downstream-regulated gene-2, which have potential roles in regulating immune and metabolic pathways. Pathway analysis predicted upstream activation of the nuclear receptors peroxisome proliferator-activated receptors involved in anti-inflammatory processes, and inhibition of nuclear factor-κB and signal transducer and activator of transcription 1. CONCLUSIONS: PNLA has immune-metabolic effects on monocytes and PBMCs that are pathogenic in RA and atherosclerosis. Dietary PNLA supplementation may be beneficial in RA.

Pro-atherogenic actions of signal transducer and activator of transcription 1 serine 727 phosphorylation in LDL receptor deficient mice via modulation of plaque inflammation.

Atherosclerosis is a chronic inflammatory disorder of the vasculature regulated by cytokines. We have previously shown that extracellular signal-regulated kinase-1/2 (ERK1/2) plays an important role in serine 727 phosphorylation of signal transducer and activator of transcription-1 (STAT1) transactivation domain, which is required for maximal interferon-γ signaling, and the regulation of modified LDL uptake by macrophages in vitro. Unfortunately, the roles of ERK1/2 and STAT1 serine 727 phosphorylation in atherosclerosis are poorly understood and were investigated using ERK1 deficient mice (ERK2 knockout mice die in utero) and STAT1 knock-in mice (serine 727 replaced by alanine; STAT1 S727A). Mouse Atherosclerosis RT² Profiler PCR Array analysis showed that ERK1 deficiency and STAT1 S727A modification produced significant changes in the expression of 18 and 49 genes, respectively, in bone marrow-derived macrophages, with 17 common regulated genes that included those that play key roles in inflammation and cell migration. Indeed, ERK1 deficiency and STAT1 S727A modification attenuated chemokine-driven migration of macrophages with the former also impacting proliferation and the latter phagocytosis. In LDL receptor deficient mice fed a high fat diet, both ERK1 deficiency and STAT1 S727A modification produced significant reduction in plaque lipid content, albeit at different time points. The STAT1 S727A modification additionally caused a significant reduction in plaque content of macrophages and CD3 T cells and diet-induced cardiac hypertrophy index. In addition, there was a significant increase in plasma IL-2 levels and a trend toward increase in plasma IL-5 levels. These studies demonstrate important roles of STAT1 S727 phosphorylation in particular in the regulation of atherosclerosis-associated macrophage processes in vitro together with plaque lipid content and inflammation in vivo, and support further assessment of its therapeutical potential.

The Lab4P Consortium of Probiotics Attenuates Atherosclerosis in LDL Receptor Deficient Mice Fed a High Fat Diet and Causes Plaque Stabilization by Inhibiting Inflammation and Several Pro-Atherogenic Processes.

SCOPE: Previous studies show that Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 (Lab4P) reduces diet-induced weight gain and plasma cholesterol levels in C57BL/6J mice fed a high fat diet (HFD). The effect of Lab4P on atherosclerosis is not known and is therefore investigated. METHODS AND RESULTS: Atherosclerosis-associated parameters are analyzed in LDL receptor deficient mice fed HFD for 12 weeks alone or supplemented with Lab4P. Lab4P increases plasma HDL and triglyceride levels and decreases LDL/VLDL levels. Lab4P also reduces plaque burden and content of lipids and macrophages, indicative of dampened inflammation, and increases smooth muscle cell content, a marker of plaque stabilization. Atherosclerosis arrays show that Lab4P alters the liver expression of 19 key disease-associated genes. Lab4P also decreases the frequency of macrophages and T-cells in the bone marrow. In vitro assays using conditioned media from probiotic bacteria demonstrates attenuation of several atherosclerosis-associated processes in vitro such as chemokine-driven monocytic migration, proliferation of monocytes and macrophages, foam cell formation and associated changes in expression of key genes, and proliferation and migration of vascular smooth muscle cells. CONCLUSION: This study provides new insights into the anti-atherogenic actions of Lab4P together with the underlying mechanisms and supports further assessments in human trials.

Protective effects of a unique combination of nutritionally active ingredients on risk factors and gene expression associated with atherosclerosis in C57BL/6J mice fed a high fat diet.

Atherosclerosis, an inflammatory disorder of the vasculature and the underlying cause of cardiovascular disease, is responsible for one in three global deaths. Consumption of active food ingredients such as omega-3 polyunsaturated fatty acids, flavanols and phytosterols has many beneficial effects on cardiovascular disease. However, their combined actions on the risk factors for atherosclerosis remains poorly understood. We have previously shown that a formulation containing each of these active components at physiologically relevant doses modulated several monocyte/macrophage processes associated with atherosclerosis in vitro, including inhibition of cytokine-induced pro-inflammatory gene expression, chemokine-driven monocyte migration, expression of M1 phenotype markers, and promotion of cholesterol efflux. The objectives of the present study were to investigate whether the protective actions of the formulation extended in vivo and to delineate the potential underlying mechanisms. The formulation produced several favourable changes, including higher plasma levels of HDL and reduced levels of macrophages and myeloid-derived suppressor cells in the bone marrow. The mRNA expression of liver-X-receptor-α, peroxisome proliferator-activated receptor-γ and superoxide dismutase-1 was induced in the liver and that of interferon-γ and the chemokine (C-X-C motif) ligand 1 decreased, thereby suggesting the potential mechanisms for many beneficial effects. Other changes were also observed such as increased plasma levels of triglycerides and lipid peroxidation that may reflect potential activation of brown fat. This study provides new insights into the protective actions and the potential underlying mechanisms of the formulation in vivo, particularly in relation to risk factors together with changes in systemic inflammation and hepatic lipid alterations associated with atherosclerosis and metabolic syndrome, and supports further assessments in human trials.

A perspective on targeting inflammation and cytokine actions in atherosclerosis.

Atherosclerosis, a chronic inflammatory disorder of the vasculature that results in cardiovascular disease, continues to pose a significant health and economic burden on modern society. Whilst inflammation has generally been accepted as the key driver of all stages of the disease, it was not until recently that inhibition of a specific proinflammatory cytokine (IL-1ß) yielded successful results in the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study trial. This article offers a perspective on targeting inflammation for atherosclerosis, focusing on results of recent Phase III clinical trials, and discusses other potential candidates together with future challenges and prospects.