Polyunsaturated fatty acids and atherosclerosis: insights from pre-clinical studies

Atherosclerosis and its complications are responsible for more global deaths than any other disease. Atherosclerosis is a chronic inflammatory disease of medium and large arteries that can cause clinical complications such as myocardial infarction and cerebrovascular accidents. Current therapies against atherosclerosis mainly target the dyslipidemia associated with the disease and are associated with considerable residual risk for cardiovascular disease together with various side effects. In addition, the outcomes of clinical trials on many pharmaceutical agents against promising therapeutic targets have been disappointing. This has resulted in considerable recent interest on nutraceuticals in the prevention of atherosclerosis and as add‐on with current pharmaceutical therapies. However, nutraceutical research has considerably lagged those on pharmaceuticals on two key aspects, large clinical trials and mechanistic insights. The latter forms the focus of this review in relation to the potential beneficial actions of polyunsaturated fatty acids as identified from pre‐clinical studies

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

A critical role for the Sp1-binding sites in the transforming growth factor-β-mediated inhibition of lipoprotein lipase gene expression in macrophages

Increasing evidence suggests that the cytokine transforming growth factor-β (TGF-β) inhibits the development of atherosclerosis. The lipoprotein lipase (LPL) enzyme expressed by macrophages has been implicated in the pathogenesis of atherosclerosis by stimulating the uptake of lipoprotein particles. Unfortunately, the action of TGF-β on the expression of LPL in macrophages remains largely unclear. We show that TGF-β inhibits LPL gene expression at the transcriptional level. Transient transfection assays reveal that the −31/+187 sequence contains the minimal TGF-β-responsive elements. Electrophoretic mobility shift assays show that Sp1 and Sp3 interact with two regions in the −31/+187 sequence. Mutations of these Sp1/Sp3 sites abolish the TGF-β-mediated suppression whereas multimers of the sequence impart the response to a heterologous promoter. TGF-β has no effect on the binding or steady-state polypeptide levels of Sp1 and Sp3. These results, therefore, suggest a novel mechanism for the TGF-β-mediated repression of LPL gene transcription that involves regulation of the action of Sp1 and Sp3

Regulation of ADAMTS-1, -4 and -5 expression in human macrophages: differential regulation by key cytokines implicated in atherosclerosis and novel synergism between TL1A and IL-17

Atherosclerosis is an inflammatory disease of the vasculature regulated by cytokines. Macrophages play a crucial role at all stages of this disease, including regulation of foam cell formation, the inflammatory response and stability of atherosclerotic plaques. For example, matrix metalloproteinases produced by macrophages play an important role in modulating plaque stability. More recently, the ADAMTS proteases, which are known to play a key role in the control of cartilage degradation during arthritis, have been found to be expressed in atherosclerotic lesions and suggested to have potentially important functions in the control of plaque stability. Unfortunately, the action of cytokines on the expression of ADAMTS family in macrophages is poorly understood. We have investigated the effect of classical cytokines (IFN-γ and TGF-β) and those that have been recently identified (TL1A and IL-17) on the expression of ADAMTS-1, -4 and -5 in human macrophages. The expression of all three ADAMTS members was induced during differentiation of monocytes into macrophages. TGF-β had a differential action with induction of ADAMTS-1 and -5 expression and attenuation in the levels of ADAMTS-4. In contrast, IFN-γ suppressed the expression of ADAMTS-1 without having an effect on ADAMTS-4 and -5. Although TL-1A or IL-17A alone had little effect on the expression of all the members, they induced their expression synergistically when present together. These studies provide new insight into the regulation of key ADAMTS family members in human macrophages by major cytokines in relation to atherosclerosis

The role of mitogen-activated protein kinases and sterol receptor coactivator-1 in TGF-β-regulated expression of genes implicated in macrophage cholesterol uptake

The anti-atherogenic cytokine TGF-β inhibits macrophage foam cell formation by suppressing the expression of key genes implicated in the uptake of modified lipoproteins. We have previously shown a critical role for p38 MAPK and JNK in the TGF-β-mediated regulation of apolipoprotein E expression in human monocytes. However, the roles of these two MAPK pathways in the control of expression of key genes involved in the uptake of modified lipoproteins in human macrophages is poorly understood and formed the focus of this study. TGF-β activated both p38 MAPK and JNK, and knockdown of p38 MAPK or c-Jun, a key downstream target of JNK action, demonstrated their requirement in the TGF-β-inhibited expression of several key genes implicated in macrophage lipoprotein uptake. The potential role of c-Jun and specific co-activators in the action of TGF-β was investigated further by studies on the lipoprotein lipase gene. c-Jun did not directly interact with the minimal promoter region containing the TGF-β response elements and a combination of transient transfection and knock down assays revealed an important role for SRC-1. These studies provide novel insights into the mechanisms underlying the TGF-β-mediated inhibition of macrophage gene expression associated with the control of cholesterol homeostasis

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 rheumatoid arthritis (RA) patients and healthy controls (HCs). Methods Modified Boyden chamber was used to assess chemokine-induced migration of THP-1 monocytes. Macropinocytosis was assessed using lucifer yellow and ox-LDL uptake using Dil-oxLDL in THP-1 macrophages and human monocyte-derived macrophages (HMDM). IL6, TNFα and PGE2 release by lipopolysaccharide (LPS) stimulated PBMCs from RA patients and HCs were 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 Dil-oxLDL uptake were reduced by 50% (p< 0.001) and 40% (p< 0.01) in THP-1 macrophages and 40% (p< 0.01) and 25% (p< 0.05) in HMDM, respectively. PNLA reduced IL6 and TNFα release from LPS stimulated PBMCs from RA by 60% (p< 0.001) and by 50% and 35% respectively (p< 0.01) for HCs. PNLA also reduced PGE2 levels in such PBMCs from RA patients and HCs (p< 0.0001). Differentially expressed genes included upregulated expression of pyruvate dehydrogenase kinase-4, plasminogen activator inhibitor-1, fructose bisphosphatase 1 and N-Myc downstream-regulated gene, which have potential roles in regulating immune and metabolic pathways. Pathway analysis predicted upstream activation of nuclear receptors peroxisome proliferator-activated receptors involved in anti-inflammatory processes, and inhibition of NF-κB and STAT1. Conclusions PNLA has immune-metabolic effects on monocytes and PBMC which are pathogenic in RA and atherosclerosis. Dietary PNLA supplementation may be beneficial in RA

The interleukin-33-mediated inhibition of expression of two key genes implicated in atherosclerosis in human macrophages requires MAP kinase, phosphoinositide 3-kinase and nuclear factor-κB signaling pathways

Atherosclerosis, a chronic inflammatory disorder of the walls of arteries, causes more deaths worldwide than any other disease. Cytokines, which are present at high levels in atherosclerotic plaques, play important roles in regulating the initiation and the progression of the disease. Previous studies using animal and cell culture model systems revealed protective, anti-atherogenic effects of the cytokine interleukin-33 (IL-33). The action of this cytokine involves both the induction and suppression of expression of many genes. Unfortunately, the signaling pathways that are responsible for the inhibition of gene expression by this cytokine are poorly understood. Further studies are required given the important roles of genes whose expression is inhibited by IL-33 in key cellular processes associated with atherosclerosis such as monocyte recruitment, foam cell formation and lipoprotein metabolism. We have investigated here the roles of various known IL-33 activated signaling pathways in such inhibitory actions using RNA interference-mediated knockdown assays and monocyte chemotactic protein-1 and intercellular adhesion molecule-1 as model genes. Key roles were identified for extracellular signal-regulated kinase-1/2, p38α kinase, c-Jun N-terminal kinase-1/2, phosphoinositide 3-kinase-γ, and p50 and p65 nuclear factor-κB in such inhibitory action of IL-33. These studies provide new insights on the signaling pathways through which IL-33 inhibits the macrophage expression of key atherosclerosis-associated genes

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 showed that Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 (Lab4P) reduced 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 was therefore investigated. Methods and results Atherosclerosis-associated parameters were analyzed in LDL receptor deficient mice fed HFD for 12 weeks alone or supplemented with Lab4P. Lab4P increased plasma HDL and triglyceride levels and decreased LDL/VLDL levels. Lab4P also reduced plaque burden and content of lipids and macrophages, indicative of dampened inflammation, and increased smooth muscle cell content, a marker of plaque stabilization. Atherosclerosis arrays showed that Lab4P altered the liver expression of 19 key disease-associated genes. Lab4P also decreased the frequency of macrophages and T-cells in the bone marrow. In vitro assays using conditioned media from probiotic bacteria demonstrated 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

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

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