Development of nitroalkene-based inhibitors to target STING-dependent inflammation.

Stimulator of Interferon Genes (STING) is essential for the inflammatory response to cytosolic DNA. Despite that aberrant activation of STING is linked to an increasing number of inflammatory diseases, the development of inhibitors has been challenging, with no compounds in the pipeline beyond the preclinical stage. We previously identified endogenous nitrated fatty acids as novel reversible STING inhibitors. With the aim of improving the specificity and efficacy of these compounds, we developed and tested a library of nitroalkene-based compounds for in vitro and in vivo STING inhibition. The structure-activity relationship study revealed a robustly improved electrophilicity and reduced degrees of freedom of nitroalkenes by conjugation with an aromatic moiety. The lead compounds CP-36 and CP-45, featuring a ß-nitrostyrene moiety, potently inhibited STING activity in vitro and relieved STING-dependent inflammation in vivo. This validates the potential for nitroalkene compounds as drug candidates for STING modulation to treat STING-driven inflammatory diseases, providing new robust leads for preclinical development.

Optimization of ozone concentration and storage time in green asparagus (Asparagus officinalis L. ) using response surface methodology / Optimización de concentración de ozono y tiempo de almacenamiento en espárrago (Asparagus officinalis L. ) verde utilizando metodología de superficie de respuesta

Background: Asparagus (Asparagus officinalis L.) green is a vegetable with a great demand worldwide, and likewise, it is highly perishable, due to its high respiration rate that accelerates its senescence. Disinfection of vegetables after their harvest is an obligatory practice that can reduce losses by decomposition due to the attack of microorganisms. Therefore, it is vital to preserving its microbiological and sensory characteristics to reach the final consumer. Objective: to evaluate the effect of gaseous ozone (0 to 10 ppm) and storage time (0 to 30 days) on phenol content, overall appearance, count of molds, psychrophilic bacteria, and viable mesophilic aerobes. Methods: the response surface methodology was used, applying a rotatable central composite design. Results: the results indicated that there was a significant influence (p <0.05) of the independent variables on the characteristics studied, as well as an adequate lack of fit of the quadratic regression model (p> 0.05). By means of the contour superposition technique, it was determined that the optimal conditions for the highest retention of phenol content (16.99 mg/g) and overall appearance (7.61 points) and lower counts of viable aerobic mesophilic bacteria (5.3 x 103 CFU/g) they corresponded to 10 ppm of gaseous ozone up to 25.91 days of storage, with adequate quality characteristics in the spears. Conclusion: the region of interest was determined for optimal retention of phenol content and overall appearance, and a lower count of viable aerobic mesophilic bacteria in green asparagus during postharvest, suggesting to use the initial application of ozone gas at 10 ppm allowing 25.9 days storage at 1 °C. The results indicate that this technology is a good alternative in the conservation of fresh vegetables

Antecedentes: El espárrago (Asparagus officinalis L.) verde; es una hortaliza con una gran demanda a nivel mundial, y, asimismo, es altamente perecible, por su elevada velocidad de respiración que, acelera su proceso de senescencia. La desinfección de los vegetales después de su cosecha es una práctica obligada que puede disminuir las pérdidas por descomposición debido al ataque de microrganismos. Por lo tanto, es muy importante conservar sus características microbiológicas y sensoriales para llegar al consumidor final. Objetivo: evaluar el efecto del ozono gaseoso (0 a 10 ppm) y tiempo de almacenamiento (0 a 30 días) sobre el contenido de fenoles, apariencia general, recuento de mohos, bacterias psicrófilas y aerobias mesófilas viables. Métodos: se utilizó la metodología de superficie de respuesta, aplicando un diseño compuesto central rotable. Resultados: los resultados indicaron que existió influencia significativa (p<0.05) de las variables independientes sobre las características estudiadas, así como, una adecuada bondad de ajuste del modelo de regresión cuadrático (p>0.05). Mediante la técnica de superposición de contornos se determinó que las condiciones óptimas para la mayor retención de contenido de fenoles (16.99 mg/g) y apariencia general (7.61 puntos) y menor recuentos de bacterias aerobias mesófilas viables (5.3 x 103 UFC/g) correspondieron a 10 ppm de ozono gaseoso hasta los 25.91 días de almacenamiento, con adecuadas características de calidad en los turiones. Conclusión: se determinó la región de interés para una óptima retención de contenido de fenoles y apariencia general, así como, menor recuento de bacterias aerobias mesófilas viables en el espárrago verde durante la postcosecha, sugiriendo utilizar la aplicación inicial de ozono gaseoso a 10 ppm permitiendo 25.9 días de almacenamiento a 1 °C. Los resultados indican que esta tecnología es una buena alternativa en la conservación de hortalizas frescas

Functional beverage design based on fresh milk, tarwi (Lupinus mutabilis) beverage and oatmeal (Avena sativa)

ABSTRACT The tarwi is an Andean legume with a high nutritional value from which a vegetable beverage can be obtained. Cereals, like oat, have good characteristics as a prebiotic for the production of functional drinks, whose consumption is currently increasing. The objective of the research was to design a probiotic fermented beverage based on fresh milk, tarwi beverage, and oatmeal. An optimal treatment consisted of 1.9% oatmeal, 39.9% tarwi beverage, 46.2% fresh milk, 10.0% honey, and 2.0% probiotic culture; determined by applying a rotatable central composite design of surface response methodology. It had a probiotic count of 3.47x108 cfu/mL, a protein content of 3.75%, and overall acceptability of 7 points, which corresponds to "I like it very much". The result was experimentally validated. Likewise, the shelf life of the optimal beverage was 20 days at 5 °C with appropriate functional, nutritional, and sensory characteristics.

RESUMEN El tarwi es una leguminosa andina con un alto valor nutricional a partir de la cual se puede obtener una bebida vegetal, los cereales como la avena tienen mejores características como prebióticos para la producción de bebidas funcionales, cuyo consumo está aumentando actualmente. El objetivo de la investigación fue diseñar la formulación de una bebida fermentada probiótica a base de leche fresca, bebida de tarwi y avena. Se determinó un tratamiento óptimo que consistió en 1,9% de avena, 39,9% de bebida de tarwi, 46,2% de leche fresca, 10,0% de miel y 2,0% de cultivo probiótico; mediante la aplicación de un diseño compuesto central rotable de metodología de respuesta de superficie. Se reportó un recuento de probióticos de 3,47x108 ufc/mL, un contenido de proteínas de 3,8% y una aceptabilidad general de 7 puntos, que corresponde a "Me gusta mucho"; el resultado fue validado experimentalmente. Asimismo, la vida útil de la bebida óptima fue de 20 días de almacenamiento a 5 °C con características funcionales, nutricionales y sensoriales apropiadas.

Suppression of Vascular Macrophage Activation by Nitro-Oleic Acid and its Implication for Abdominal Aortic Aneurysm Therapy.

PURPOSE: Abdominal aortic aneurysm (AAA) is one of the leading causes of death in the developed world and is currently undertreated due to the complicated nature of the disease. Herein, we aimed to address the therapeutic potential of a novel class of pleiotropic mediators, specifically a new drug candidate, nitro-oleic acid (NO2-OA), on AAA, in a well-characterized murine AAA model. METHODS: We generated AAA using a mouse model combining AAV.PCSK9-D377Y induced hypercholesterolemia with angiotensin II given by chronic infusion. Vehicle control (PEG-400), oleic acid (OA), or NO2-OA were subcutaneously delivered to mice using an osmotic minipump. We characterized the effects of NO2-OA on pathophysiological responses and dissected the underlying molecular mechanisms through various in vitro and ex vivo strategies. RESULTS: Subcutaneous administration of NO2-OA significantly decreased the AAA incidence (8/28 mice) and supra-renal aorta diameters compared to mice infused with either PEG-400 (13/19, p = 0.0117) or OA (16/23, p = 0.0078). In parallel, the infusion of NO2-OA in the AAA model drastically decreased extracellular matrix degradation, inflammatory cytokine levels, and leucocyte/macrophage infiltration in the vasculature. Administration of NO2-OA reduced inflammation, cytokine secretion, and cell migration triggered by various biological stimuli in primary and macrophage cell lines partially through activation of the peroxisome proliferator-activated receptor-gamma (PPARγ). Moreover, the protective effect of NO2-OA relies on the inhibition of macrophage prostaglandin E2 (PGE2)-induced PGE2 receptor 4 (EP4) cAMP signaling, known to participate in the development of AAA. CONCLUSION: Administration of NO2-OA protects against AAA formation and multifactorial macrophage activation. With NO2-OA currently undergoing FDA approved phase II clinical trials, these findings may expedite the use of this nitro-fatty acid for AAA therapy.

Loss-of-function genomic variants highlight potential therapeutic targets for cardiovascular disease.

Pharmaceutical drugs targeting dyslipidemia and cardiovascular disease (CVD) may increase the risk of fatty liver disease and other metabolic disorders. To identify potential novel CVD drug targets without these adverse effects, we perform genome-wide analyses of participants in the HUNT Study in Norway (n = 69,479) to search for protein-altering variants with beneficial impact on quantitative blood traits related to cardiovascular disease, but without detrimental impact on liver function. We identify 76 (11 previously unreported) presumed causal protein-altering variants associated with one or more CVD- or liver-related blood traits. Nine of the variants are predicted to result in loss-of-function of the protein. This includes ZNF529:p.K405X, which is associated with decreased low-density-lipoprotein (LDL) cholesterol (P = 1.3 × 10-8) without being associated with liver enzymes or non-fasting blood glucose. Silencing of ZNF529 in human hepatoma cells results in upregulation of LDL receptor and increased LDL uptake in the cells. This suggests that inhibition of ZNF529 or its gene product should be prioritized as a novel candidate drug target for treating dyslipidemia and associated CVD.

Glycine-based treatment ameliorates NAFLD by modulating fatty acid oxidation, glutathione synthesis, and the gut microbiome.

Nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH) has reached epidemic proportions with no pharmacological therapy approved. Lower circulating glycine is consistently reported in patients with NAFLD, but the causes for reduced glycine, its role as a causative factor, and its therapeutic potential remain unclear. We performed transcriptomics in livers from humans and mice with NAFLD and found suppression of glycine biosynthetic genes, primarily alanine-glyoxylate aminotransferase 1 (AGXT1). Genetic (Agxt1 -/- mice) and dietary approaches to limit glycine availability resulted in exacerbated diet-induced hyperlipidemia and steatohepatitis, with suppressed mitochondrial/peroxisomal fatty acid ß-oxidation (FAO) and enhanced inflammation as the underlying pathways. We explored glycine-based compounds with dual lipid/glucose-lowering properties as potential therapies for NAFLD and identified a tripeptide (Gly-Gly-L-Leu, DT-109) that improved body composition and lowered circulating glucose, lipids, transaminases, proinflammatory cytokines, and steatohepatitis in mice with established NASH induced by a high-fat, cholesterol, and fructose diet. We applied metagenomics, transcriptomics, and metabolomics to explore the underlying mechanisms. The bacterial genus Clostridium sensu stricto was markedly increased in mice with NASH and decreased after DT-109 treatment. DT-109 induced hepatic FAO pathways, lowered lipotoxicity, and stimulated de novo glutathione synthesis. In turn, inflammatory infiltration and hepatic fibrosis were attenuated via suppression of NF-κB target genes and TGFß/SMAD signaling. Unlike its effects on the gut microbiome, DT-109 stimulated FAO and glutathione synthesis independent of NASH. In conclusion, impaired glycine metabolism may play a causative role in NAFLD. Glycine-based treatment attenuates experimental NAFLD by stimulating hepatic FAO and glutathione synthesis, thus warranting clinical evaluation.

Novel gene regulatory networks identified in response to nitro-conjugated linoleic acid in human endothelial cells.

Endothelial cell (EC) dysfunction is a crucial initiation event in the development of atherosclerosis and is associated with diabetes mellitus, hypertension, and heart failure. Both digestive and oxidative inflammatory conditions lead to the endogenous formation of nitrated derivatives of unsaturated fatty acids (FAs) upon generation of the proximal nitrating species nitrogen dioxide (·NO2) by nitric oxide (·NO) and nitrite-dependent reactions. Nitro-FAs (NO2-FAs) such as nitro-oleic acid (NO2-OA) and nitro-linoleic acid (NO2-LA) potently inhibit inflammation and oxidative stress, regulate cellular functions, and maintain cardiovascular homeostasis. Recently, conjugated linoleic acid (CLA) was identified as the preferential FA substrate of nitration in vivo. However, the functions of nitro-CLA (NO2-CLA) in ECs remain to be explored. In the present study, a distinct transcriptome regulated by NO2-CLA was revealed in primary human coronary artery endothelial cells (HCAECs) through RNA sequencing. Differential gene expression and pathway enrichment analysis identified numerous regulatory networks including those related to the modulation of inflammation, oxidative stress, cell cycle, and hypoxic responses by NO2-CLA, suggesting a diverse impact of NO2-CLA and other electrophilic nitrated FAs on cellular processes. These findings extend the understanding of the protective actions of NO2-CLA in cardiovascular diseases and provide new insight into the underlying mechanisms that mediate the pleiotropic cellular responses to NO2-CLA.

Nitro-fatty acids protect against steatosis and fibrosis during development of nonalcoholic fatty liver disease in mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) and resulting nonalcoholic steatohepatitis (NASH) are reaching global epidemic proportions. Lack of non-invasive diagnostic tools and effective therapies constitute two of the major hurdles for a bona fide treatment and a reversal of NASH progression and/or regression of the disease. Nitro-oleic acid (OA-NO2) has been proven effective in multiple experimental models of inflammation and fibrosis. Thus, the potential benefit of in vivo administration of OA-NO2 to treat advanced NAFLD was tested herein in a model of long-term NASH diet-induced liver damage. METHODS: Non-invasive imaging (e.g. photoacustic-ultrasound (PA-US)) was pursued to establish advanced experimental model of NASH in mice in which both steatosis and fibrosis were diagnosed prior experimental therapy with OA-NO2. Experimental controls included equimolar amounts of the non-nitrated oleic acid (OA). CLAMS and NMR-based analysis was used for energy metabolism. FINDINGS: CLAMS and NMR-based analysis demonstrates that OA-NO2 improves body composition and energy metabolism and inhibits hepatic triglyceride (TG) accumulation. Photoacoustic-ultrasound imaging revealed a robust inhibition of liver steatosis and fibrosis by OA-NO2. RNA-sequencing analysis uncovered inflammation and fibrosis as major pathways suppressed by OA-NO2 administration, as well as regulation of lipogenesis and lipolysis pathways, with a robust inhibition of SREBP1 proteolytic activation and subsequent lipogenesis gene expression by OA-NO2. These results were further supported by histological analysis and quantification of lipid accumulation, lobular inflammation (F4/80 staining) and fibrosis (collagen deposition, αSMA staining) as well as established parameters of liver damage (ALT). In vitro studies indicate that OA-NO2 inhibits TG biosynthesis and accumulation in hepatocytes and inhibits fibrogenesis in human stellate cells. INTERPRETATION: OA-NO2 improve steatohepatitis and fibrosis and may constitute an effective therapeutic approach against advanced NAFLD that warrants further clinical evaluation.

Brown Adipocyte-Specific PPARγ (Peroxisome Proliferator-Activated Receptor γ) Deletion Impairs Perivascular Adipose Tissue Development and Enhances Atherosclerosis in Mice.

Objective- Perivascular adipose tissue (PVAT) contributes to vascular homeostasis by producing paracrine factors. Previously, we reported that selective deletion of PPARγ (peroxisome proliferator-activated receptor γ) in vascular smooth muscle cells resulted in concurrent loss of PVAT and enhanced atherosclerosis in mice. To address the causal relationship between loss of PVAT and atherosclerosis, we used BA-PPARγ-KO (brown adipocyte-specific PPARγ knockout) mice. Approach and Results- Deletion of PPARγ in brown adipocytes did not affect PPARγ in white adipocytes or vascular smooth muscle cells or PPARα and PPARδ expression in brown adipocytes. However, development of PVAT and interscapular brown adipose tissue was remarkably impaired, associated with reduced expression of genes encoding lipogenic enzymes in the BA-PPARγ-KO mice. Thermogenesis in brown adipose tissue was significantly impaired with reduced expression of thermogenesis genes in brown adipose tissue and compensatory increase in subcutaneous and gonadal white adipose tissues. Remarkably, basal expression of inflammatory genes and macrophage infiltration in PVAT and brown adipose tissue were significantly increased in the BA-PPARγ-KO mice. BA-PPARγ-KO mice were crossbred with ApoE KO (apolipoprotein E knockout) mice to investigate the development of atherosclerosis. Flow cytometry analysis confirmed increased systemic and PVAT inflammation. Consequently, atherosclerotic lesions were significantly increased in mice with impaired PVAT development, thus indicating that the lack of normal PVAT is sufficient to drive increased atherosclerosis. Conclusions- PPARγ is required for functional PVAT development. PPARγ deficiency in PVAT, while still expressed in vascular smooth muscle cell, enhances atherosclerosis and results in vascular and systemic inflammation, providing new insights on the specific roles of PVAT in atherosclerosis and cardiovascular disease at large.

Inflammatory signaling and metabolic regulation by nitro-fatty acids.

The addition of nitrogen dioxide (NO2) to the double bond of unsaturated fatty acids yields an array of electrophilic nitro-fatty acids (NO2-FA) with unique biochemical and signaling properties. During the last decade, NO2-FA have been shown to exert a protective role in various inflammatory and metabolic disorders. NO2-FA exert their biological effects primarily by regulating two central physiological adaptive responses: the canonical inflammatory signaling and metabolic pathways. In this mini-review, we summarize current knowledge on the regulatory role of NO2-FA in the inflammatory and metabolic response via regulation of nuclear factor kappa B (NF-κB) and peroxisome proliferator-activated receptor γ (PPARγ), master regulators of inflammation and metabolism. Moreover, the engagement of novel signaling and metabolic pathways influenced by NO2-FA, beyond NF-κB and PPAR signaling, is discussed herein.

Transcriptomic sequencing reveals diverse adaptive gene expression responses of human vascular smooth muscle cells to nitro-conjugated linoleic acid.

Nitro-conjugated linoleic acid (NO2-CLA) is formed by metabolic and inflammatory reactions of nitric oxide and nitrite, and represents the most abundant nitro-fatty acid species in humans. These electrophilic fatty acid nitroalkene derivatives mediate pleiotropic cell signaling responses. Here, we report a systematic approach to investigate the effect of NO2-CLA on human coronary artery smooth muscle cells (hCASMC), based on the RNA-Seq and bioinformatics analysis. There were extensive differentially expressed genes in NO2-CLA vs. control (510) and NO2-CLA vs. CLA (272) treatment groups, respectively. Notably, only minimal alterations were observed in CLA vs. control conditions, indicating that the electrophilic character of NO2-CLA is requited to induce differential gene expression responses independently from native CLA. Functional enrichment analysis of differentially expressed genes reveals multiple cellular processes to be affected under NO2-CLA treatment, including cell proliferation, lipid metabolism, antioxidant and inflammatory-related gene expression responses. These findings reveal that nitro-fatty acid derivatives such as NO2-CLA regulate a broad array of adaptive gene expression responses by hCASMC.

In situ generation, metabolism and immunomodulatory signaling actions of nitro-conjugated linoleic acid in a murine model of inflammation.

Conjugated linoleic acid (CLA) is a prime substrate for intra-gastric nitration giving rise to the formation of nitro-conjugated linoleic acid (NO2-CLA). Herein, NO2-CLA generation is demonstrated within the context of acute inflammatory responses both in vitro and in vivo. Macrophage activation resulted in dose- and time-dependent CLA nitration and also in the production of secondary electrophilic and non-electrophilic derivatives. Both exogenous NO2-CLA as well as that generated in situ, attenuated NF-κB-dependent gene expression, decreased pro-inflammatory cytokine production and up-regulated Nrf2-regulated proteins. Importantly, both CLA nitration and the corresponding downstream anti-inflammatory actions of NO2-CLA were recapitulated in a mouse peritonitis model where NO2-CLA administration decreased pro-inflammatory cytokines and inhibited leukocyte recruitment. Taken together, our results demonstrate that the formation of NO2-CLA has the potential to function as an adaptive response capable of not only modulating inflammation amplitude but also protecting neighboring tissues via the expression of Nrf2-dependent genes.

Modulation of cAMP levels by high-fat diet and curcumin and regulatory effects on CD36/FAT scavenger receptor/fatty acids transporter gene expression.

Curcumin, a polyphenol from turmeric (Curcuma longa), reduces inflammation, atherosclerosis, and obesity in several animal studies. In Ldlr-/- mice fed a high-fat diet (HFD), curcumin reduces plasma lipid levels, therefore contributing to a lower accumulation of lipids and to reduced expression of fatty acid transport proteins (CD36/FAT, FABP4/aP2) in peritoneal macrophages. In this study, we analyzed the molecular mechanisms by which curcumin (500, 1000, 1500 mg/kg diet, for 4 months) may influence plasma and tissue lipid levels in Ldlr-/- mice fed an HFD. In liver, HFD significantly suppressed cAMP levels, and curcumin restored almost normal levels. Similar trends were observed in adipose tissues, but not in brain, skeletal muscle, spleen, and kidney. Treatment with curcumin increased phosphorylation of CREB in liver, what may play a role in regulatory effects of curcumin in lipid homeostasis. In cell lines, curcumin increased the level of cAMP, activated the transcription factor CREB and the human CD36 promoter via a sequence containing a consensus CREB response element. Regulatory effects of HFD and Cur on gene expression were observed in liver, less in skeletal muscle and not in brain. Since the cAMP/protein kinase A (PKA)/CREB pathway plays an important role in lipid homeostasis, energy expenditure, and thermogenesis by increasing lipolysis and fatty acid ß-oxidation, an increase in cAMP levels induced by curcumin may contribute to its hypolipidemic and anti-atherosclerotic effects. © 2016 BioFactors, 43(1):42-53, 2017.

Cardiomyocyte Overexpression of FABP4 Aggravates Pressure Overload-Induced Heart Hypertrophy.

Fatty acid binding protein 4 (FABP4) is a member of the intracellular lipid-binding protein family, responsible for the transportation of fatty acids. It is considered to express mainly in adipose tissues, and be strongly associated with inflammation, obesity, diabetes and cardiovasculardiseases. Here we report that FABP4 is also expressed in cardiomyocytes and plays an important role in regulating heart function under pressure overload. We generated heart-specific transgenic FABP4 (FABP4-TG) mice using α myosin-heavy chain (α-MHC) promoter and human FABP4 sequence, resulting in over-expression of FABP4 in cardiomyocytes. The FABP4-TG mice displayed normal cardiac morphology and contractile function. When they were subjected to the transverse aorta constriction (TAC) procedure, the FABP4-TG mice developed more cardiac hypertrophy correlated with significantly increased ERK phosphorylation, compared with wild type controls. FABP4 over-expression in cardiomyocytes activated phosphor-ERK signal and up-regulate the expression of cardiac hypertrophic marker genes. Conversely, FABP4 induced phosphor-ERK signal and hypertrophic gene expressions can be markedly inhibited by an ERK inhibitor PD098059 as well as the FABP4 inhibitor BMS309403. These results suggest that FABP4 over-expression in cardiomyocytes can aggravate the development of cardiac hypertrophy through the activation of ERK signal pathway.

Nitro-fatty acids in cardiovascular regulation and diseases: characteristics and molecular mechanisms.

Electrophilic nitro-fatty acids (NO2-FAs) are endogenously formed by redox reactions of nitric oxide ((.)NO)- and nitrite ((.)NO2)- derived nitrogen dioxide with unsaturated fatty acids. Nitration preferentially occurs on polyunsaturated fatty acids with conjugated dienes under physiological or pathophysiological conditions such as during digestion, metabolism and as adaptive inflammatory processes. Nitro-fatty acids are present in free and esterified forms achieving broad biodistribution in humans and experimental models. Structural, functional and biological characterization of NO2-FAs has revealed clinically relevant protection from inflammatory injury in a number of cardiovascular, renal and metabolic experimental models. NO2-FAs are engaged in posttranslational modifications (PTMs) of a selective redox sensitive pool of proteins and regulate key adaptive signaling pathways involved in cellular homeostasis and inflammatory response. Here, we review and update the biosynthesis, metabolism and signaling actions of NO2-FAs, highlighting their diverse protective roles relevant to the cardiovascular system.

The role of perivascular adipose tissue in vasoconstriction, arterial stiffness, and aneurysm.

Since the "rediscovery" of brown adipose tissue in adult humans, significant scientific efforts are being pursued to identify the molecular mechanisms to promote a phenotypic change of white adipocytes into brown-like cells, a process called "browning". It is well documented that white adipose tissue (WAT) mass and factors released from WAT influence the vascular function and positively correlate with cardiac arrest, stroke, and other cardiovascular complications. Similar to other fat depots, perivascular adipose tissue (PVAT) is an active endocrine organ and anatomically surrounds vessels. Both brown-like and white-like PVAT secrete various adipokines, cytokines, and growth factors that either prevent or promote the development of cardiovascular diseases (CVDs) depending on the relative abundance of each type and their bioactivity in the neighboring vasculature. Notably, pathophysiological conditions, such as obesity, hypertension, or diabetes, induce the imbalance of PVAT-derived vasoactive products that promote the infiltration of inflammatory cells. This then triggers derangements in vascular smooth muscle cells and endothelial cell dysfunction, resulting in the development of vascular diseases. In this review, we discuss the recent advances on the contribution of PVAT in CVDs. Specifically, we summarize the current proposed roles of PVAT in relationship with vascular contractility, endothelial dysfunction, neointimal formation, arterial stiffness, and aneurysm.

Electrophilic nitro-fatty acids inhibit vascular inflammation by disrupting LPS-dependent TLR4 signalling in lipid rafts.

AIMS: Electrophilic fatty acid nitroalkene derivatives, products of unsaturated fatty acid nitration, exert long-term cardiovascular protection in experimental models of metabolic and cardiovascular diseases. The goal of this study is to examine the effects of nitro-fatty acids in the regulation of upstream signalling events in nuclear factor-κB (NF-κB) activation and determine whether low-dose acute administration of nitro-fatty acids reduces vascular inflammation in vivo. METHODS AND RESULTS: Using NF-κB-luciferase transgenic mice, it was determined that pre-emptive treatment with nitro-oleic acid (OA-NO2), but not oleic acid (OA) inhibits lipopolysaccharide (LPS)-induced NF-κB activation both in vivo and in isolated macrophages. Acute intravenous administration of OA-NO2 was equally effective to inhibit leukocyte recruitment to the vascular endothelium assessed by intravital microscopy and significantly reduces aortic expression of adhesion molecules. An acute treatment with OA-NO2 in vivo yielding nanomolar concentrations in plasma, is sufficient to inhibit LPS-induced Toll-like receptor 4 (TLR4)-induced cell surface expression in leukocytes and NF-κB activation. In vitro experiments reveal that OA-NO2 suppresses LPS-induced TLR4 signalling, inhibitor of κB (IκBα) phosphorylation and ubiquitination, phosphorylation of the IκB kinase (IKK), impairing the recruitment of the TLR4 and TNF receptor associated factor 6 (TRAF6) to the lipid rafts compartments. CONCLUSION: These studies demonstrate that acute administration of nitro-fatty acids is effective to reduce vascular inflammation in vivo. These findings reveal a direct role of nitro-fatty acids in the disruption of the TLR4 signalling complex in lipid rafts, upstream events of the NF-κB pathway, leading to resolution of pro-inflammatory activation of NF-κB in the vasculature.

Novel keto-phospholipids are generated by monocytes and macrophages, detected in cystic fibrosis, and activate peroxisome proliferator-activated receptor-γ.

12/15-Lipoxygenases (LOXs) in monocytes and macrophages generate novel phospholipid-esterified eicosanoids. Here, we report the generation of two additional families of related lipids comprising 15-ketoeicosatetraenoic acid (KETE) attached to four phosphatidylethanolamines (PEs). The lipids are generated basally by 15-LOX in IL-4-stimulated monocytes, are elevated on calcium mobilization, and are detected at increased levels in bronchoalveolar lavage fluid from cystic fibrosis patients (3.6 ng/ml of lavage). Murine peritoneal macrophages generate 12-KETE-PEs, which are absent in 12/15-LOX-deficient mice. Inhibition of 15-prostaglandin dehydrogenase prevents their formation from exogenous 15-hydroxyeicosatetraenoic acid-PE in human monocytes. Both human and murine cells also generated analogous hydroperoxyeicosatetraenoic acid-PEs. The electrophilic reactivity of KETE-PEs is shown by their Michael addition to glutathione and cysteine. Lastly, both 15-hydroxyeicosatetraenoic acid-PE and 15-KETE-PE activated peroxisome proliferator-activated receptor-γ reporter activity in macrophages in a dose-dependent manner. In summary, we demonstrate novel peroxisome proliferator-activated receptor-γ-activating oxidized phospholipids generated enzymatically by LOX and 15-prostaglandin dehydrogenase in primary monocytic cells and in a human Th2-related lung disease. The lipids are a new family of bioactive mediators from the 12/15-LOX pathway that may contribute to its known anti-inflammatory actions in vivo.

Loss of perivascular adipose tissue on peroxisome proliferator-activated receptor-γ deletion in smooth muscle cells impairs intravascular thermoregulation and enhances atherosclerosis.

BACKGROUND: Perivascular adipose tissue (PVAT) surrounds most vessels and shares common features with brown adipose tissue (BAT). Although adaptive thermogenesis in BAT increases energy expenditure and is beneficial for metabolic diseases, little is known about the role of PVAT in vascular diseases such as atherosclerosis. We hypothesize that the thermogenic function of PVAT regulates intravascular temperature and reduces atherosclerosis. METHODS AND RESULTS: PVAT shares similar structural and proteomics with BAT. We demonstrated that PVAT has thermogenic properties similar to BAT in response to cold stimuli in vivo. Proteomics analysis of the PVAT from mice housed in a cold environment identified differential expression in proteins highly related to cellular metabolic processes. In a mouse model deficient in peroxisome proliferator-activated receptor-γ in smooth muscle cells (SMPG KO mice), we uncovered a complete absence of PVAT surrounding the vasculature, likely caused by peroxisome proliferator-activated receptor-γ deletion in the perivascular adipocyte precursor cells as well. Lack of PVAT, which results in loss of its thermogenic activity, impaired vascular homeostasis, which caused temperature loss and endothelial dysfunction. We further showed that cold exposure inhibits atherosclerosis and improves endothelial function in mice with intact PVAT but not in SMPG KO mice as a result of impaired lipid clearance. Proinflammatory cytokine expression in PVAT is not altered on exposure to cold. Finally, prostacyclin released from PVAT contributes to the vascular protection against endothelial dysfunction. CONCLUSIONS: PVAT is a vasoactive organ with functional characteristics similar to BAT and is essential for intravascular thermoregulation of cold acclimation. This thermogenic capacity of PVAT plays an important protective role in the pathogenesis of atherosclerosis.