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BACKGROUND Metabolic endotoxemia is considered a cause for high-fat diet (HFD)-induced inflammation. However, convincing experimental evidence in humans is scant. OBJECTIVE We determined whether a HFD or moderately HFD increases LPS and LPS-mediated cytokine production in the postprandial blood (PPB). METHODS Ninety-eight volunteers (age: 37.3 ± 1.5 y) from the cross-sectional phenotyping study (PS) and 62 volunteers (age: 26.8 ± 1.2 y) from the intervention study (IS) consumed a breakfast containing 60% kcal fat (HF) and 36% kcal fat (moderately HF), respectively. For the IS, only the results from the placebo group are presented. Blood samples were probed for LPS-mediated cytokine production by incubating them with LPS inhibitor polymyxin B (PMB) for 24 h at 37°C besides the Limulus amebocyte lysate (LAL) assay. Repeated-measures ANOVA was used to compare the temporal changes of metabolic profiles and treatment outcomes. RESULTS At least 87.5% of the plasma LPS measurements in 32 PS volunteers from each time point were below the LAL assay sensitivity (0.002 EU/mL). PMB suppressed IL-1β (P = 0.035) and IL-6 (P = 0.0487) production in the 3 h PPB of the PS after 24 h incubation at 37°C compared to the vehicle control, suggesting the presence of LPS. However, the amount of LPS did not increase the cytokine concentrations in the 3 h PPB above the fasting concentrations. Such suppression was not detected in the PPB of the IS. Treating whole blood with lipoprotein lipase (LPL) significantly (P

Oxylipins are the oxidation products of polyunsaturated fatty acids and have been implicated in neurodegenerative disorders, including dementia. Soluble epoxide hydrolase (sEH) converts epoxy-fatty acids to their corresponding diols, is found in the brain, and its inhibition is a treatment target for dementia. In this study, male and female C57Bl/6J mice were treated with an sEH inhibitor (sEHI), trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), for 12 weeks to comprehensively study the effect of sEH inhibition on the brain oxylipin profile, and modulation by sex. Ultra-high-performance liquid chromatography–tandem mass spectrometry was used to measure the profile of 53 free oxylipins in the brain. More oxylipins were modified by the inhibitor in males than in females (19 versus 3, respectively) and favored a more neuroprotective profile. Most were downstream of lipoxygenase and cytochrome p450 in males, and cyclooxygenase and lipoxygenase in females. The inhibitor-associated oxylipin changes were unrelated to serum insulin, glucose, cholesterol, or female estrous cycle. The inhibitor affected behavior and cognitive function as measured by open field and Y-maze tests in males, but not females. These findings are novel and important to our understanding of sexual dimorphism in the brain’s response to sEHI and may help inform sex-specific treatment targets.

International audience; Diet is a modifiable risk factor for cardiovascular disease (CVD) and dementia, yet relatively little is known about the effect of a high glycemic diet (HGD) on the brain’s microvasculature. The objective of our study was to determine the molecular effects of an HGD on hippocampal microvessels and cognitive function and determine if a soluble epoxide hydrolase (sEH) inhibitor (sEHI), known to be vasculoprotective and anti-inflammatory, modulates these effects. Wild type male mice were fed a low glycemic diet (LGD, 12% sucrose/weight) or an HGD (34% sucrose/weight) with/without the sEHI, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), for 12 weeks. Brain hippocampal microvascular gene expression was assessed by microarray and data analyzed using a multi-omic approach for differential expression of protein and non-protein-coding genes, gene networks, functional pathways, and transcription factors. Global hippocampal microvascular gene expression was fundamentally different for mice fed the HGD vs. the LGD. The HGD response was characterized by differential expression of 608 genes involved in cell signaling, neurodegeneration, metabolism, and cell adhesion/inflammation/oxidation effects reversible by t-AUCB and hence sEH inhibitor correlated with protection against Alzheimer’s dementia. Ours is the first study to demonstrate that high dietary glycemia contributes to brain hippocampal microvascular inflammation through sEH.

Oxylipins have been implicated in many biological processes and diseases. Dysregulation of cerebral lipid homeostasis and altered lipid metabolites have been associated with the onset and progression of dementia. Although most dietary interventions have focused on modulation of dietary fats, the impact of a high sucrose diet on the brain oxylipin profile is unknown.Male and female C57BL/6J mice were fed a high sucrose diet (HSD, 34%) in comparison to a control low sucrose diet (LSD, 12%) for 12 weeks beginning at 20 weeks of age. The profile of 53 free oxylipins was then measured in brain by ultra-high performance liquid chromatography tandem mass spectrometry. Serum glucose and insulin were measured enzymatically. We first assessed whether there were any effects of the diet on the brain oxylipin profile, then assessed for sex differences.There were no differences in fasting serum glucose between the sexes for mice fed a HSD or in fasting serum insulin levels for mice on either diet. The HSD altered the brain oxylipin profile in both sexes in distinctly different patterns: there was a reduction in three oxylipins (by 47-61%) and an increase in one oxylipin (16%) all downstream of lipoxygenase enzymes in males and a reduction in eight oxylipins (by 14-94%) mostly downstream of cyclooxygenase activity in females. 9-oxo-ODE and 6-trans-LTB4 were most influential in the separation of the oxylipin profiles by diet in male mice, whereas 5-HEPE and 12-HEPE were most influential in the separation by diet in female mice. Oxylipins 9‑hydroxy-eicosatetraenoic acid (HETE), 11-HETE, and 15-HETE were higher in the brains of females, regardless of diet.A HSD substantially changes brain oxylipins in a distinctly sexually dimorphic manner. Results are discussed in terms of potential mechanisms and links to metabolic disease. Sex and diet effects on brain oxylipin composition may provide future targets for the management of neuroinflammatory diseases, such as dementia.

Elevation of blood triglycerides, primarily triglyceride-rich lipoproteins (TGRL), is an independent risk factor for cardiovascular disease and vascular dementia (VaD). Accumulating evidence indicates that both atherosclerosis and VaD are linked to vascular inflammation. However, the role of TGRL in vascular inflammation, which increases risk for VaD, remains largely unknown and its underlying mechanisms are still unclear. We strived to determine the effects of postprandial TGRL exposure on brain microvascular endothelial cells, the potential risk factor of vascular inflammation, resulting in VaD. We showed in Aung et al., J Lipid Res., 2016 that postprandial TGRL lipolysis products (TL) activate mitochondrial reactive oxygen species (ROS) and increase the expression of the stress-responsive protein, activating transcription factor 3 (ATF3), which injures human brain microvascular endothelial cells (HBMECs) in vitro. In this study, we deployed high-throughput sequencing (HTS)-based RNA sequencing methods and mito stress and glycolytic rate assays with an Agilent Seahorse XF analyzer and profiled the differential expression of transcripts, constructed signaling pathways, and measured mitochondrial respiration, ATP production, proton leak, and glycolysis of HBMECs treated with TL. Conclusions: TL potentiate ROS by mitochondria which activate mitochondrial oxidative stress, decrease ATP production, increase mitochondrial proton leak and glycolysis rate, and mitochondria DNA damage. Additionally, CPT1A1 siRNA knockdown suppresses oxidative stress and prevents mitochondrial dysfunction and vascular inflammation in TL treated HBMECs. TL activates ATF3-MAPKinase, TNF, and NRF2 signaling pathways. Furthermore, the NRF2 signaling pathway which is upstream of the ATF3-MAPKinase signaling pathway, is also regulated by the mitochondrial oxidative stress. We are the first to report differential inflammatory characteristics of transcript variants 4 (ATF3-T4) and 5 (ATF3-T5) of the stress responsive gene ATF3 in HBMECs induced by postprandial TL. Specifically, our data indicates that ATF3-T4 predominantly regulates the TL-induced brain microvascular inflammation and TNF signaling. Both siRNAs of ATF3-T4 and ATF3-T5 suppress cells apoptosis and lipotoxic brain microvascular endothelial cells. These novel signaling pathways triggered by oxidative stress-responsive transcript variants, ATF3-T4 and ATF3-T5, in the brain microvascular inflammation induced by TGRL lipolysis products may contribute to pathophysiological processes of vascular dementia.

Diet-induced obesity (DIO) is associated with glucose intolerance, insulin resistance (IR), and an increase in intramyocellular lipids (IMCL), which may lead to disturbances in glucose and protein metabolism. To this matter, it has been speculated that chronic obesity and elevated IMCL may contribute to skeletal muscle loss and deficits in muscle function and growth capacity. Thus, we hypothesized that diets with elevated fat content would induce obesity and insulin resistance, leading to a decrease in muscle mass and an attenuated growth response to increased external loading in adult male mice. Male C57BL/6 mice (8 wk of age) were subjected to five different diets, namely, chow, low-dat-diet (LFD), high-fat-diet (HFD), sucrose, or Western diet, for 28 wk. At 25 wk, HFD and Western diets induced a 60.4% and 35.9% increase in body weight, respectively. Interestingly, HFD, but not Western or sucrose, induced glucose intolerance and insulin resistance. Measurement of isometric torque (ankle plantar flexor and ankle dorsiflexor muscles) revealed no effect of DIO on muscle function. At 28 wk of intervention, muscle area and protein synthesis were similar across all diet groups, despite insulin resistance and increased IMCL being observed in HFD and Western diet groups. In response to 30 days of functional overload, an attenuated growth response was observed in only the HFD group. Nevertheless, our results show that DIO alone is not sufficient to induce muscle atrophy and contractile dysfunction in adult male C57BL/6 mice. However, diet composition does have an impact on muscle growth in response to increased external loading. NEW & NOTEWORTHY The effects of diet-induced obesity on skeletal muscle mass are complex and dependent on diet composition and diet duration. The present study results show that chronic exposure to high levels of fatty acids does not affect muscle mass, contractile function, or protein synthesis in obese C57BL/6 mice compared with the consumption of chow. Obesity did result in a delay in load-induced growth; however, only a 45% HFD resulted in attenuated growth following 30 days of functional overload.

Cardiovascular risk factors and biologic sex play a role in vascular dementia which is characterized by progressive reduction in cognitive function and memory. Yet, we lack understanding about the role sex plays in the molecular mechanisms whereby lipid stress contributes to cognitive decline. Five-week-old low-density lipoprotein deficient (LDL-R&minus, /&minus, ) male and female mice and C57BL/6J wild types (WT) were fed a control or Western Diet for 8 weeks. Differential expression of protein coding and non-protein coding genes (DEG) were determined in laser captured hippocampal microvessels using genome-wide microarray, followed by bioinformatic analysis of gene networks, pathways, transcription factors and sex/gender-based analysis (SGBA). Cognitive function was assessed by Y-maze. Bioinformatic analysis revealed more DEGs in females (2412) compared to males (1972). Hierarchical clusters revealed distinctly different sex-specific gene expression profiles irrespective of diet and genotype. There were also fewer and different biologic responses in males compared to females, as well as different cellular pathways and gene networks (favoring greater neuroprotection in females), together with sex-specific transcription factors and non-protein coding RNAs. Hyperlipidemic stress also resulted in less severe cognitive dysfunction in females. This sex-specific pattern of differential hippocampal microvascular RNA expression might provide therapeutic targets for dementia in males and females.

The Western diet (WD) and hyperlipidemia are risk factors for vascular disease, dementia, and cognitive impairment. However, the molecular mechanisms are poorly understood. This pilot study investigated the genomic pathways by which the WD and hyperlipidemia regulate gene expression in brain microvessels. Five-week-old C57BL/6J wild type (WT) control and low-density lipoprotein receptor deficient (LDL-R&minus, /&minus, ) male mice were fed the WD for eight weeks. Differential gene expression, gene networks and pathways, transcription factors, and non-protein coding RNAs were evaluated by a genome-wide microarray and bioinformatics analysis of laser-captured hippocampal microvessels. The WD resulted in the differential expression of 1972 genes. Much of the differentially expressed gene (DEG) was attributable to the differential regulation of cell signaling proteins and their transcription factors, approximately 4% was attributable to the differential expression of miRNAs, and 10% was due to other non-protein coding RNAs, primarily long non-coding RNAs (lncRNAs) and small nucleolar RNAs (snoRNAs) not previously described to be modified by the WD. Lipotoxic injury resulted in complex and multilevel molecular regulation of the hippocampal microvasculature involving transcriptional and post-transcriptional regulation and may provide a molecular basis for a better understanding of hyperlipidemia-associated dementia risk.

Our lab previously demonstrated that triglyceride-rich lipoprotein (TGRL) lipolysis products induce lipid droplet formation and pro-inflammatory gene expression in monocytes. We hypothesized that the inhibition of perilipin 2 expression in THP-1 monocytes would reduce lipid droplet formation and suppress pro-inflammatory gene expression induced by TGRL lipolysis products. In the current study, we use microarray analysis to identify gene expression altered by TGRL lipolysis products in THP-1 monocytes. We confirmed the expression of selected genes by quantitative reverse transcription PCR and characterized lipid droplet formation in these cells after exposure to TGRL lipolysis products. Using siRNA inhibition of perilipin 2 expression, we examined the role of perilipin 2 in the response of THP-1 monocytes to TGRL lipolysis products. We found that perilipin 2 siRNA increased the intracellular triglyceride content, increased the size of lipid droplets, and reduced pro-atherogenic and pro-inflammatory gene expression. We saw a reduction of serum/glucocorticoid kinase 1, v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (avian), chemokine (C-C motif) ligand 3, and interleukin 8 gene expression induced by TGRL lipolysis products. This study supports previous findings that reduction of perilipin 2 expression is protective against atherogenesis, while finding an unexpected increase in lipid droplet size with reduced perilipin 2 expression.

To better understand how lipoproteins interact and enter endothelium and participate in cellular processes, we investigated preferential lipid partitioning of triglyceride rich lipoproteins (TGRL), chylomicrons (CM), low density lipoproteins (LDL), very low density lipoproteins (VLDL) and their lipolysis products using supported phospholipid raft membrane (SPRM) patterns. We prepared SPRM patterns with Texas red labeled phospholipid patterns and Marina blue labeled raft patterns and added Atto-520 labeled lipoproteins (TGRL, CM, VLDL, LDL) and their lipolysis products in separate experiments and characterized these interactions using fluorescence microscopy. We observed that VLDL and LDL preferentially interacted with raft patterns. In contrast the TGRL and lipolysed products of TGRL interacted with both the patterns, slightly elevated preference for raft patterns and CM and its lipolysis products showed greater affinity to phospholipid patterns. The clear preference of VLDL and LDL for raft patterns suggests that these lipoproteins associate with cholesterol and sphingomyelin rich lipid micro-domains during their early interactions with endothelial cells, leading to atherosclerosis.

Our objective was to determine whether consumption of a single meal has the potential to alter brain oxylipin content. We examined the cerebrum of mice fed a single high-fat/high-sucrose Western meal or a low-fat/low-sucrose control meal, as well as fasted mice. We found no changes in fatty acid composition of cerebrum across the groups. The cerebral oxylipin profile of mice fed a Western meal is distinct from the profile of mice fed a low-fat/low-sucrose meal. Cerebral gene expression of cyclooxygenase 1, cyclooxygenase 2, and epoxide hydrolase 1 were elevated in Western meal-fed mice compared to low-fat/low-sucrose meal-fed mice. Mice that consumed either meal had lower gene expression of cytochrome P450, family 2, subfamily j, polypeptide 12 than fasted mice. Our data in this hypothesis-generating study indicates that the composition of a single meal has the potential to alter brain oxylipins and the gene expression of the enzymes responsible for their production.

Background: As obesity and Type II Diabetes rise globally, it is important to understand the similarities and differences in the response of metabolic tissues between males and females. We wanted to evaluate the impact of prolonged diet induced obesity (DIO) on the skeletal muscle transcriptome of our MuRF1 KO (KO) mice. Methods: RNA was isolated from the gastrocnemius muscle of male and female WT and KO mice that were fed either standard chow (Envigo 2918) or a 45% HFD (Research Diets D12451) for 22 weeks (n = 4). RNA was enriched for mRNA prior to library preparation. RNA sequencing was performed using 150 bp paired-end reads (~ 31.6 M reads per sample). Differentially expressed genes (DEGs) were identified using DESeq2 with an FDR set to 5%. Results: At baseline (chow diet), both male and female KO mice had DEGs compared to their WT counterparts (male, 1174; female, 105). Most DEGs were found to be unique by sex (male, 1151; female, 82), though 23 genes were found to be changed in common. After obesity was induced by 22 weeks of 45% HFD feeding, KO animals showed a greater transcriptional response than their WT counterparts. Males had 1821 DEGs (v. 179 in WT) while females had 4425 DEGs (v. 2090 in WT). In males, 78 genes were changed in common between WT and KO in response to DIO, with 76 of those genes changing in the same direction (Slc282a and Gm15427 did not). In females, 1445 genes were changed in common between WT and KO, with all but 2 genes (Pla2g7 and Zfp385b) changing in the same direction. In both male and female KO animals, oxidative phosphorylation and ribosomal pathways were most significant, though the direction of change in the DEGs was opposite. Conclusion: In skeletal muscle, sex highly influences the genes and pathways changed in response to DIO. Even among common pathways identified, the response between males and females differed. Loss of MuRF1 results in common and unique transcript changes in and between males and females under normal conditions and in DIO.

Hyperlipidemia is a risk factor for dementia, and chronic consumption of a Western Diet (WD) is associated with cognitive impairment. However, the molecular mechanisms underlying the development of microvascular disease in the memory centers of the brain are poorly understood. This pilot study investigated the nutrigenomic pathways by which the WD regulates gene expression in hippocampal brain microvessels of female mice. Five-week-old female low-density lipoprotein receptor deficient (LDL-R−/−) and C57BL/6J wild type (WT) mice were fed a chow or WD for 8 weeks. Metabolics for lipids, glucose and insulin were determined. Differential gene expression, gene networks and pathways, transcription factors, and non-protein coding RNAs were evaluated by genome-wide microarray and bioinformatics analysis of laser captured hippocampal microvessels. The WD resulted in differential expression of 2,412 genes. The majority of differential gene expression was attributable to differential regulation of cell signaling proteins and their transcription factors, approximately 7% was attributable to differential expression of miRNAs, and a lesser proportion was due to other non-protein coding RNAs, primarily long non-coding RNAs (lncRNAs) and small nucleolar RNAs (snoRNAs) not previously described to be modified by the WD in females. Our findings revealed that chronic consumption of the WD resulted in integrated multilevel molecular regulation of the hippocampal microvasculature of female mice and may provide one of the mechanisms underlying vascular dementia.

Purpose Previous studies indicated hyperlipidemia may be a risk factor for Alzheimer's disease, but the contributions of postprandial triglyceride-rich lipoprotein (TGRL) are not known. In this study, changes in blood–brain barrier diffusional transport following exposure to human TGRL lipolysis products were studied using MRI in a rat model. Methods Male Sprague-Dawley rats (∼180–250 g) received an i.v. injection of lipoprotein lipase (LpL)-hydrolyzed TGRL (n = 8, plasma concentration ≈ 150 mg human TGRL/dL). Controls received i.v. injection of either saline (n = 6) or LpL only (n = 6). The 1H longitudinal relaxation rate R1 = 1/T1 was measured over 18 min using a rapid-acquired refocus-echo (RARE) sequence after each of three injections of the contrast agent Gd-DTPA. Patlak plots were generated for each pixel yielding blood-to-brain transfer coefficients, Ki, chosen for best fit to impermeable, uni-directional influx or bi-directional flux models using the F-test. Results Analysis from a 2-mm slice, 2-mm rostral to the bregma showed a 275% increase of mean Ki during the first 20 min after infusion of human TGRL lipolysis product that differed significantly compared with saline and LpL controls. This difference disappeared by 40 min mark. Conclusion These results suggest human TGRL lipolysis products can lead to a transient increase in rat BBB permeability. Magn Reson Med, 2015. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Dynamic Contrast Enhanced (DCE) MRI is increasingly being used to assess changes in capillary permeability. Most quantitative techniques used to measure capillary permeability are based on the Fick equation that requires measurement of signal reflecting both plasma and tissue concentrations of the solute being tested. To date, most Magnetic Resonance Imaging (MRI) methods for acquiring appropriate data quickly rely on gradient recalled echo (GRE) type acquisitions, which work well in clinical low field settings. However, acquiring this type of data on high field small animal preclinical MRIs is problematic due to geometrical distortions from susceptibility mismatch. This problem can be exacerbated when using small animal models to measure blood brain barrier (BBB) permeability, where precise sampling from the superior sagittal sinus (SSS) is commonly used to determine the plasma concentration of the contrast agent. Here we present results demonstrating that a standard saturation recovery rapid acquisition refocused echo (RARE) method is capable of acquiring T1 maps with good spatial and temporal resolution for Patlak analysis (Patlak, 1983) to assess changes in BBB Gd-DTPA permeability following middle cerebral artery occlusion with reperfusion in the rat. This method limits known problems with magnetic susceptibility mismatch and may thus allow greater accuracy in BBB permeability measurement in small animals.

In our previous work in mice, we have shown that chronic consumption of a Western diet (WD; 42% kcal fat, 0.2% total cholesterol and 34% sucrose) is correlated with impaired cognitive function. Cognitive decline has also been associated with alterations in DNA methylation. Additionally, although there have been many studies analyzing the effect of maternal consumption of a WD on DNA methylation in the offspring, few studies have analyzed how an individual's consumption of a WD can impact his/her DNA methylation. Since the frontal cortex is involved in the regulation of cognitive function and is often affected in cases of cognitive decline, this study aimed to examine how chronic consumption of a WD affects DNA methylation in the frontal cortex of mice. Eight-week-old male mice were fed either a control diet (CD) or a WD for 12 weeks, after which time alterations in DNA methylation were analyzed. Assessment of global DNA methylation in the frontal cortex using dot blot analysis revealed that there was a decrease in global DNA methylation in the WD-fed mice compared with the CD-fed mice. Bioinformatic analysis identified several networks and pathways containing genes displaying differential methylation, particularly those involved in metabolism, cell adhesion and cytoskeleton integrity, inflammation and neurological function. In conclusion, the results from this study suggest that consumption of a WD alters DNA methylation in the frontal cortex of mice and could provide one of the mechanisms by which consumption of a WD impairs cognitive function.

Dementia has become a major health concern for the aging population of the United States. Studies indicate that participation in moderate exercise, with training, has been shown to have a beneficial impact on cognition. Thus, exercise and its effects on cognitive function has become an important area of research. This review summarizes the current literature on the potential mechanisms of the benefits of exercise for cognitive function.

The observation that Alzheimer’s disease (AD) patients with similar and even identical genetic backgrounds often present with heterogeneous pathologies has prompted the hypothesis that epigenetics may contribute to AD. While the study of epigenetics encompasses a variety of modifications including histone modifications and non-coding RNAs, much of the research on how epigenetics might impact AD pathology has been focused on DNA methylation. To this end, several studies have characterized DNA methylation alterations in various brain regions of individuals with AD, with conflicting results. This review examines the results of studies analyzing both global and gene-specific DNA methylation changes in AD and also assesses the results of studies analyzing DNA hydroxymethylation in patients with AD.

The Western diet, causative in the development of atherosclerotic cardiovascular disease, has recently been associated with the development of diffuse white matter disease (WMD) and other subcortical changes. Yet, little is known about the pathophysiological mechanisms by which a high-fat diet can cause WMD. Mechanistic studies of deep brain regions in mice have been challenging due to a lack of non-invasive, high-resolution, and deep imaging technologies. Here we used Optical Coherence Tomography to study mouse cortical/subcortical structures noninvasively and in vivo. To better understand the role of Western Diet in the development of WMD, intensity and Doppler flow OCT images, obtained using a 1300 nm spectral / Fourier domain OCT system, were used to observe the structural and functional alterations in the cortex and corpus callosum of Western Diet and control diet mouse models. Specifically, we applied segmentation to the OCT images to identify the boundaries of the cortex/corpus callosum, and further quantify the layer thicknesses across animals between the two diet groups. Furthermore, microvasculature alterations such as changes in spatiotemporal flow profiles within diving arterioles, arteriole diameter, and collateral tortuosity were analyzed. In the current study, while the arteriole vessel diameters between the two diet groups was comparable, we show that collateral tortuosity was significantly higher in the Western diet group, compared to control diet group, possibly indicating remodeling of brain vasculature due to dietary changes. Moreover, there is evidence showing that the corpus callosum is thinner in Western diet mice, indicative of tissue atrophy.

Activating transcription factor 3 (ATF3) is a member of the mammalian activation transcription factor/cAMP, physiologically important in the regulation of pro- and anti-inflammatory target genes. We compared the induction of ATF3 protein as measured by Western blot analysis with single-molecule localization microscopy dSTORM to quantify the dynamics of accumulation of intranuclear ATF3 of triglyceride-rich (TGRL) lipolysis product-treated HAEC (Human Aortic Endothelial Cells). The ATF3 expression rate within the first three hours after treatment with TGRL lipolysis products is about 3500 h(-1). After three hours we detected 33 090 3491 single-molecule localizations of ATF3. This was accompanied by significant structural changes in the F-actin network of the cells at similar to 3-fold increased localization precision compared to widefield microscopy after treatment. Additionally, we discovered a cluster size of approximately 384 nanometers of ATF3 molecules. We show for the first time the time course of ATF3 accumulation in the nucleus undergoing lipotoxic injury. Furthermore, we demonstrate ATF3 accumulation associated with increased concentrations of TGRL lipotysis products occurs in large aggregates.

Postprandial lipemia is characterized by a transient increase in circulating triglyceride-rich lipoproteins such as very low-density lipoprotein (VLDL) and has been shown to activate monocytes in vivo. Lipolysis of VLDL releases remnant particles, phospholipids, monoglycerides, diglycerides, and fatty acids in close proximity to endothelial cells and monocytes. We hypothesized that postprandial VLDL lipolysis products could activate and recruit monocytes by increasing monocyte expression of proinflammatory cytokines and adhesion molecules, and that such activation is related to the development of lipid droplets. Freshly isolated human monocytes were treated with VLDL lipolysis products (2.28 mmol/l triglycerides + 2 U/ml lipoprotein lipase), and monocyte adhesion to a primed endothelial monolayer was observed using a parallel plate flow chamber coupled with a CCD camera. Treated monocytes showed more rolling and adhesion than controls, and an increase in transmigration between endothelial cells. The increased adhesive events were related to elevated expression of key integrin complexes including Mac-1 [αm-integrin (CD11b)/β2-integrin (CD18)], CR4 [αx-integrin (CD11c)/CD18] and VLA-4 [α4-integrin (CD49d)/β1-integrin (CD29)] on treated monocytes. Treatment of peripheral blood mononuclear cells (PBMCs) and THP-1 monocytes with VLDL lipolysis products increased expression of TNFα, IL-1β, and IL-8 over controls, with concurrent activation of NFkB and AP-1. NFκB and AP-1-induced cytokine and integrin expression was dependent on ERK and Akt phosphorylation. Additionally, fatty acids from VLDL lipolysis products induced ERK2-dependent lipid droplet formation in monocytes, suggesting a link to inflammatory signaling pathways. These results provide novel mechanisms for postprandial monocyte activation by VLDL lipolysis products, suggesting new pathways and biomarkers for chronic, intermittent vascular injury.

Diabetes mellitus (DM) is caused by inadequate insulin secretion or an inability to respond appropriately to secreted insulin, which leads to chronic hyperglycemia. An estimated 171 million people worldwide have DM, and the prevalence of DM will more than double over the next 2 decades.1 Patients with DM have a 2- to 4-fold increased risk of coronary artery disease (CAD) over nondiabetic patients,2 and 75% of diabetic patients die as a result of a cardiovascular cause.3 Diabetic patients commonly undergo percutaneous revascularization procedures; 25% to 30% of all percutaneous coronary interventions (PCIs) are performed in patients with DM.4 A diagnosis of DM is also considered equivalent to having CAD because diabetic patients without a history of CAD have a 5-year cardiovascular mortality that is similar to that of nondiabetic patients who have a history of myocardial infarction (MI).5 According to current American College of Cardiology/American Heart Association guidelines, patients with DM are therefore treated as having a CAD equivalent.6,7 Previous review articles have summarized specific medical therapies for patients with DM.8,9 This review focuses on mechanisms of accelerated atherosclerosis, percutaneous and surgical revascularization strategies, and outcomes among patients with DM and CAD. Diabetic patients have increased rates, extent, and complexity of atherosclerotic CAD.8 After coronary revascularization, diabetics have an increased risk of target vessel failure (TVF) and need for repeat interventions.10 Altered inflammatory pathways stemming from the effects of hyperglycemia, insulin resistance, and altered free fatty acid metabolism predispose diabetic patients to endothelial dysfunction, thrombogenesis, monocyte activation, foam cell transformation, and altered smooth muscle cell migration.11–13 These mechanisms converge to create increased coronary artery plaque burden and more complex CAD (Figure 1A). Figure 1. Mechanisms of atherosclerosis and restenosis in diabetes mellitus. A , The combination …

Background The physiological actions of magnesium within the cardiac conduction system and myocytes have yet to be fully elucidated. Because concurrent hypocalcemia or hypokalemia were also present in previous human reports, specific electrocardiographic effects of isolated hypomagnesemia have not been clearly delineated. Objective We report a case in which dynamic electrocardiogram (ECG) changes were demonstrated in isolated hypomagnesemia. Case Report A 37-year-old man with history of heavy alcohol use was admitted for syncope. The ECG showed global T-wave inversions with prolonged corrected QT (QTc) duration on ECG. Extensive work-up including cardiac catheterization was unremarkable. His serum magnesium was noted to be low at 1.1 mg/dL, and his serum calcium and potassium were within normal limits. The patient received magnesium infusion with subsequent ECGs showing resolution of his global T-wave inversions and prolonged QTc. Conclusion This case is unique because it reports dynamic ECG changes in a patient with isolated hypomagnesemia. Although isolated hypomagnesemia is commonly believed to result in dysrhythmia, we were unaware of any previous cases of ECG abnormalities in humans. Clinically, we advise checking serum magnesium and correcting hypomagnesemia when prolonged QTc duration and global T-wave inversions are seen on ECG.

Elevation of blood triglycerides, primarily as triglyceride-rich lipoproteins (TGRL), has been linked to cerebrovascular inflammation, vascular dementia, and Alzheimer’s disease (AD). Brain microvascular endothelial cells and astrocytes, two cell components of the neurovascular unit, participate in controlling blood-brain barrier (BBB) permeability and regulating neurovascular unit homeostasis. Our studies showed that infusion of high physiological concentrations of TGRL lipolysis products (TGRL + lipoprotein lipase) activate and injure brain endothelial cells and transiently increase the BBB transfer coefficient ( Ki = permeability × surface area/volume) in vivo. However, little is known about how blood lipids affect astrocyte lipid accumulation and inflammation. To address this, we first demonstrated TGRL lipolysis products increased lipid droplet formation in cultured normal human astrocytes. We then evaluated the transcriptional pathways activated in astrocytes by TGRL lipolysis products and found upregulated stress and inflammatory-related genes including activating transcription factor 3 (ATF3), macrophage inflammatory protein-3α (MIP-3α), growth differentiation factor-15 (GDF15), and prostaglandin-endoperoxide synthase 2 (COX2). TGRL lipolysis products also activated the JNK/cJUN/ATF3 pathway, induced endoplasmic reticulum stress protein C/EBP homologous protein (CHOP), and the NF-κB pathway, while increasing secretion of MIP-3α, GDF15, and IL-8. Thus our results demonstrate TGRL lipolysis products increase the BBB transfer coefficient ( Ki), induce astrocyte lipid droplet formation, activate cell stress pathways, and induce secretion of inflammatory cytokines. Our observations are consistent with evidence for lipid-induced neurovascular injury and inflammation, and we, therefore, speculate that lipid-induced astrocyte injury could play a role in cognitive decline.

Wilson disease (WD), a genetic disorder affecting copper transport, is characterized by hepatic and neurological manifestations with variable and often unpredictable presentation. Global DNA methylation in liver was previously modified by dietary choline in tx-j mice, a spontaneous mutant model of WD. We therefore hypothesized that the WD phenotype and hepatic gene expression of tx-j offspring could be modified by maternal methyl supplementation during pregnancy. In an initial experiment, female tx-j mice or wild type mice were fed control or choline-supplemented diets 2weeks prior to mating through embryonic day 17. Transcriptomic analysis (RNA-seq) on embryonic livers revealed tx-j-specific differences in genes related to oxidative phosphorylation, mitochondrial dysfunction, and the neurological disorders Huntington's disease and Alzheimer disease. Maternal choline supplementation restored the transcript levels of a subset of genes to wild type levels. In a separate experiment, a group of tx-j offspring continued to receive choline-supplemented or control diets, with or without the copper chelator penicillamine (PCA) for 12weeks until 24weeks of age. Combined choline supplementation and PCA treatment of 24-week-old tx-j mice was associated with increased liver transcript levels of methionine metabolism and oxidative phosphorylation-related genes. Sex differences in gene expression within each treatment group were also observed. These results demonstrate that the transcriptional changes in oxidative phosphorylation and methionine metabolism genes in WD that originate during fetal life are, in part, prevented by prenatal maternal choline supplementation, a finding with potential relevance to preventive treatments of WD.

White Matter Disease is increasingly being recognized as an important cause of cognitive decline and dementia. Various investigations have linked chronic diet-related conditions to the development of white matter lesions, which appear as white matter hyperintensities on T2-weighted magnetic resonance imaging (MRI) scans of the brain. Thus, it can be postulated that the metabolic, inflammatory, and microvascular changes accompanying a western diet, hyperlipidemia, hypertension, and diabetes mellitus type II (DMII) are potential mediators in the development and progression of white matter disease, which in turn contributes to the development and progression of cognitive decline. This review will examine evidence for potential metabolic, inflammatory, and microvascular determinants of white matter disease and cognitive decline. Specifically, we will focus on the effects of altered insulin signaling in diabetes, obesity-induced oxidative stress, neuroinflammation, arterial stiffness due to hypertension, ischemia secondary to cerebral small vessel disease, and blood brain barrier disturbances.

Background: Saturated fatty acids (FAs) released from triglyceride-rich lipoproteins (TGRLs) activate Toll-like receptor 2 (TLR-2) and induce the expression of proinflammatory cytokines in monocytes. Certain plant polyphenols inhibit TLR-mediated signaling pathways. Objective: We determined whether plasma free FAs (FFAs) after a moderately high-fat (MHF, 40% kcal from fat) breakfast modulate the inflammatory status of postprandial blood, and whether blueberry intake suppresses FFA-induced inflammatory responses in healthy humans. Methods: Twenty-three volunteers with a mean ± SEM age and body mass index (in kg/m2) of 30 ± 3 y and 21.9 ± 0.4, respectively, consumed an MHF breakfast with either a placebo powder or 2 or 4 servings of blueberry powder in a randomized crossover design. The placebo powder was provided on the first test day and the blueberry powder doses were randomized with a 2-wk washout period. Plasma concentrations of lipids, glucose, and cytokines were determined. To determine whether FFAs derived from TGRL stimulate monocyte activation, and whether this is inhibited by blueberry intake, whole blood was treated with lipoprotein lipase (LPL). Results: The median concentrations of FFAs and cytokines [tumor necrosis factor-α, interleukin (IL)-6 and IL-8] in postprandial plasma (3.5 h) decreased compared with fasting plasma regardless of the blueberry intake (P < 0.001 for FFAs and P < 0.05 for cytokines). However, concentrations of FFAs and cytokines including IL-1β increased in LPL-treated whole blood compared with untreated blood samples from participants who consumed the placebo powder. Blueberry intake suppressed IL-1β and IL-6 production in LPL-treated postprandial blood compared with the placebo control when fasting changes were used as a covariate. Conclusions: The plasma FFA concentration may be an important determinant affecting inflammatory cytokine production in blood. Supplementation with blueberry powder did not affect plasma FFA and cytokine concentrations; however, it attenuated the cytokine production induced by ex vivo treatment of whole blood with LPL. This trial was registered at clinicaltrials.gov as {"type":"clinical-trial","attrs":{"text":"NCT01594008","term_id":"NCT01594008"}}NCT01594008.

Dysfunction of the cerebrovasculature plays an important role in vascular cognitive impairment (VCI). Lipotoxic injury of the systemic endothelium in response to hydrolyzed triglyceride-rich lipoproteins (TGRLs; TGRL lipolysis products) or a high-fat Western diet (WD) suggests similar mechanisms may be present in brain microvascular endothelium. We investigated the hypothesis that TGRL lipolysis products cause lipotoxic injury to brain microvascular endothelium by generating increased mitochondrial superoxide radical generation, upregulation of activating transcription factor 3 (ATF3)-dependent inflammatory pathways, and activation of cellular oxidative stress and apoptotic pathways. Human brain microvascular endothelial cells were treated with human TGRL lipolysis products that induced intracellular lipid droplet formation, mitochondrial superoxide generation, ATF3-dependent transcription of proinflammatory, stress response, and oxidative stress genes, as well as activation of proapoptotic cascades. Male apoE knockout mice were fed a high-fat/high-cholesterol WD for 2 months, and brain microvessels were isolated by laser capture microdissection. ATF3 gene transcription was elevated 8-fold in the hippocampus and cerebellar brain region of the WD-fed animals compared with chow-fed control animals. The microvascular injury phenotypes observed in vitro and in vivo were similar. ATF3 plays an important role in mediating brain microvascular responses to acute and chronic lipotoxic injury and may be an important preventative and therapeutic target for endothelial dysfunction in VCI.

BackgroundPrevious studies indicated that nonpurified and purified commercially available control murine diets have different metabolic effects with potential consequences on hepatic methionine metabolism and liver histology.MethodsWe compared the metabolic and histological effects of commercial nonpurified (13% calories from fat; 57% calories from carbohydrates with 38 grams/kg of sucrose) and purified control diets (12% calories from fat; 69% calories from carbohydrates with ∼500 grams/kg of sucrose) with or without choline supplementation administered to C3H mice with normal lipid and methionine metabolism. Diets were started 2 weeks before mating, continued through pregnancy and lactation, and continued in offspring until 24 weeks of age when we collected plasma and liver tissue to study methionine and lipid metabolism.ResultsCompared to mice fed nonpurified diets, the liver/body weight ratio was significantly higher in mice fed either purified diet, which was associated with hepatic steatosis and inflammation. Plasma alanine aminotransferase levels were higher in mice receiving the purified diets. The hepatic S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio was higher in female mice fed purified compared to nonpurified diet (4.6 ± 2 vs. 2.8 ± 1.9; P

As the population of people living with HIV ages, atherosclerotic cardiovascular disease (ASCVD) has become an increasing cause of morbidity and mortality. Traditional cardiovascular risk factors are common among those with HIV. In addition, some antiretroviral therapy (ART) regimens contribute to conditions such as hyperlipidemia and insulin resistance. However, inflammation is increasingly recognized as a key contributor to ASCVD. HIV infection induces immune activation and inflammation through several mechanisms. Co-infections such as hepatitis C and cytomegalovirus along with HIV itself likely initiate immune activation and inflammation. Translocation of bacterial products across a compromised epithelial barrier as a result of HIV infection is another mechanism by which the immune system is activated. In this article we summarize the current understanding of drivers of immune activation and inflammation among those with HIV and the contribution of each to ASCVD.

Background The relationship between gut microbial community composition at the higher-taxonomic order level and local and systemic immunologic abnormalities in HIV disease may provide insight into how bacterial translocation impacts HIV disease. Methods Antiretroviral-naive patients with HIV underwent upper endoscopy before and 9 months after starting antiretroviral treatment. Duodenal tissue was paraffin-embedded for immunohistochemical analysis and digested for fluorescence activated cell sorting for T-cell subsets and immune activation (CD38+/HLA-DR+) enumeration. Stool samples were provided from patients and control subjects for comparison. Metagenomic microbial DNA was extracted from feces for optimized 16S ribosomal RNA gene (rDNA) real-time quantitative polymerase chain reaction assays designed to quantify panbacterial loads and the relative abundances of proinflammatory Enterobacteriales order and the dominant Bacteroidales and Clostridiales orders. Results Samples from 10 HIV subjects before initiating and from six subjects receiving antiretroviral treatment were available for analysis. There was a trend for a greater proportion of Enterobacteriales in HIV-positive subjects compared with control subjects (P = 0.099). There were significant negative correlations between total bacterial load and duodenal CD4 and CD8 T-cell activation levels (r = -0.74, P = 0.004 and r = -0.67, P = 0.013, respectively). The proportions of Enterobacteriales and Bacteroidales were significantly correlated with duodenal CD4 T-cell depletion and peripheral CD8 T-cell activation, respectively. Conclusions These data represent the first report of quantitative molecular and cellular correlations between total/universal and order-level gut bacterial populations and gastrointestinal-associated lymphoid tissue levels of immune activation in HIV-infected subjects. The correlations between lower overall 16S rDNA levels and tissue immune activation suggest that the gut microbiome may contribute to immune activation and influence HIV progression.

Epidemiologic studies associate exposure to ambient particulate matter (APM) with increased cardiovascular mortality. Since both pulmonary inflammation and systemic circulation of ultrafine particles are hypothesized as initiating cardiovascular effects, we examined responses of potential target cells in vitro. Human aortic endothelial cells (HAEC) were exposed to 10 μg/ml fine and ultrafine APM collected in an urban setting in summer 2006 or winter 2007 in the San Joaquin Valley, California. RNA isolated after 3 h was analyzed with high-density oligonucleotide arrays. Summer APM treatment affected genes involved in xenobiotic and oxidoreductase activity, transcription factors, and inflammatory responses in HAEC, while winter APM had a robust xenobiotic but lesser inflammatory response. Real-time polymerase chain reaction analysis confirmed that particulate matter (PM)-treated HAEC increased mRNA levels of xenobiotic response enzymes CYP1A1, ALDH1A3, and TIPARP and cellular stress response transcription factor ATF3. Inflammatory response genes included E-selectin, PTGS2, CXCL-2 (MIP-2α), and CCL-2 (MCP-1). Multiplex protein assays showed secretion of IL-6 and MCP-1 by HAEC. Since induction of CYP1A1 is mediated through the ligand-activated aryl hydrocarbon receptor (AhR), we demonstrated APM induced AhR nuclear translocation by immunofluorescence and Western blotting and activation of the AhR response element using a luciferase reporter construct. Inhibitor studies suggest differential influences of polycyclic aromatic hydrocarbon signaling, ROS-mediated responses and endotoxin alter stress and proinflammatory endothelial cell responses. Our findings demonstrate gene responses correlated with current concepts that systemic inflammation drives cardiovascular effects of particulate air pollution. We also demonstrate a unique pattern of gene responses related to xenobiotic metabolism in PM-exposed HAEC.

Psoriasis and atherosclerosis are diseases in which effector T lymphocytes such as Helper T cells type 1 (Th1) and 17 (Th17) play integral roles in disease pathogenesis and progression. Regulatory T cells (Treg) also exert clinically important anti-inflammatory effects that are pathologically altered in psoriasis and atherosclerosis. We review the immunological pathways involving Th1, Th17 and Treg cells that are common to psoriasis and atherosclerosis. These shared pathways provide the basis for mechanisms that may explain the epidemiologic observation that patients with psoriasis have an increased risk of heart disease. Improved understanding of these pathways will guide future experiments and may lead to the development of therapeutics that prevent or treat cardiovascular complications in patients with psoriasis.

Coronary disease risk increases inversely with high-density lipoprotein (HDL) level. The measurement of the biodistribution and clearance of HDL in vivo, however, has posed a technical challenge. This study presents an approach to the development of a lipoprotein MRI agent by linking gadolinium methanethiosulfonate (Gd[MTS-ADO3A]) to a selective cysteine mutation in position 55 of apo AI, the major protein of HDL. The contrast agent targets both liver and kidney, the sites of HDL catabolism, whereas the standard MRI contrast agent, gadolinium-diethylenetriaminepentaacetic acid-bismethylamide (GdDTPA-BMA, gadodiamide), enhances only the kidney image. Using a modified apolipoprotein AI to create an HDL contrast agent provides a new approach to investigate HDL biodistribution, metabolism and regulation in vivo.

Necrotizing enterocolitis (NEC) is the most common intestinal emergency among premature infants. Risk factors in premature infants include immature intestinal immunity and an intestinal microbiota dominated by hospital-acquired bacteria. Some probiotics have been shown to decrease the incidence of NEC in premature infants. Among term infants, NEC is rare. However, among term infants with cyanotic congenital heart disease (CCHD), the incidence of NEC is similar to that of premature infants but with even greater mortality rates. Mechanisms by which NEC occurs in term infants with CCHD are unknown. Of central interest is the potential role of changes in the intestinal microbiota and whether these can be modified with probiotic bacteria; accordingly, we review the literature, propose hypotheses and present the rationale for future studies involving preliminary probiotic clinical trials.

Maternal mainstream tobacco smoking is known to have adverse outcomes on fetal respiratory function; however, no data is currently available on the effects of passive exposure to tobacco smoking and environmental tobacco smoke (ETS) on fetal systemic arterial structure and function. Eight pregnant rhesus macaque monkeys were studied at the California Regional Primate Research Center breeding colony. The estimated gestational age for each dam was established by sonography performed before gestational day 40. Two inhalation chambers were used, each with an air capacity of 3.5 m(3), and each housed two dams. Aged and diluted sidestream smoke was used as a surrogate for ETS. Exposure to ETS (1 mg/m(3)) occurred for 6 h/day, 5 days/week, beginning on gestational day 100. All dams were allowed to give birth spontaneously and then ETS exposure continued 70-80 days postnatally with the chamber containing both the mother and infant. Carotid arteries from four control (C) and four ETS-treated newborns were analyzed for mRNA by gene macroarray and for protein by Western blotting. A total of 588 cardiovascular genes were studied. Four genes were upregulated by ETS compared to C, and nine genes were downregulated (≥2-fold change). Three genes were selected for further study. Following ETS exposure, neonatal carotid arteries of non-human primates manifested evidence of inflammation with increased gene and protein expression of LFA-1 and RANTES, proteins that are recognized to be important in vascular adhesion and inflammation, and downregulation of expression for the receptor for VEGF, which has a key role in angiogenesis. Prenatal and postnatal exposure to ETS increases expression of pro-inflammatory genes and may be responsible for early arterial vascular remodeling that is predisposing to a subsequent vascular disease.

Our previous work indicated that apolipoprotein (apo) E4 assumes a more expanded conformation in the postprandial period. The postprandial state is characterized by increased VLDL lipolysis. In this article, we tested the hypothesis that VLDL lipolysis products increase VLDL particle fluidity, which mediates expansion of apoE4 on the VLDL particle. Plasma from healthy subjects was collected before and after a moderately high-fat meal and incubated with nitroxyl-spin labeled apoE. ApoE conformation was examined by electron paramagnetic resonance spectroscopy using targeted spin probes on cysteines introduced in the N-terminal (S76C) and C-terminal (A241C) domains. Further, we synthesized a novel nitroxyl spin-labeled cholesterol analog, which gave insight into lipoprotein particle fluidity. Our data revealed that the order of lipoprotein fluidity was HDL approximately LDL

We describe a 47-year-old African American woman affected by a rapidly progressing thromboangiitis associated with high serum levels of lipoprotein(a) (Lp(a)).

The incidence of obesity and obesity-related conditions, such as metabolic syndrome and insulin resistance, is on the increase. The effect of obesity on skeletal muscle function, especially the regulation of muscle mass, is poorly understood. In this study we investigated the effect of diet-induced obesity on the ability of skeletal muscle to respond to an imposed growth stimulus, such as increased load. Male C57BL/6 mice were randomized into two diet groups: a low fat, high carbohydrate diet (LFD) and a high fat, low carbohydrate diet (HFD) fed ad libitum for 14 weeks. Mice from each diet group were divided into two treatment groups: sedentary control or bilateral functional overload (FO) of the plantaris muscle. Mice were evaluated at 3, 7, 14 or 30 days following FO. By 14 days of FO, there was a 10% reduction (P < 0.05) in absolute growth of the plantaris in response to overload in HFD mice vs. LFD mice. By 30 days the attenuation in growth increased to 16% in HFD mice compared to LFD mice. Following FO, there was a reduction in the formation of polysomes in the HFD mice relative to the LFD mice, suggesting a decrease in protein translation. Further, activation of Akt and S6K1, in response to increased mechanical loading, was significantly attenuated in the HFD mice relative to the LFD mice. In conclusion, chronic high fat feeding impairs the ability of skeletal muscle to hypertrophy in response to increased mechanical load. This failure coincided with a failure to activate key members of the Akt/mTOR signalling pathway and increase protein translation.

Macrophages are activated by IFN-γ, a proinflammatory and proatherogenic cytokine that mediates its downstream effects primarily through STAT1. IFN-γ signaling induces phosphorylation of two STAT1 residues: Tyr701 (Y701), which facilitates dimerization, nuclear translocation, and DNA binding; and Ser727 (S727), which enables maximal STAT1 transcription activity. Immunosuppressive molecules such as adenosine in the cellular microenvironment can reduce macrophage inflammatory and atherogenic functions through receptor-mediated signaling pathways. We hypothesized that adenosine achieves these protective effects by interrupting IFN-γ signaling in activated macrophages. This investigation demonstrates that adding adenosine to IFN-γ-stimulated murine RAW 264.7 and human THP-1 macrophages results in unique modulation of STAT1 serine and tyrosine phosphorylation events. We show that adenosine inhibits IFN-γ-induced STAT1 S727 phosphorylation by >30% and phosphoserine-mediated transcriptional activity by 58% but has no effect on phosphorylation of Y701 or receptor-associated JAK tyrosine kinases. Inhibition of the adenosine A3 receptor with a subtype-specific antagonist (MRS 1191 in RAW 264.7 cells and MRS 1220 in THP-1 cells) reverses this adenosine suppressive effect on STAT1 phosphoserine status by 25–50%. Further, RAW 264.7 A3 receptor stimulation with Cl-IB-MECA reduces IFN-γ-induced STAT1 transcriptional activity by 45% and STAT1-dependent gene expression by up to 80%. These data suggest that A3 receptor signaling is key to adenosine-mediated STAT1 modulation and anti-inflammatory action in IFN-γ-activated mouse and human macrophages. Because STAT1 plays a key role in IFN-γ-induced inflammation and foam cell transformation, a better understanding of the mechanisms underlying STAT1 deactivation by adenosine may improve preventative and therapeutic approaches to vascular disease.

The ‘response to retention’ hypothesis of atherogenesis proposes that proteoglycans bind and retain low-density lipoproteins (LDL) in the vessel wall. Platelet-derived growth factor (PDGF) is strongly implicated in atherosclerosis and stimulates proteoglycan synthesis. Here we investigated the action of the PDGF receptor inhibitor imatinib on PDGF-mediated proteoglycan biosynthesis in vitro, lipid deposition in the aortic wall in vivo and the carotid artery ex vivo. In human vSMCs, imatinib inhibited PDGF mediated 35S-SO4 incorporation into proteoglycans by 31% (P < 0.01) and inhibited PDGF-mediated size increases in both chemically cleaved and xyloside associated glycosaminoglycan (GAG) chains by 19%, P < 0.05 and 27%, P < 0.05, respectively. Imatinib decreased PDGF stimulation of the 6:4 position sulphation ratio of disaccharides. The half maximal saturation value for LDL binding for proteoglycans from PDGF stimulated cells in the presence of imatinib was approximately 2.5-fold higher than for PDGF treatment alone. In high fat fed ApoE−/– mice, imatinib reduced total lipid staining area by ∼31% (P < 0.05). Carotid artery lipid accumulation in imatinib treated mice was also reduced. Furthermore, we demonstrate that imatinib inhibits phosphorylation of tyrosine 857, the autophosphorylation site of the PDGF receptor, in vSMCs. Thus imatinib inhibits GAG synthesis on vascular proteoglycans and reduces LDL binding in vitro and in vivo and this effect is mediated via the PDGF receptor. These findings validate a novel mechanism to prevent cardiac disease.