Short-Term, High-Dose Fish Oil Supplementation Increases the Production of Omega-3 Fatty Acid-Derived Mediators in Patients With Peripheral Artery Disease (the OMEGA-PAD I Trial).

BACKGROUND: Patients with peripheral artery disease (PAD) experience significant morbidity and mortality. The OMEGA-PAD I Trial, a randomized, double-blinded, placebo-controlled trial, addressed the hypothesis that short-duration, high-dose n-3 polyunsaturated fatty acids (n-3 PUFA) oral supplementation improves endothelial function and inflammation in PAD. METHODS AND RESULTS: Eighty patients with stable claudication received 4.4 g of fish oil or placebo for 1 month. The primary end point was endothelial function as measured by brachial artery flow-mediated vasodilation. Secondary end points included biomarkers of inflammation, n-3 polyunsaturated fatty acids metabolome changes, lipid profile, and walking impairment questionnaires. Although there was a significant increase in FMD in the fish oil group following treatment (0.7±1.8% increase from baseline, P=0.04), this response was not different then the placebo group (0.6±2.5% increase from baseline, P=0.18; between-group P=0.86) leading to a negative finding for the primary endpoint. There was, however, a significant reduction in triglycerides (fish oil: -34±46 mg/dL, P<0.001; placebo -10±43 mg/dL, P=0.20; between-group differential P-value: 0.02), and an increase in the omega-3 index of 4±1% (P<0.001) in the fish oil group (placebo 0.1±0.9%, P=0.49; between-group P<0.0001). We observed a significant increase in the production of pathway markers of specialized pro-resolving mediators generated from n-3 polyunsaturated fatty acids in the fish oil group. CONCLUSIONS: High-dose, short-duration fish oil supplementation did not lead to a different response in the primary end point of endothelial function between the treatment and placebo group, but improved serum triglycerides and increased the production of downstream n-3 polyunsaturated fatty acids-derived products and mediators in patients with PAD. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov/. Unique identifier: NCT01310270.

The Rel/NF-κB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity.

This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in µg. Immunosuppression during spaceflight is a major barrier to safe, long-term human space habitation and travel. The goals of these experiments were to prove that µg was the cause of impaired T cell activation during spaceflight, as well as understand the mechanisms controlling early T cell activation. T cells from four human donors were stimulated with Con A and anti-CD28 on board the ISS. An on-board centrifuge was used to generate a 1g simultaneous control to isolate the effects of µg from other variables of spaceflight. Microarray expression analysis after 1.5 h of activation demonstrated that µg- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly, differentially down-regulated in µg. Importantly, several key immediate early genes were inhibited in µg. In particular, transactivation of Rel/NF-κB, CREB, and SRF gene targets were down-regulated. Expression of cREL gene targets were significantly inhibited, and transcription of cREL itself was reduced significantly in µg and upon anti-CD3/anti-CD28 stimulation in simulated µg. Analysis of gene connectivity indicated that the TNF pathway is a major early downstream effector pathway inhibited in µg and may lead to ineffective proinflammatory host defenses against infectious pathogens during spaceflight. Results from these experiments indicate that µg was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes.

Effects of fatty acids on endothelial cells: inflammation and monocyte adhesion.

BACKGROUND: Diet is known to have an important impact on cardiovascular health. n-3 Fatty acids (FAs), found in high quantity in fish oil, have demonstrated beneficial effects in patients with coronary artery disease. The role of n-6 FAs remains more controversial. The objective of this study was to examine the effect of arachidonic acid (AA), an n-6 FA, and eicosapentanoic acid (EPA), an n-3 FA, on the interaction between monocytes and endothelial cells (ECs). DESIGN: We used a cellular model of ECs (EA.hy.926) and monocytes (human leukemic myelomonocytic U937). Confluent ECs were treated with AA or EPA, in the presence of tumor necrosis factor-alpha (TNF-α) or vehicle alone for either 4 or 24h. Adhesion of monocytes to the endothelial monolayer was performed. For gene expression, reverse transcription, followed by real-time quantitative polymerase chain reaction, was performed. RESULTS: There was a significant increase in adhesion of monocytes to the endothelial monolayer in the presence of n-6 FAs, both in the presence and in the absence of TNF-α at 4 and 24h. The adhesion of monocytes to the endothelial monolayer was decreased with n-3 FAs at 24h. Intercellular adhesion molecule 1, vascular cell adhesion molecule 1, E-Selectin, Interleukin 6, and TNF-α were significantly increased in ECs treated with n-6 FAs. CONCLUSIONS: We conclude that AA increases inflammation and enhances the ability of ECs to bind monocytes in vitro. EPA leads to a decrease in the ability of EA.hy.926 to bind monocytes, although the effect appears more modest. Taken together, these data indicate that the n-6 FA AA could potentiate inflammation and early events of atherosclerosis.

Gene expression and biological pathways in tissue of men with prostate cancer in a randomized clinical trial of lycopene and fish oil supplementation.

BACKGROUND: Studies suggest that micronutrients may modify the risk or delay progression of prostate cancer; however, the molecular mechanisms involved are poorly understood. We examined the effects of lycopene and fish oil on prostate gene expression in a double-blind placebo-controlled randomized clinical trial. METHODS: Eighty-four men with low risk prostate cancer were stratified based on self-reported dietary consumption of fish and tomatoes and then randomly assigned to a 3-month intervention of lycopene (n = 29) or fish oil (n = 27) supplementation or placebo (n = 28). Gene expression in morphologically normal prostate tissue was studied at baseline and at 3 months via cDNA microarray analysis. Differential gene expression and pathway analyses were performed to identify genes and pathways modulated by these micronutrients. RESULTS: Global gene expression analysis revealed no significant individual genes that were associated with high intake of fish or tomato at baseline or after 3 months of supplementation with lycopene or fish oil. However, exploratory pathway analyses of rank-ordered genes (based on p-values not corrected for multiple comparisons) revealed the modulation of androgen and estrogen metabolism in men who routinely consumed more fish (p = 0.029) and tomato (p = 0.008) compared to men who ate less. In addition, modulation of arachidonic acid metabolism (p = 0.01) was observed after 3 months of fish oil supplementation compared with the placebo group; and modulation of nuclear factor (erythroid derived-2) factor 2 or Nrf2-mediated oxidative stress response for either supplement versus placebo (fish oil: p = 0.01, lycopene: p = 0.001). CONCLUSIONS: We did not detect significant individual genes associated with dietary intake and supplementation of lycopene and fish oil. However, exploratory analyses revealed candidate in vivo pathways that may be modulated by these micronutrients. TRIAL REGISTRATION: ClinicalTrials.gov NCT00402285.

The role of FGF-2 and BMP-2 in regulation of gene induction, cell proliferation and mineralization.

INTRODUCTION: The difficulty in re-growing and mineralizing new bone after severe fracture can result in loss of ambulation or limb. Here we describe the sequential roles of FGF-2 in inducing gene expression, cell growth and BMP-2 in gene expression and mineralization of bone. MATERIALS AND METHODS: The regulation of gene expression was determined using real-time RTPCR (qRTPCR) and cell proliferation was measured by thymidine incorporation or fluorescent analysis of DNA content in MC3T3E1 osteoblast-like cells. Photomicroscopy was used to identify newly mineralized tissue and fluorescence was used to quantify mineralization. RESULTS: Fibroblast growth factor-2 (FGF-2) had the greatest ability to induce proliferation after 24 hours of treatment when compared to transforming growth factor beta (TGFß, insulin-like growth factor-1 (IGF-1), bone morphogenic protein (BMP-2), platelet derived growth factor (PDGF) or prostaglandin E2 (PGE2). We found that FGF-2 caused the most significant induction of expression of early growth response-1 (egr-1), fgf-2, cyclo-oxygenase-2 (cox-2), tgfß and matrix metalloproteinase-3 (mmp-3) associated with proliferation and expression of angiogenic genes like vascular endothelial growth factor A (vegfA) and its receptor vegfr1. We found that FGF-2 significantly reduced gene expression associated with mineralization, e.g. collagen type-1 (col1a1), fibronectin (fn), osteocalcin (oc), IGF-1, noggin, bone morphogenic protein (bmp-2) and alkaline phosphatase (alp). In contrast, BMP-2 significantly stimulated expression of the mineralization associated genes but had little or no effect on gene expression associated with growth. CONCLUSIONS: The ability of FGF-2 to re-program a mineralizing gene expression profile to one of proliferation suggests that FGF-2 plays a critical role of osteoblast growth in early fracture repair while BMP-2 is instrumental in stimulating mineralization.

Nutritional supplements, COX-2 and IGF-1 expression in men on active surveillance for prostate cancer.

BACKGROUND: Nutritional factors are associated with reduced risk of prostate cancer progression, yet mechanisms remain unclear. We examined the effects of lycopene and fish oil supplements versus placebo on the normal prostate microenvironment, among men pursuing active surveillance for low-burden prostate cancer. We hypothesized that lycopene or fish oil supplements would down-regulate insulin-like growth factor-1 (IGF-1) and cyclooxygenase 2 (COX-2) gene expression, respectively, reflecting putative proliferation (IGF-1) and inflammatory (COX-2) pathways relevant to carcinogenesis. METHODS: We conducted a 3-month randomized, double-blinded, clinical trial comparing prostate tissue gene expression profiles (assessed by qRT-PCR) among men with favorable-risk prostate cancer receiving either 30 mg/day lycopene, 3 g/day fish oil (including 1,098 mg eicosapentaenoic and 549 mg docosahexaenoic fatty acids) or placebo. RESULTS: Among 69 men (22 assigned to lycopene, 21 to fish, and 26 to placebo), there was no difference in the change from baseline to the 3 months in IGF-1 expression level between the placebo and lycopene arms (p = 0.93) nor in COX-2 expression between the placebo and fish arms (p = 0.99). CONCLUSION: Compared to placebo, 3-month intervention with lycopene or fish oil did not significantly change IGF-1 and COX-2 gene expression in the normal prostate microenvironment in men with low-burden prostate cancer. Further analysis of global gene expression profiles may shed light on the bioactivity and relevance of these nutrients in prostate cancer.

Monolayer and spheroid culture of human liver hepatocellular carcinoma cell line cells demonstrate distinct global gene expression patterns and functional phenotypes.

Understanding cell biology of three-dimensional (3D) biological structures is important for more complete appreciation of in vivo tissue function and advancing ex vivo organ engineering efforts. To elucidate how 3D structure may affect hepatocyte cellular responses, we compared global gene expression of human liver hepatocellular carcinoma cell line (HepG2) cells cultured as monolayers on tissue culture dishes (TCDs) or as spheroids within rotating wall vessel (RWV) bioreactors. HepG2 cells grown in RWVs form spheroids up to 100 mum in diameter within 72 h and up to 1 mm with long-term culture. The actin cytoskeleton in monolayer cells show stress fiber formation while spheroids have cortical actin organization. Global gene expression analysis demonstrates upregulation of structural genes such as extracellular matrix, cytoskeletal, and adhesion molecules in monolayers, whereas RWV spheroids show upregulation of metabolic and synthetic genes, suggesting functional differences. Indeed, liver-specific functions of cytochrome P450 activity and albumin production are higher in the spheroids. Enhanced liver functions require maintenance of 3D structure and environment, because transfer of spheroids to a TCD results in spheroid disintegration and subsequent loss of function. These findings illustrate the importance of physical environment on cellular organization and its effects on hepatocyte processes.

Arachidonic acid activates phosphatidylinositol 3-kinase signaling and induces gene expression in prostate cancer.

Essential fatty acids are not only energy-rich molecules; they are also an important component of the membrane bilayer and recently have been implicated in induction of fatty acid synthase and other genes. Using gene chip analysis, we have found that arachidonic acid, an omega-6 fatty acid, induced 11 genes that are regulated by nuclear factor-kappaB (NF-kappaB). We verified gene induction by omega-6 fatty acid, including COX-2, IkappaBalpha, NF-kappaB, GM-CSF, IL-1beta, CXCL-1, TNF-alpha, IL-6, LTA, IL-8, PPARgamma, and ICAM-1, using quantitative reverse transcription-PCR. Prostaglandin E(2) (PGE(2)) synthesis was increased within 5 minutes of addition of arachidonic acid. Analysis of upstream signal transduction showed that within 5 minutes of fatty acid addition, phosphatidylinositol 3-kinase (PI3K) was significantly activated followed by activation of Akt at 30 minutes. Extracellular signal-regulated kinase 1 and 2, p38 and stress-activated protein kinase/c-Jun-NH(2)-kinase were not phosphorylated after omega-6 fatty acid addition. Thirty minutes after fatty acid addition, we found a significant 3-fold increase in translocation of NF-kappaB transcription factor to the nucleus. Addition of a nonsteroidal anti-inflammatory drug (NSAID) caused a decrease in COX-2 protein synthesis, PGE(2) synthesis, as well as inhibition of PI3K activation. We have previously shown that NSAIDs cause an inhibition of arachidonic acid-induced proliferation; here, we have shown that arachidonic acid-induced proliferation is also blocked (P < 0.001) by PI3K inhibitor LY294002. LY294002 also significantly inhibited the arachidonic acid-induced gene expression of COX-2, IL-1beta, GM-CSF, and ICAM1. Taken together, the data suggest that arachidonic acid via conversion to PGE(2) plays an important role in stimulation of growth-related genes and proliferation via PI3K signaling and NF-kappaB translocation to the nucleus.

Early immune response and regulation of IL-2 receptor subunits.

Affymetrix oligonucleotide arrays were used to monitor expression of 8796 genes and probe sets in activated T-cells; analysis revealed that 217 genes were significantly upregulated within 4 h. Induced genes included transcription factors, cytokines and their receptor genes. Analysis by semi-quantitative RT-PCR confirmed the significant induction of IL-2, IL-2R(gamma) and IL-2R(alpha). Forty-eight of the 217 induced genes are known to or predicted to be regulated by a CRE promoter/enhancer. We found that T-cell activation caused a significant increase in CREB phosphorylation furthermore, inhibition of the PKC pathway by GF109203 reduced CREB activation by 50% and inhibition of the PKA pathway caused a total block of CREB phosphorylation and significantly reduced IFN(gamma), IL-2 and IL-2R(alpha) gene expression by approximately 40% (p<0.001). PKC(theta) plays a major role in T-cell activation: inhibition of PKC significantly reduced the expression of IFN(gamma), IL-2 and IL-2R(alpha). Since PKC blocked activation of CREB, we studied potential cross-talk between the PKC and the PKA/MAPK pathways, PMA-stimulated Jurkat cells were studied with specific signal pathway inhibitors. Extracellular signal-regulated kinase-2 (ERK2) pathway was found to be significantly activated greater than seven-fold within 30 min; however, there was little activation of ERK-1 and no activation of JNK or p38 MAPK. Inhibition of the PKA pathway, but not the PKC pathway, resulted in inhibition of ERK1/2 activation at all time points, inhibition of MEK1 and 2 significantly blocked expression of IL-2 and IL-2R(alpha). Gene expression of IL-2R(alpha) and IFN(gamma) was dependent on PKA in S49 wt cells but not in kin- mutants. Using gel shift analysis, we found that forskolin activation of T-cells resulted in activation of AP1 sites; this increase in nuclear extract AP1 was significantly blocked by MEK1 inhibitor U0126. Taken together, these results suggest that the PKA in addition to PKC and MAPK pathways plays a role in early T-cell activation and induction of IL-2, IL-2R(alpha) and IFN(gamma) gene expression.

Arachidonic acid, an omega-6 fatty acid, induces cytoplasmic phospholipase A2 in prostate carcinoma cells.

For the past 60 years, dietary intake of essential fatty acids has increased. Moreover, the omega-6 fatty acids have recently been found to play an important role in regulation of gene expression. Proliferation of human prostate cells was significantly increased 48 h after arachidonic acid (AA) addition. We have analyzed initial uptake using nile red fluorescence and we found that the albumin conjugated AA is endocytosed into the cells followed by the induction of RNA within minutes, protein and PGE2 synthesis within hours. Here we describe that AA induces expression of cytosolic phospholipase A2 (cPLA2) in a dose-dependent manner and that this upregulation is dependent upon downstream synthesis of PGE2. The upregulation of cox-2 and cPLA2 was inhibited by flurbiprofen, a cyclooxygenase (COX) inhibitor, making this a second feed-forward enzyme in the eicosanoid pathway. Cox-2 specific inhibitors are known to inhibit colon and prostate cancer growth in humans; however, recent findings show that some of these have cardiovascular complications. Since cPLA2 is upstream in the eicosanoid pathway, it may be a good alternative for a pharmaceutical target for the treatment of cancer.