Assessing Anti-Adipogenic Effects of Mango Leaf Tea and Mangiferin within Cultured Adipocytes.

Obesity is a condition caused by surplus adipose tissue and is a risk factor for several diet-related diseases. Obesity is a global epidemic that has also been challenging to treat effectively. However, one promoted therapy to safely treat obesity is anti-adipogenic therapeutics. Therefore, identifying potent anti-adipogenic bioactive compounds that can safely be used clinically may effectively treat obesity in humans. Mango leaf has potential medicinal properties due to its many bioactive compounds that may enhance human health. Mangiferin (MGF) is a primary constituent in mango plants, with many health-promoting qualities. Therefore, this study investigated the effect of MGF, and tea brewed with mango leaves in cultured adipocytes. The anti-adipogenic efficacy of mango leaf tea (MLT) and MGF in 3T3-L1 cells were assessed, along with cell viability, triglyceride levels, adiponectin secretion, and glucose uptake analyzed. In addition, changes in the mRNA expression of genes involved in lipid metabolism within 3T3-L1 cells were determined using quantitative real-time PCR. Our results showed while both MLT and MGF increased glucose uptake in adipocytes, only MLT appeared to inhibit adipogenesis, as determined by decreased triglyceride accumulation. We also observed increased secretory adiponectin levels, reduced ACC mRNA expression, and increased FOXO1 and ATGL gene expression in 3T3-L1 cells treated with MLT but not MGF. Together, these results suggest that MLT may exhibit anti-adipogenic properties independent of MGF content.

Polyploid giant cancer cells are dependent on cholesterol for progeny formation through amitotic division.

Polyploid Giant Cancer Cells (PGCC) are increasingly being recognized as drivers of cancer recurrence. Therapy stress promotes the formation of these cells, which upon stress cessation often successfully generate more aggressive progeny that repopulate the tumor. Therefore, identification of potential PGCC vulnerabilities is key to preventing therapy failure. We have previously demonstrated that PGCC progeny formation depends on the lysosomal enzyme acid ceramidase (ASAH1). In this study, we compared transcriptomes of parental cancer cells and PGCC in the absence or presence of the ASAH1 inhibitor LCL521. Results show that PGCC express less INSIG1, which downregulates cholesterol metabolism and that inhibition of ASAH1 increased HMGCR which is the rate limiting enzyme in cholesterol synthesis. Confocal microscopy revealed that ceramide and cholesterol do not colocalize. Treatment with LCL521 or simvastatin to inhibit ASAH1 or HMGCR, respectively, resulted in accumulation of ceramide at the cell surface of PGCC and prevented PGCC progeny formation. Our results suggest that similarly to inhibition of ASAH1, disruption of cholesterol signaling is a potential strategy to interfere with PGCC progeny formation.

MOVAS Cells: A Versatile Cell Line for Studying Vascular Smooth Muscle Cell Cholesterol Metabolism.

Cholesterol metabolism is paramount to cells. Aberrations to cholesterol metabolism affects cholesterol homeostasis, which may impact the risk of several diseases. Recent evidence has suggested that vascular smooth muscle cell (VSMC) cholesterol metabolism may play a role in atherosclerosis. However, there is scant in vitro mechanistic data involving primary VSMC that directly tests how VSMC cholesterol metabolism may impact atherosclerosis. One reason for this lack of data is due to the impracticality of gene manipulation studies in primary VSMC, as cultured primary VSMC become senescent and lose their morphology rapidly. However, there are no immortalized VSMC lines known to be suitable for studying VSMC cholesterol metabolism. The purpose of this study was to determine whether MOVAS cells, a commercially available VSMC line, are suitable to use for studying VSMC cholesterol metabolism. Using immunoblotting and immunofluorescence, we showed that MOVAS cells express ABCA1, ABCG1, and SREBP-2. We also determined that MOVAS cells efflux cholesterol to apoAI and HDL, which indicates functionality of ABCA1/ABCG1. In serum-starved MOVAS cells, SREBP-2 target gene expression was increased, confirming SREBP-2 functionality. We detected miR-33a expression in MOVAS cells and determined this microRNA can silence ABCA1 and ABCG1 via identifying conserved miR-33a binding sites within ABCA1/ABCG1 3'UTR in MOVAS cells. We showed that cholesterol-loading MOVAS cells results in this cell line to transdifferentiate into a macrophage-like cell, which also occurs when VSMC accumulate cholesterol. Our characterization of MOVAS cells sufficiently demonstrates that they are suitable to use for studying VSMC cholesterol metabolism in the context of atherosclerosis.

Exosome-Mediated Transfer of Anti-miR-33a-5p from Transduced Endothelial Cells Enhances Macrophage and Vascular Smooth Muscle Cell Cholesterol Efflux.

Atherosclerosis is a disease of large- and medium-sized arteries that is caused by cholesterol accumulation in arterial intimal cells, including macrophages and smooth muscle cells (SMC). Cholesterol accumulation in these cells can be prevented or reversed in preclinical models-and atherosclerosis reduced-by transgenesis that increases expression of molecules that control cholesterol efflux, including apolipoprotein AI (apoAI) and ATP-binding cassette subfamily A, member 1 (ABCA1). In a previous work, we showed that transduction of arterial endothelial cells (EC)-with a helper-dependent adenovirus (HDAd) expressing apoAI-enhanced EC cholesterol efflux in vitro and decreased atherosclerosis in vivo. Similarly, overexpression of ABCA1 in cultured EC increased cholesterol efflux and decreased inflammatory gene expression. These EC-targeted gene-therapy strategies might be improved by concurrent upregulation of cholesterol-efflux pathways in other intimal cell types. Here, we report modification of this strategy to enable delivery of therapeutic nucleic acids to cells of the sub-endothelium. We constructed an HDAd (HDAdXMoAntimiR33a5p) that expresses an antagomiR directed at miR-33a-5p (a microRNA that suppresses cholesterol efflux by silencing ABCA1). HDAdXMoAntimiR33a5p contains a sequence motif that enhances uptake of anti-miR-33a-5p into exosomes. Cultured EC release exosomes containing small RNA, including miR-33a-5p. After transduction with HDAdXMoAntimiR33a5p, EC-derived exosomes containing anti-miR-33a-5p accumulate in conditioned medium (CM). When this CM is added to macrophages or SMC, anti-miR-33a-5p is detected in these target cells. Exosome-mediated transfer of anti-miR-33a-5p reduces miR-33a-5p by ∼65-80%, increases ABCA1 protein by 1.6-2.2-fold, and increases apoAI-mediated cholesterol efflux by 1.4-1.6-fold (all p ≤ 0.01). These effects were absent in macrophages and SMC incubated in exosome-depleted CM. EC transduced with HDAdXMoAntimiR33a5p release exosomes that can transfer anti-miR-33a-5p to other intimal cell types, upregulating cholesterol efflux from these cells. This strategy provides a platform for genetic modification of intimal and medial cells, using a vector that transduces only EC.

ABCA1 Overexpression in Endothelial Cells In Vitro Enhances ApoAI-Mediated Cholesterol Efflux and Decreases Inflammation.

Atherosclerosis, a disease of blood vessels, is driven by cholesterol accumulation and inflammation. Gene therapy that removes cholesterol from blood vessels and decreases inflammation is a promising approach for prevention and treatment of atherosclerosis. In previous work, we reported that helper-dependent adenoviral (HDAd) overexpression of apolipoprotein A-I (apoAI) in endothelial cells (ECs) increases cholesterol efflux in vitro and reduces atherosclerosis in vivo. However, the effect of HDAdApoAI on atherosclerosis is partial. To improve this therapy, we considered concurrent overexpression of ATP-binding cassette subfamily A, member 1 (ABCA1), a protein that is required for apoAI-mediated cholesterol efflux. Before attempting combined apoAI/ABCA1 gene therapy, we tested whether an HDAd that expresses ABCA1 (HDAdABCA1) increases EC cholesterol efflux, whether increased cholesterol efflux alters normal EC physiology, and whether ABCA1 overexpression in ECs has anti-inflammatory effects. HDAdABCA1 increased EC ABCA1 protein (∼3-fold; p < 0.001) and apoAI-mediated cholesterol efflux (2.3-fold; p = 0.007). Under basal culture conditions, ABCA1 overexpression did not alter EC proliferation, metabolism, migration, apoptosis, nitric oxide production, or inflammatory gene expression. However, in serum-starved, apoAI-treated EC, ABCA1 overexpression had anti-inflammatory effects: decreased inflammatory gene expression (∼50%; p ≤ 0.02 for interleukin [IL]-6, tumor necrosis factor [TNF]-α, and vascular cell adhesion protein-1); reduced lipid-raft Toll-like receptor 4 (80%; p = 0.001); and a trend towards increased nitric oxide production (∼55%; p = 0.1). In ECs stimulated with lipopolysaccharide, ABCA1 overexpression markedly decreased inflammatory gene expression (∼90% for IL-6 and TNF-α; p < 0.001). Therefore, EC ABCA1 overexpression has no toxic effects and counteracts the two key drivers of atherosclerosis: cholesterol accumulation and inflammation. In vivo testing of HDAdABCA1 is warranted.

Apo A-I (Apolipoprotein A-I) Vascular Gene Therapy Provides Durable Protection Against Atherosclerosis in Hyperlipidemic Rabbits.

OBJECTIVE: Gene therapy that expresses apo A-I (apolipoprotein A-I) from vascular wall cells has promise for preventing and reversing atherosclerosis. Previously, we reported that transduction of carotid artery endothelial cells with a helper-dependent adenoviral (HDAd) vector expressing apo A-I reduced early (4 weeks) fatty streak development in fat-fed rabbits. Here, we tested whether the same HDAd could provide long-term protection against development of more complex lesions. APPROACH AND RESULTS: Fat-fed rabbits (n=25) underwent bilateral carotid artery gene transfer, with their left and right common carotids randomized to receive either a control vector (HDAdNull) or an apo A-I-expressing vector (HDAdApoAI). Twenty-four additional weeks of high-fat diet yielded complex intimal lesions containing lipid-rich macrophages as well as smooth muscle cells, often in a lesion cap. Twenty-four weeks after gene transfer, high levels of apo A-I mRNA (median ≥250-fold above background) were present in all HDAdApoAI-treated arteries. Compared with paired control HDAdNull-treated arteries in the same rabbit, HDAdApoAI-treated arteries had 30% less median intimal lesion volume (P=0.03), with concomitant reductions (23%-32%) in intimal lipid, macrophage, and smooth muscle cell content (P≤0.05 for all). HDAdApoAI-treated arteries also had decreased intimal inflammatory markers. VCAM-1 (vascular cell adhesion molecule-1)-stained area was reduced by 36% (P=0.03), with trends toward lower expression of ICAM-1 (intercellular adhesion molecule-1), MCP-1 (monocyte chemoattractant protein 1), and TNF-α (tumor necrosis factor-α; 13%-39% less; P=0.06-0.1). CONCLUSIONS: In rabbits with severe hyperlipidemia, transduction of vascular endothelial cells with an apo A-I-expressing HDAd yields at least 24 weeks of local apo A-I expression that durably reduces atherosclerotic lesion growth and intimal inflammation.

Tissue Specific Effects of Dietary Carbohydrates and Obesity on ChREBPα and ChREBPß Expression.

Carbohydrate response element binding protein (ChREBP) regulates insulin-independent de novo lipogenesis. Recently, a novel ChREBPß isoform was identified. The purpose of the current study was to define the effect of dietary carbohydrates (CHO) and obesity on the transcriptional activity of ChREBP isoforms and their respective target genes. Mice were subjected to fasting-refeeding of high-CHO diets. In all three CHO-refeeding groups, mice failed to induce ChREBPα, yet ChREBPß increased 10- to 20-fold. High-fat fed mice increased hepatic ChREBPß mRNA expression compared to chow-fed along with increased protein expression. To better assess the independent effect of fructose on ChREBPα/ß activity, HepG2 cells were treated with fructose ± a fructose-1,6-bisphosphatase inhibitor to suppress gluconeogenesis. Fructose treatment in the absence of gluconeogenesis resulted in increased ChREBP activity. To confirm the existence of ChREBPß in human tissue, primary hepatocytes were incubated with high-glucose and the expression of ChREBPα and -ß was determined. As with the animal models, glucose induced ChREBPß expression while ChREBPα was decreased. Taken together, ChREBPß is more responsive to changes in dietary CHO availability than the -α isoform. Diet-induced obesity increases basal expression of ChREBPß, which may increase the risk of developing hepatic steatosis, and fructose-induced activation is independent of gluconeogenesis.

Drinking orange juice increases total antioxidant status and decreases lipid peroxidation in adults.

Cardiovascular disease (CVD) is the leading cause of death in the world and is the primary cause of mortality among Americans. One of the many reasons for the pathogenesis of CVD is attributed to eating diets high in saturated fat and refined carbohydrates and low in fruits and vegetables. Epidemiological evidence has supported a strong association between eating diets rich in fruits and vegetables and cardiovascular health. An experiment was conducted utilizing 24 adults with hypercholesterolemia and hypertriglyceridemia to evaluate the impact of drinking 20 fl oz of freshly squeezed orange juice daily for 90 days on blood pressure, lipid panels, plasma antioxidant capacity, metabolic hormones, lipid peroxidation, and inflammatory markers. Except for addition of drinking orange juice, subjects did not modify their eating habits. The findings suggested that drinking orange juice does not affect (P>.1) blood pressure, lipid panels, metabolic hormones, body fat percentage, or inflammatory markers. However, total plasma antioxidant capacity was significantly increased (P<.05) and lipid peroxidation was significantly decreased (P<.05) after orange juice consumption. Drinking orange juice may protect the cardiovascular system by increasing total plasma antioxidant status and by lowering lipid peroxidation independent of other cardiovascular risk markers evaluated in this study.

Drinking carrot juice increases total antioxidant status and decreases lipid peroxidation in adults.

BACKGROUND: High prevalence of obesity and cardiovascular disease is attributable to sedentary lifestyle and eating diets high in fat and refined carbohydrate while eating diets low in fruit and vegetables. Epidemiological studies have confirmed a strong association between eating diets rich in fruits and vegetables and cardiovascular health. The aim of this pilot study was to determine whether drinking fresh carrot juice influences antioxidant status and cardiovascular risk markers in subjects not modifying their eating habits. METHODS: An experiment was conducted to evaluate the effects of consuming 16 fl oz of daily freshly squeezed carrot juice for three months on cardiovascular risk markers, C-reactive protein, insulin, leptin, interleukin-1α, body fat percentage, body mass index (BMI), blood pressure, antioxidant status, and malondialdehyde production. Fasting blood samples were collected pre-test and 90 days afterward to conclude the study. RESULTS: Drinking carrot juice did not affect (P > 0.1) the plasma cholesterol, triglycerides, Apo A, Apo B, LDL, HDL, body fat percentage, insulin, leptin, interleukin-1α, or C-reactive protein. Drinking carrot juice decreased (P = 0.06) systolic pressure, but did not influence diastolic pressure. Drinking carrot juice significantly (P < 0.05) increased the plasma total antioxidant capacity and decreased (P < 0.05) the plasma malondialdehyde production. CONCLUSION: Drinking carrot juice may protect the cardiovascular system by increasing total antioxidant status and by decreasing lipid peroxidation independent of any of the cardiovascular risk markers measured in the study.