The Influence of the FFAR4 Agonist TUG-891 on Liver Steatosis in ApoE-Knockout Mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) constitutes an independent risk factor for the development of coronary heart disease. Low-grade inflammation has been shown to play an important role in the development of atherosclerosis and NAFLD. Free fatty acid receptor 4 (FFAR4/GPR120), which is involved in damping inflammatory reactions, may represent a promising target for the treatment of inflammatory diseases. Our objective was to evaluate the effect of TUG-891, the synthetic agonist of FFAR4/GPR120, on fatty liver in vivo. METHODS: The effect of TUG-891 on fatty liver was investigated in apoE-/- mice fed a high-fat diet (HFD), using microscopic, biochemical, molecular, and proteomic methods. RESULTS: Treatment with TUG-891 inhibited the progression of liver steatosis in apoE-/- mice, as evidenced by histological analysis, and reduced the accumulation of TG in the liver. This action was associated with a decrease in plasma AST levels. TUG-891 decreased the expression of liver genes and proteins involved in de novo lipogenesis (Srebp-1c, Fasn and Scd1) and decreased the expression of genes related to oxidation and uptake (Acox1, Ehhadh, Cd36, Fabp1). Furthermore, TUG-891 modified the levels of selected factors related to glucose metabolism (decreased Glut2, Pdk4 and Pklr, and increased G6pdx). CONCLUSION: Pharmacological stimulation of FFAR4 may represent a promising lead in the search for drugs that inhibit NAFLD.

Inhaled silica nanoparticles exacerbate atherosclerosis through skewing macrophage polarization towards M1 phenotype.

BACKGROUND AND AIMS: Exposure to environmental nanoparticles is related to the adverse impact on health, including cardiovascular system. Various forms of nanoparticles have been reported to interact with endothelium and induce inflammation. However, the potential role of nanoparticles in the pathogenesis of atherosclerosis and their mechanisms of action are still unclear. The aim of this study was to investigate the effect of two broadly used nanomaterials, which also occur in natural environment - silicon oxide (SiO2) and ferric oxide (Fe2O3) in the form of nanoparticles (NPs) - on the development of atherosclerosis. METHODS: We used apolipoprotein E-knockout mice exposed to silica and ferric oxide nanoparticles in a whole body inhalation chamber. RESULTS: Inhaled silica nanoparticles augmented the atherosclerotic lesions and increased the percentage of pro-inflammatory M1 macrophages in both the plaque and the peritoneum in apoE-/- mice. Exposure to ferric oxide nanoparticles did not enhance atherogenesis process, however, it caused significant changes in the atherosclerotic plaque composition (elevated content of CD68-positive macrophages and enlarged necrotic core accompanied by the decreased level of M1 macrophages). Both silica and ferric oxide NPs altered the phenotype of T lymphocytes in the spleen by promoting polarization towards Th17 cells. CONCLUSIONS: Exposure to silica and ferric oxide nanoparticles exerts impact on atherosclerosis development and plaque composition. Pro-atherogenic abilities of silica nanoparticles are associated with activation of pro-inflammatory macrophages.

Impact of Polypyridyl Ru Complexes on Angiogenesis-Contribution to Their Antimetastatic Activity.

The use of polypyridyl Ru complexes to inhibit metastasis is a novel approach, and recent studies have shown promising results. We have reported recently that Ru (II) complexes gathering two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and the one being 2,2'-bipyridine (bpy) or its derivative with a 4-[3-(2-nitro-1H-imidazol-1-yl)propyl (bpy-NitroIm) or 5-(4-{4'-methyl-[2,2'-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone (bpy-SC) moieties can alter the metastatic cascade, among others, by modulating cell adhesion properties. In this work, we show further studies of this group of complexes by evaluating their effect on HMEC-1 endothelial cells. While all the tested complexes significantly inhibited the endothelial cell migration, Ru-bpy additionally interrupted the pseudovessels formation. Functional changes in endothelial cells might arise from the impact of the studied compounds on cell elasticity and expression of proteins (vinculin and paxillin) involved in focal adhesions. Furthermore, molecular studies showed that complexes modulate the expression of cell adhesion molecules, which has been suggested to be one of the factors that mediate the activation of angiogenesis. Based on the performed studies, we can conclude that the investigated polypyridyl Ru (II) complexes can deregulate the functionality of endothelial cells which may lead to the inhibition of angiogenesis.

The Anti-Atherosclerotic Action of FFAR4 Agonist TUG-891 in ApoE-Knockout Mice Is Associated with Increased Macrophage Polarization towards M2 Phenotype.

Background: Over the past few years, a better understanding of the biology of G-protein coupled receptors (GPRs) has led to the identification of several receptors as novel targets for free fatty acids (FFAs). FFAR4 has received special attention in the context of chronic inflammatory diseases, including atherosclerosis, obesity and NAFLD, through to its anti-inflammatory effect. Methods: The present study investigates the influence of prolonged treatment with TUG-891-FFAR4 agonist on the development of atherosclerosis plaque in apoE-knockout mice, using morphometric and molecular methods. Results: TUG-891 administration has led to the reduction of atherosclerotic plaque size and necrotic cores in an apoE-knockout mice model. TUG-891-treated mice were administered subcutaneously at a dose of 20 mg/kg three times a week for 4 months. The FFAR4 agonist reduced the content of pro-inflammatory M1-like macrophages content in atherosclerotic plaques, as evidenced by immunohistochemical phenotyping and molecular methods. In atherosclerotic plaque, the population of smooth muscle cells increased as evidenced by α-SMA staining. We observed changes in G-CSF and eotaxin markers in the plasma of mice; changes in the levels of these markers in the blood may be related to macrophage differentiation. Importantly, we observed a significant increase in M2-like macrophage cells in atherosclerotic plaque and peritoneum. Conclusions: Prolonged administration of TUG-891 resulted in significant amelioration of atherogenesis, providing evidence that the strategy based on macrophage phenotype switching toward an M2-like activation state via stimulation of FFAR4 receptor holds promise for a new approach in the prevention or treatment of atherosclerosis.

Inhibition of Atherosclerosis and Liver Steatosis by Agmatine in Western Diet-Fed apoE-Knockout Mice Is Associated with Decrease in Hepatic De Novo Lipogenesis and Reduction in Plasma Triglyceride/High-Density Lipoprotein Cholesterol Ratio.

Atherosclerosis and NAFLD are the leading causes of death worldwide. The hallmark of NAFLD is triglyceride accumulation caused by an imbalance between lipogenesis de novo and fatty acid oxidation. Agmatine, an endogenous metabolite of arginine, exerts a protective effect on mitochondria and can modulate fatty acid metabolism. In the present study, we investigate the influence of agmatine on the progression of atherosclerotic lesions and the development of hepatic steatosis in apoE-/- mice fed with a Western high-fat diet, with a particular focus on its effects on the DNL pathway in the liver. We have proved that treatment of agmatine inhibits the progression of atherosclerosis and attenuates hepatic steatosis in apoE-/- mice on a Western diet. Such effects are associated with decreased total macrophage content in atherosclerotic plaque as well as a decrease in the TG levels and the TG/HDL ratio in plasma. Agmatine also reduced TG accumulation in the liver and decreased the expression of hepatic genes and proteins involved in lipogenesis de novo such as SREBP-1c, FASN and SCD1. In conclusion, agmatine may present therapeutic potential for the treatment of atherosclerosis and fatty liver disease. However, an exact understanding of the mechanisms of the advantageous actions of agmatine requires further study.

Diminazene Aceturate Stabilizes Atherosclerotic Plaque and Attenuates Hepatic Steatosis in apoE-Knockout Mice by Influencing Macrophages Polarization and Taurine Biosynthesis.

Atherosclerosis and nonalcoholic fatty liver disease are leading causes of morbidity and mortality in the Western countries. The renin-angiotensin system (RAS) with its two main opposing effectors, i.e., angiotensin II (Ang II) and Ang-(1-7), is widely recognized as a major regulator of cardiovascular function and body metabolic processes. Angiotensin-converting enzyme 2 (ACE2) by breaking-down Ang II forms Ang-(1-7) and thus favors Ang-(1-7) actions. Therefore, the aim of our study was to comprehensively evaluate the influence of prolonged treatment with ACE2 activator, diminazene aceturate (DIZE) on the development of atherosclerotic lesions and hepatic steatosis in apoE-/- mice fed a high-fat diet (HFD). We have shown that DIZE stabilized atherosclerotic lesions and attenuated hepatic steatosis in apoE-/- mice fed an HFD. Such effects were associated with decreased total macrophages content and increased α-smooth muscle actin levels in atherosclerotic plaques. Moreover, DIZE changed polarization of macrophages towards increased amount of anti-inflammatory M2 macrophages in the atherosclerotic lesions. Interestingly, the anti-steatotic action of DIZE in the liver was related to the elevated levels of HDL in the plasma, decreased levels of triglycerides, and increased biosynthesis and concentration of taurine in the liver of apoE-/- mice. However, exact molecular mechanisms of both anti-atherosclerotic and anti-steatotic actions of DIZE require further investigations.

The Influence of Trehalose on Atherosclerosis and Hepatic Steatosis in Apolipoprotein E Knockout Mice.

Atherosclerosis and nonalcoholic fatty liver disease (NAFLD) are frequent causes of death in the Western countries. Recently, it has been shown that autophagy dysfunction plays an important role in the pathogenesis of both atherosclerosis and NAFLD; thus, activators of autophagy might be useful for novel therapeutic interventions. Trehalose-a naturally occuring disaccharide present in plants, bacteria, fungi, insects, and certain types of shrimps-is a known inducer of autophagy. However, according to the literature, its anti-atherosclerotic and anti-steatotic potential seem to depend on the experimental setting. The aim of our study was to comprehensively describe the influence of a prolonged treatment with orally administered trehalose on the development of atherosclerotic lesions and hepatic steatosis in apolipoprotein E knockout (apoE-/-) mice in an experimental set up reflecting both moderate and severe proatherogenic conditions: male apoE-/- mice on a chow diet (CD) and female apoE-/- mice fed with a high-fat diet (HFD). We found that exogenous trehalose inhibited atherosclerosis and attenuated hepatic steatosis in apoE-/- mice. Such effects of trehalose were not associated with changes of plasma cholesterol, low-density lipoproteins (LDL), or high-density lipoproteins (HDL). Moreover, the anti-steatotic action of trehalose in the liver was associated with the induction of autophagy. The exact molecular mechanisms of both the anti-atherosclerotic action of trehalose and its inhibitory effect on liver steatosis require further clarification.

Air pollution and atherosclerosis - a brief review of mechanistic links between atherogenesis and biological actions of inorganic part of particulate matter.

Atherosclerosis is considered as a chronic, low-grade inflammatory process involving the aorta and the medium-sized arteries. Exposure to air pollutants, especially particulate matter, is highly related to cardiovascular diseases including atherosclerosis. Many studies confirm that proatherogenic potential of particulate matter is determined by its ability to induce inflammation, oxidative stress and thrombosis formation. Recently, an important role in the pathogenesis of atherosclerosis has been attributed to autoimmune response. Moreover, harmful effects of PM particles strongly depend on their physicochemical properties. It is still not known what exact role air pollutants, and in particular their inorganic part, play in the development of atherosclerotic lesions. In this article, we will briefy discuss the different aspects of particulate matter activity and its implication with atherosclerosis progression.

The antiatherosclerotic action of 1G244 - An inhibitor of dipeptidyl peptidases 8/9 - is mediated by the induction of macrophage death.

BACKGROUND: Targeting cell death to induce favorable functional and morphological changes within atherosclerotic plaques has long been postulated as a promising anti-atherosclerotic strategy. In this regard, inhibition of dipeptidyl peptidases 8/9 has received special attention in the context of chronic inflammatory diseases due to its regulatory role in macrophage death in vivo. METHODS: The present study investigates the influence of prolonged treatment with 1G244 - an inhibitor of dipeptidyl peptidases 8/9 - on the development of the advanced atherosclerosis plaque in apoE-knockout mice, using morphometric and molecular methods. RESULTS: 1G244 administration has led to a reduction in atherosclerotic plaque size in an apoE-knockout mice model. Moreover, it reduced the content of in-plaque macrophages, attributed by immunohistochemical phenotyping to the pro-inflammatory M1-like activation state of these cells. Inhibition of dipeptidyl peptidases 8/9 augmented the lytic form of death response of activated macrophages in-vitro. CONCLUSIONS: In summary, inhibition of DPP 8/9 elicited an anti-atherosclerotic effect in apoE-/- mice, which can be attributed to the lytic form of death induction in activated macrophages, as assessed by the in vitro BMDM model. This, in turn, results in a reduction of the plaque area without its transformation towards a rupture-prone morphology.

Changes in the liver proteome in apoE knockout mice exposed to inhalation of silica nanoparticles indicate mitochondrial damage and impairment of ER stress responses associated with microvesicular steatosis.

The adverse effects of air pollution on the cardiovascular system have been well documented. Nonalcoholic fatty liver disease (NAFLD) is an independent risk factor for cardiovascular events. However, the influence of exposure to airborne particles on the development of NAFLD is less recognised. The aim of this study was to investigate the impact of silica nanoparticles (SiNPs) on the development of liver steatosis. We used molecular and proteomic SWATH-MS methods to investigate the changes in the liver proteome of apolipoprotein E-knockout mice (apoE-/- mice) exposed to SiNPs for 4 months in a whole-body exposure chamber. Exposure to SiNPs evoked microvesicular liver steatosis in apoE-/- mice. Quantitative liver proteomics showed significant downregulation of ribosomal proteins and endoplasmic reticulum proteins. Gene expression analysis revealed a reduced level of proteins related to endoplasmic reticulum stress. Treatment with SiNPs decreased mitochondrial membrane potential and increased the production of reactive oxygen species in cultured HepG2 cells. This is the first report that inhalation exposure to SiNPs induces microvesicular steatosis and significant changes in the liver proteome in vivo. Our results highlight the important role of silica and point to the ER stress response and mitochondrial dysfunction as potential mechanisms responsible for the increase in fatty liver by SiNPs.

Anti-Atherosclerotic Potential of Free Fatty Acid Receptor 4 (FFAR4).

Fatty acids (FAs) are considered not only as a basic nutrient, but are also recognized as signaling molecules acting on various types of receptors. The receptors activated by FAs include the family of rhodopsin-like receptors: GPR40 (FFAR1), GPR41 (FFAR3), GPR43 (FFAR2), GPR120 (FFAR4), and several other, less characterized G-protein coupled receptors (GPR84, GPR109A, GPR170, GPR31, GPR132, GPR119, and Olfr78). The ubiquitously distributed FFAR4 can be activated by saturated and unsaturated medium- and long-chain fatty acids (MCFAs and LCFAs), as well as by several synthetic agonists (e.g., TUG-891). The stimulation of FFAR4 using selective synthetic agonists proved to be promising strategy of reduction of inflammatory reactions in various tissues. In this paper, we summarize the evidence showing the mechanisms of the potential beneficial effects of FFAR4 stimulation in atherosclerosis. Based partly on our own results, we also suggest that an important mechanism of such activity may be the modulatory influence of FFAR4 on the phenotype of macrophage involved in atherogenesis.

Decrease of the pro-inflammatory M1-like response by inhibition of dipeptidyl peptidases 8/9 in THP-1 macrophages - quantitative proteomics of the proteome and secretome.

BACKGROUND: Cellular peptidases are an emerging target of novel pharmacological strategies in inflammatory diseases and cancer. In this context, the dipeptidyl peptidases 8 and 9 (DPP8/9) have gained special attention due to their activities in the immune cells. However, in spite of more than hundred protein substrates identified to date by mass spectrometry-based analysis, the cellular DPP8/9 functions are still elusive. METHODS: We applied the proteomic approach (iTRAQ-2DLC-MS/MS) to comprehensively analyze the role of DPP8/9 in the regulation of macrophage activation by in-depth protein quantitation of THP-1 proteome and secretome. RESULTS: Cells pre-incubated with DPP8/9 inhibitor (1G244) prior activation (LPS or IL-4/IL-13) diminished the expression levels of M1-like response markers, but not M2-like phenotype features. This was accompanied by multiple intra- and extra-cellular protein abundance changes in THP-1 cells, related to cellular metabolism, mitochondria and endoplasmic reticulum function, as well as those engaged with inflammatory and apoptotic processes, including previously reported and novel DPP8/9 targets. CONCLUSIONS: Inhibition of DPP 8/9 had a profound effect on the THP-1 macrophage proteome and secretome, evidencing the decrease of the pro-inflammatory M1-like response. Presented results are to our best knowledge the first which, among others, highlight the metabolic effects of DPP8/9 inhibition in macrophages.

Anti-atherosclerotic action of GW9508 - Free fatty acid receptors activator - In apoE-knockout mice.

BACKGROUND: In the past two decades, enhanced understanding of the biology of G-protein-coupled receptors (GPRs) has led to the identification of several such receptors as novel targets for free fatty acids (FFAs). Two GPRs, FFAR1 and FFAR4, have received special attention in the context of chronic inflammatory diseases, thanks to their anti-inflammatory activities. METHODS: The present study investigates the influence of prolonged treatment with GW9508 - agonist of FFAR1 and FFAR4 - on the development of atherosclerosis plaque in apoE-knockout mice, using morphometric and molecular methods. RESULTS: GW9508 administration has led to the reduction of atheroscletoric plaque size in an apoE-knockout mice model. Moreover, a FFAR1/FFAR4 agonist reduced the content of macrophages by almost 20%, attributed by immunohistochemical phenotyping to the pro-inflammatory M1-like activation state macrophages. CONCLUSIONS: Prolonged administration of GW9508 resulted in significant amelioration of atherogenesis, providing evidence that the strategy based on macrophage phenotype switching toward an M2-like activation state via stimulation of FFAR1/FFAR4 receptors holds promise for a new approach to the prevention or treatment of atherosclerosis.