Effects of Cigarette Smoke and Opium on the Expression of CD9, CD36, and CD68 at mRNA and Protein Levels in Human Macrophage Cell Line THP-1.

Cigarette smoking and opium use are risk factors for coronary artery disease (CAD). It has been known that scavenger receptors such as CD36 and CD68 play critical roles in the pathogenesis of CAD. CD9, as a member of the tetraspanin, has been shown to interact with scavenger receptors. The aim of this study was to investigate the effects of these risk factors on expression levels of CD9, CD36, and CD68 on the THP-1 cell line. The THP-1 cell line treated with cigarette smoke extract (CSE( and opium, both individually and combinatory, in 24 h incubation. The protein and mRNA levels of CD9, CD36, and CD68 were evaluated by flow cytometry and quantitative reverse transcription-Polymerase Chain Reaction (qRT-PCR) techniques, respectively. CD36 and CD68 mRNA and protein expression levels were significantly increased in the cells treated with cigarette smoke extract compared to the control (p<0.001 in mRNA expression levels and p=0.016 and p=0.012 in protein expression levels, respectively). The CSE increased the level of CD9 protein expression compared to the control group (p=0.041) on the human macrophage cell line THP-1. No significant differences were observed in the CD9, CD36, and CD68 gene expression and at the protein levels between opium-treated THP-1 cells and controls. In conclusion, cigarettes by increasing the levels of CD36, CD68, and CD9 can be a risk factor in the development of many inflammatory diseases, including cardiovascular diseases, chronic obstructive pulmonary disease (COPD) and lung carcinoma.

Expression of neural markers of gustatory signaling are differentially altered by continuous and intermittent feeding patterns.

Food intake patterns are regulated by signals from the gustatory neural circuit, a complex neural network that begins at the tongue and continues to homeostatic and hedonic brain regions involved in eating behavior. The goal of the current study was to investigate the short-term effects of continuous access to a high fat diet (HFD) versus limited access to dietary fat on the gustatory neural circuit. Male Sprague-Dawley rats were fed a chow diet, a HFD (56% kcal from fat), or provided limited, daily (2 h/day) or limited, intermittent (2 h/day, 3 times/week) access to vegetable shortening for 2 weeks. Real time PCR was used to determine mRNA expression of markers of fat sensing/signaling (e.g. CD36) on the circumvallate papillae, markers of homeostatic eating in the mediobasal hypothalamus (MBH) and markers of hedonic eating in the nucleus accumbens (NAc). Continuous HFD increased mRNA levels of lingual CD36 and serotonin signaling, altered markers of homeostatic and hedonic eating. Limited, intermittent access to dietary fat selectively altered the expression of genes associated with the regulation of dopamine signaling. Overall, these data suggest that short-term, continuous access to HFD leads to altered fat taste and decreased expression of markers of homeostatic and hedonic eating. Limited, intermittent access, or binge-like, consumption of dietary fat led to an overall increase in markers of hedonic eating, without altering expression of lingual fat sensors or homeostatic eating. These data suggest that there are differential effects of meal patterns on gustatory neurocircuitry which may regulate the overconsumption of fat and lead to obesity.

Three-dimensional spheroid cell model of in vitro adipocyte inflammation.

To improve treatment of obesity, a contributing factor to multiple systemic and metabolic diseases, a better understanding of metabolic state and environmental stress at the cellular level is essential. This work presents development of a three-dimensional (3D) in vitro model of adipose tissue displaying induced lipid accumulation as a function of fatty acid supplementation that, subsequently, investigates cellular responses to a pro-inflammatory stimulus, thereby recapitulating key stages of obesity progression. Three-dimensional spheroid organization of adipose cells was induced by culturing 3T3-L1 mouse preadipocytes on an elastin-like polypeptide-polyethyleneimine (ELP-PEI)-coated surface. Results indicate a more differentiated phenotype in 3D spheroid cultures relative to two-dimensional (2D) monolayer analogues based on triglyceride accumulation, CD36 and CD40 protein expression, and peroxisome proliferator-activated receptor-γ (PPAR-γ) and adiponectin mRNA expression. The 3T3-L1 adipocyte spheroid model was then used to test the effects of a pro-inflammatory microenvironment, namely maturation in the presence of elevated fatty acid levels followed by acute exposure to tumor necrosis factor alpha (TNF-α). Under these conditions, we demonstrate that metabolic function was reduced across all cultures exposed to TNF-α, especially so when pre-exposed to linoleic acid. Further, in response to TNF-α, enhanced lipolysis, monitored as increased extracellular glycerol and fatty acids levels, was observed in adipocytes cultured in the presence of exogenous fatty acids. Taken together, our 3D spheroid model showed enhanced adipogenic differentiation and presents a platform for elucidating the key phenotypic responses that occur in pro-inflammatory microenvironments that characterize obesogenic states.

Relevance of vascular peroxisome proliferator-activated receptor γ coactivator-1α to molecular alterations in atherosclerosis.

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is emerging as a novel factor that plays a critical role in integrating signalling pathways in the control of cellular and systemic metabolism. We investigated the role of vascular expression of PGC-1α and related factors, such as sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ (PPARγ) and adiponectin, during the atherosclerotic process. Endothelial function, vascular superoxide anion production and inflammatory mediators were also evaluated. This study was carried out in male New Zealand rabbits fed a diet containing 0.5% cholesterol and 14% coconut oil for 8 weeks. Animals developed mixed dyslipidaemia and atherosclerotic lesions, which were associated with endothelial dysfunction, aortic overproduction of superoxide anions and inflammation. Expression of PGC-1α, SIRT1, PPARγ and adiponectin was reduced (P<0.05) in aorta from atherosclerotic rabbits. Levels of PGC-1α were correlated negatively (P<0.05) with total cholesterol levels, aortic superoxide anion production and tumour necrosis factor-α expression, and positively (P<0.05) with maximal relaxation in response to acetylcholine. The observed results suggest that PGC-1α could be considered to be a link between the main atherosclerotic processes (endothelial dysfunction, oxidation and inflammation) and alterations of other factors involved in vascular wall integrity, such as SIRT1, PPARγ and adiponectin.

An image-based drug susceptibility assay targeting the placental sequestration of Plasmodium falciparum-infected erythrocytes.

Placental malaria is a significant cause of all malaria-related deaths globally for which no drugs have been developed to specifically disrupt its pathogenesis. To facilitate the discovery of antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes in the placenta microvasculature, we have developed an automated image-based assay for high-throughput screening for potent cytoadherence inhibitors in vitro. Parasitized erythrocytes were drug-treated for 24 h and then allowed to adhere on a monolayer of placental BeWo cells prior to red blood cell staining with glycophorin A antibodies. Upon image-acquisition, drug effects were quantified as the proportion of treated parasitized erythrocytes to BeWo cells compared to the binding of untreated iRBCs. We confirmed the reliability of this new assay by comparing the binding ratios of CSA- and CD36-panned parasites on the placental BeWo cells, and by quantifying the effects of chondroitin sulfate A, brefeldin A, and artemisinin on the binding. By simultaneously examining the drug effects on parasite viability, we could discriminate between cytoadherence-specific inhibitors and other schizonticidal compounds. Taken together, our data establish that the developed assay is highly suitable for drug studies targeting placental malaria, and will facilitate the discovery and rapid development of new therapies against malaria.

Curcumin enhances non-opsonic phagocytosis of Plasmodium falciparum through up-regulation of CD36 surface expression on monocytes/macrophages.

OBJECTIVES: Curcumin is a natural plant product with antimalarial activity and immunomodulatory properties. In this study we aimed to investigate its effects on CD36 expression and CD36-mediated Plasmodium falciparum phagocytosis as well as the role played by reactive oxygen species (ROS) and the peroxisome proliferator-activated receptor γ retinoid X receptor (PPARγ-RXR) in these processes. METHODS: In vitro antimalarial activity was evaluated by the [³H]hypoxanthine assay. ROS production and surface CD36 in human monocyte/macrophages were measured by flow cytometry. PPARγ and CD36 mRNA expression was determined by the QuantiGene Plex® assay and RT-qPCR. Nuclear PPARγ activation was analysed by a DNA-binding ELISA while nuclear erythroid-related factor 2 (Nrf2) expression was analysed by western blotting. P. falciparum phagocytosis was assessed by light microscopy. RESULTS: Curcumin's antimalarial activity was confirmed and did not differ between drug-susceptible and -resistant P. falciparum strains. Curcumin increased monocyte ROS production and expression of PPARγ and CD36 at the mRNA and protein levels. Although PPARγ activation was blocked by the PPARγ antagonist GW9662, CD36 expression and CD36-mediated P. falciparum phagocytosis were only inhibited by N-acetylcysteine (NAC), suggesting a PPARγ-independent CD36 expression pathway. We then identified seven putative Nrf2 antioxidant response elements on the CD36 gene promoter and showed that NAC inhibited curcumin-induced Nrf2 protein expression. CONCLUSIONS: CD36 expression and CD36-mediated P. falciparum phagocytosis by curcumin are dependent on ROS production and probably involve the Nrf2 pathway. The dual immunomodulatory and antimalarial mechanisms of curcumin action may mean that curcumin has potential as an adjuvant treatment limiting the risk of recrudescence following standard antimalarial therapy.

Artemisinic acid is a regulator of adipocyte differentiation and C/EBP δ expression.

Adipocyte dysfunction is associated with the development of obesity. In this study, artemisinic acid, which was isolated from Artemisia annua L., inhibited adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs) and its mechanism of action was determined. The mRNA levels of peroxidase proliferation-activated receptor (PPAR) γ and CCAAT/enhancer binding protein (C/EBP) α, late adipogenic factors, were reduced by artemisinic acid. Moreover, the mRNA levels of the PPAR γ target genes lipoprotein lipase, CD36, adipocyte protein, and liver X receptor were down-regulated by artemisinic acid. Artemisinic acid reduced expression of the C/EBP δ gene without impacting C/EBP ß. In addition, attempts to elucidate a possible mechanism underlying the artemisinic acid-mediated effects revealed that reduced expression of the C/EBP δ gene was mediated by inhibiting Jun N-terminal kinase (JNK). Additionally, artemisinic acid also reduced the expression of the adipogenesis-associated genes glucose transporter-4 and vascular endothelial growth factor. In addition to the interference of artemisinic acid with adipogenesis, artemisinic acid significantly attenuated tumor necrosis factor-α-induced secretion of interleukin-6 by undifferentiated hAMSCs, thus influencing insulin resistance and the inflammatory state characterizing obesity. Taken together, these findings indicate that inhibiting adipogenic differentiation of hAMSCs by artemisinic acid occurs primarily through reduced expression of C/EBP δ, which is mediated by the inhibition of JNK and suggest that aremisinic acid may be used as a complementary treatment option for obesity associated with metabolic syndrome.

A high content drug screen identifies ursolic acid as an inhibitor of amyloid beta protein interactions with its receptor CD36.

A pathological hallmark of Alzheimer disease (AD) is deposition of amyloid ß (Aß) in the brain. Aß binds to microglia via a receptor complex that includes CD36 leading to production of proinflammatory cytokines and neurotoxic reactive oxygen species and subsequent neurodegeneration. Interruption of Aß binding to CD36 is a potential therapeutic strategy for AD. To identify pharmacologic inhibitors of Aß binding to CD36, we developed a 384-well plate assay for binding of fluorescently labeled Aß to Chinese hamster ovary cells stably expressing human CD36 (CHO-CD36) and screened an Food and Drug Administration-approved compound library. The assay was optimized based on the cells' tolerance to dimethyl sulfoxide, Aß concentration, time required for Aß binding, reproducibility, and signal-to-background ratio. Using this assay, we identified four compounds as potential inhibitors of Aß binding to CD36. These compounds were ursolic acid, ellipticine, zoxazolamine, and homomoschatoline. Of these compounds, only ursolic acid, a naturally occurring pentacyclic triterpenoid, successfully inhibited binding of Aß to CHO-CD36 cells in a dose-dependent manner. The ursolic acid effect reached a plateau at ~20 µm, with a maximal inhibition of 64%. Ursolic acid also blocked binding of Aß to microglial cells and subsequent ROS production. Our data indicate that cell-based high-content screening of small molecule libraries for their ability to block binding of Aß to its receptors is a useful tool to identify novel inhibitors of receptors involved in AD pathogenesis. Our data also suggest that ursolic acid is a potential therapeutic agent for AD via its ability to block Aß-CD36 interactions.

Molecular mechanism of alpha-tocopheryl-phosphate transport across the cell membrane.

alpha-Tocopheryl-phosphate (alpha-TP) is synthesized and hydrolyzed in animal cells and tissues where it modulates several functions. alpha-TP is more potent than alpha-T in inhibiting cell proliferation, down-regulating CD36 transcription, inhibiting atherosclerotic plaque formation. Administration of alpha-TP to cells or animals requires its transfer through membranes, via a transporter. We show here that alpha-TP is passing the plasma membrane via a system that is inhibited by glibenclamide and probenecid, inhibitors of a number of transporters. Glibenclamide and probenecid prevent dose-dependently alpha-TP inhibition of cell proliferation. The two inhibitors act on ATP binding cassette (ABC) and organic anion transporters (OAT). Since ABC transporters function to export solutes and alpha-TP is transported into cells, it may be concluded that alpha-TP transport may occur via an OAT family member. Due to the protection by glibenclamide and probenecid on the alpha-TP induced cell growth inhibition it appears that alpha-TP acts after its uptake inside cells.

Identification of thrombospondin 1 (TSP-1) as a novel mediator of cell injury in kidney ischemia.

Thrombospondin 1 (TSP-1) is a matricellular protein that inhibits angiogenesis and causes apoptosis in vivo and in vitro in several cancerous cells and tissues. Here we identify TSP-1 as the molecule with the highest induction level at 3 hours of IR injury in rat and mouse kidneys subjected to ischemia/reperfusion (IR) injury using the DNA microarray approach. Northern hybridizations demonstrated that TSP-1 expression was undetectable at baseline, induced at 3 and 12 hours, and returned to baseline levels at 48 hours of reperfusion. Immunocytochemical staining identified the injured proximal tubules as the predominant sites of expression of TSP-1 in IR injury and showed colocalization of TSP-1 with activated caspase-3. Addition of purified TSP-1 to normal kidney proximal tubule cells or cells subjected to ATP depletion in vitro induced injury as demonstrated by cytochrome c immunocytochemical staining and caspase-3 activity. The deleterious role of TSP-1 in ischemic kidney injury was demonstrated directly in TSP-1 null mice, which showed significant protection against IR injury-induced renal failure and tubular damage. We propose that TSP-1 is a novel regulator of ischemic damage in the kidney and may play an important role in the pathophysiology of ischemic kidney failure.

Effects of aldehydes on CD36 expression.

INTRODUCTION: During the oil frying process lipid peroxidation compounds are formed. These products can modulate gene expression and alter cellular behaviour. The cellular uptake of oxidized LDL, a key step in the development of atherosclerosis, is mediated by the CD36 scavenger receptor, whose expression is down-regulated by alpha-tocopherol. OBJECTIVE: To determine the effects of water-soluble aldehydes, obtained from thermally oxidized sunflower oil on the expression of CD36 scavenger receptor in human monocytes (THP-1 cells). We also wanted to study the effects of alpha-tocopherol on CD36 expression in the presence of water-soluble aldehydes. MATERIALS AND METHODS: Sunflower oil was heated in a frying pan, at 180--200 degrees C for 40 min, water-soluble aldehydes were isolated, and the content of thiobarbituric acid reacting substances (TBARS) was determined. THP-1 monocytes were cultured in RPMI medium during 24 h and incubated with increasing concentrations of the water-soluble aldehydes (ranging from 0.05 to 1 microM) and with or without 50 microM of alpha-tocopherol. In parallel, THP-1 cells were cultured with the same volume of an extract obtained from non-oxidized oil or distilled water. The CD36 expression at the cell surface was studied with fluorescence-activated cell sorting (FACS). RESULTS: Monocytes incubated in a medium containing water-soluble aldehydes, showed a dose dependent increase in the expression of the CD36 protein on the cell surface, compared to with the control groups. When the cells were treated simultaneously with 50 microM of alpha-tocopherol a significant reduction in the expression of the CD36 protein was observed. CONCLUSION: Water-soluble aldehydes, extracted from thermally oxidized culinary oil, increase the expression of CD36. This effect is partially decreased by the presence of alpha-tocopherol.

HIV protease inhibitors-induced atherosclerosis: prevention by alpha-tocopherol.

Prolonged treatments with inhibitors of human immunodeficiency(HIV)-encoded protease (ARPI) have been reported to induce early atherosclerotic events. Our in vitro study indicates that alpha-tocopherol may prevent drug-induced premature atherosclerosis since it interferes with CD36 scavenger receptor over-expression induced by ritonavir in monocytes. The mechanism of CD36 upregulation by ritonavir involves inhibition of the ubiquitin-proteasome system and alpha-tocopherol is able to normalize proteasome activity. These findings suggest that ARPI combined with early alpha-tocopherol supplementation may decrease the drug-induced atherosclerotic risk.

Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-BI) expression and HDL cholesteryl ester uptake in the hamster.

Diets rich in polyunsaturated fatty acids lower plasma HDL cholesterol concentrations when compared to diets rich in saturated fatty acids. We investigated the mechanistic basis for this effect in the hamster and sought to determine whether reduced plasma HDL cholesterol concentrations resulting from a high polyunsaturated fat diet are associated with a decrease in reverse cholesterol transport. Animals were fed semisynthetic diets enriched with polyunsaturated or saturated fatty acids for 6 weeks. We then determined the effect of these diets on the following parameters: 1) hepatic scavenger receptor B1 (SR-BI) mRNA and protein levels, 2) the rate of hepatic HDL cholesteryl ester uptake, and 3) the rate of cholesterol acquisition by the extrahepatic tissues (from de novo synthesis, LDL and HDL) as a measure of the rate of reverse cholesterol transport. Compared to saturated fatty acids, dietary polyunsaturated fatty acids up-regulated hepatic SR-BI expression by approximately 50% and increased HDL cholesteryl ester transport to the liver; as a consequence, plasma HDL cholesteryl ester concentrations were reduced. Although dietary polyunsaturated fatty acids increased hepatic HDL cholesteryl ester uptake and lowered plasma HDL cholesterol concentrations, there was no change in the cholesterol content or in the rate of cholesterol acquisition (via de novo synthesis and lipoprotein uptake) by the extrahepatic tissues.These studies indicate that substitution of polyunsaturated for saturated fatty acids in the diet increases SR-BI expression and lowers plasma HDL cholesteryl ester concentrations but does not affect reverse cholesterol transport.