Constructing Lipoparticles Capable of Endothelial Cell-Derived Exosome-Mediated Delivery of Anti-miR-33a-5p to Cultured Macrophages.

Atherosclerosis is driven by intimal arterial macrophages accumulating cholesterol. Atherosclerosis also predominantly occurs in areas consisting of proinflammatory arterial endothelial cells. At time of writing, there are no available clinical treatments that precisely remove excess cholesterol from lipid-laden intimal arterial macrophages. Delivery of anti-miR-33a-5p to macrophages has been shown to increase apoAI-mediated cholesterol efflux via ABCA1 upregulation but delivering transgenes to intimal arterial macrophages is challenging due to endothelial cell barrier integrity. In this study, we aimed to test whether lipoparticles targeting proinflammatory endothelial cells can participate in endothelial cell-derived exosome exploitation to facilitate exosome-mediated transgene delivery to macrophages. We constructed lipoparticles that precisely target the proinflammatory endothelium and contain a plasmid that expresses XMOTIF-tagged anti-miR-33a-5p (LP-pXMoAntimiR33a5p), as XMOTIF-tagged small RNA demonstrates the capacity to be selectively shuttled into exosomes. The cultured cells used in our study were immortalized mouse aortic endothelial cells (iMAECs) and RAW 264.7 macrophages. From our results, we observed a significant decrease in miR-33a-5p expression in macrophages treated with exosomes released basolaterally by LPS-challenged iMAECs incubated with LP-pXMoAntimiR33a5p when compared to control macrophages. This decrease in miR-33a-5p expression in the treated macrophages caused ABCA1 upregulation as determined by a significant increase in ABCA1 protein expression in the treated macrophages when compared to the macrophage control group. The increase in ABCA1 protein also simulated ABCA1-dependent cholesterol efflux in treated macrophages-as we observed a significant increase in apoAI-mediated cholesterol efflux-when compared to the control group of macrophages. Based on these findings, strategies that involve combining proinflammatory-targeting lipoparticles and exploitation of endothelial cell-derived exosomes appear to be promising approaches for delivering atheroprotective transgenes to lipid-laden arterial intimal macrophages.

Jugular Vein Injection of High-Titer Lentiviral Vectors Does Not Transduce the Aorta-Brief Report.

OBJECTIVE: Efficient gene transfer to the vascular wall via intravenous vector injection would be useful for experimental vascular biology and gene therapy. Initial studies of lentiviral vector tropism suggested that intravenously injected vectors do not transduce murine vascular tissue; however, there are also reports of highly efficient aortic transduction after jugular vein injection of high-titer lentiviral vectors. We sought to reproduce these results. Approach and Results: We injected high-titer preparations of GFP (green fluorescent protein)-expressing lentiviral vector into jugular veins of 8 mice; 6 mice received vehicle only. Four days later, samples of aorta (thoracic and abdominal), liver, spleen, and other tissues were harvested and processed for quantitative polymerase chain reaction detection of vector DNA and immunohistochemical detection of GFP. Our vector DNA assay did not detect transduction of any of the 16 aortic segments. This finding excludes an aortic transduction efficiency of >0.02 vector copies per cell. In contrast, vector DNA was detected in all 8 spleen and liver extracts (median, 0.8 and 0.1 vector copies per cell, respectively; P<0.001 versus vehicle controls). Quantitative polymerase chain reaction signals from DNA extracted from heart, lung, kidney, skeletal muscle, and femoral artery did not differ from background polymerase chain reaction signals from DNA extracted from tissues of vehicle-injected mice (P≥0.7 for all). Immunohistochemistry revealed GFP in scattered cells in spleen and liver, not in aorta. CONCLUSIONS: Injection of high-titer lentiviral vectors via the jugular vein transduces cells in the spleen and liver but does not efficiently transduce the aorta. Graphic Abstract: A graphic abstract is available for this article.

Consuming Diet Supplemented with Either Red Wheat Bran or Soy Extract Changes Glucose and Insulin Levels in Female Obese Zucker Rats.

Type 2 diabetes mellitus is characterized by the inability to regulate blood glucose levels due to insulin resistance, resulting in hyperglycemia and hyperinsulinemia. Research has shown that consuming soy and fiber may protect against type 2 diabetes mellitus. We performed a study to determine whether supplementing diet with soy extract (0.5% weight of diet) or fiber (as red wheat bran; 11.4% weight of diet) would decrease serum insulin and blood glucose levels in a pre-diabetic/metabolic syndrome animal model. In our study, female obese Zucker rats were fed either a control diet (n = 8) or control diet supplemented with either soy extract (n = 7) or red wheat bran (n = 8) for seven weeks. Compared to rats consuming control diet, rats fed treatment diets had significantly lower (p-value < 0.05) fasting serum insulin (control = 19.34±1.6; soy extract = 11.1±1.54; red wheat bran = 12.4±1.11) and homeostatic model assessment of insulin resistance values (control = 2.16±0.22; soy extract = 1.22±0.21; red wheat bran = 1.54±0.16). Non-fasted blood glucose was also significantly lower (p-value < 0.05) in rats fed treatment diets compared to rats consuming control diet at weeks four (control = 102.63±5.67; soy extract = 80.14±2.13; red wheat bran = 82.63±3.16), six (control = 129.5±10.83; soy extract = 89.14±2.48; red wheat bran = 98.13±3.54), and seven (control = 122.25±8.95; soy extract = 89.14±4.52; red wheat bran = 84.75±4.15). Daily intake of soy extract and red wheat bran may protect against type 2 diabetes mellitus by maintaining normal glucose homeostasis.

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.

Vitamin C reverses bone loss in an osteopenic rat model of osteoporosis.

Fruits and vegetables are rich in vitamin C with antioxidant properties which are known to influence bone quality. This study evaluated whether vitamin C (1000 mg/L) added to drinking water reverses the bone loss in ovariectomized rats. Ninety-day-old female Sprague-Dawley rats were randomly assigned to either sham (n = 14) or ovariecotmized groups (n = 28). Sixty days after ovariectomy, the treatments were sham, ovariectomy (OVX), OVX + vitamin C (22 mg oral intake daily) for 60 days. Urine was collected for deoxypyridinoline (DPD) evaluation, rats were sacrificed, and antioxidant capacity, osteopontin, alkaline phosphatase (ALP), and bone specific tartrate resistant acid phosphatase (TRAP) were evaluated in the plasma. Right femur and 5th lumbar were evaluated for bone density, strength, ash, Ca, and Mg concentrations. Antioxidant capacity, ALP activity, osteopontin decreased (p-value < 0.05), while TRAP and urinary DPD increased (p-value < 0.05) with ovariectomy. In contrast, vitamin C increased (p-value < 0.05) antioxidant capacity, ALP activity, osteopontin concentration and reduced (p-value < 0.05) TRAP and urinary DPD excretion, respectively. Ovariectomy reduced (p-value < 0.05) bone quality, bone ash, Ca and Mg concentrations. Vitamin C increased (p-value < 0.05) femoral density without affecting (p-value > 0.1) femoral strength, ash, or Ca, and Mg concentrations, while it increased (p-value < 0.05) the 5th lumbar density, ash, and Ca and Mg concentrations. In conclusion, vitamin C increased bone quality and antioxidant capacity in ovariectomized rats.

Inhibition of miR-33a-5p in Macrophage-like Cells In Vitro Promotes apoAI-Mediated Cholesterol Efflux.

Atherosclerosis is caused by cholesterol accumulation within arteries. The intima is where atherosclerotic plaque accumulates and where lipid-laden foam cells reside. Intimal foam cells comprise of both monocyte-derived macrophages and macrophage-like cells (MLC) of vascular smooth muscle cell (VSMC) origin. Foam cells can remove cholesterol via apoAI-mediated cholesterol efflux and this process is regulated by the transporter ABCA1. The microRNA miR-33a-5p is thought to be atherogenic via silencing ABCA1 which promotes cholesterol retention and data has shown inhibiting miR-33a-5p in macrophages may be atheroprotective via enhancing apoAI-mediated cholesterol efflux. However, it is not entirely elucidated whether precisely inhibiting miR-33a-5p in MLC also increases ABCA1-dependent cholesterol efflux. Therefore, the purpose of this work is to test the hypothesis that inhibition of miR-33a-5p in cultured MLC enhances apoAI-mediated cholesterol efflux. In our study, we utilized the VSMC line MOVAS cells in our experiments, and cholesterol-loaded MOVAS cells to convert this cell line into MLC. Inhibition of miR-33a-5p was accomplished by transducing cells with a lentivirus that expresses an antagomiR directed at miR-33a-5p. Expression of miR-33a-5p was analyzed by qRT-PCR, ABCA1 protein expression was assessed via immunoblotting, and apoAI-mediated cholesterol efflux was measured using cholesterol efflux assays. In our results, we demonstrated that lentiviral vector-mediated knockdown of miR-33a-5p resulted in decreasing expression of this microRNA in cultured MLC. Moreover, reduction of miR-33a-5p in cultured MLC resulted in de-repression of ABCA1 expression, which caused ABCA1 protein upregulation in cultured MLC. Additionally, this increase in ABCA1 protein expression resulted in enhancing ABCA1-dependent cholesterol efflux through increasing apoAI-mediated cholesterol efflux in cultured MLC. From these findings, we conclude that inhibiting miR-33a-5p in MLC may protect against atherosclerosis by promoting ABCA1-dependent cholesterol efflux.

Cholesterol Efflux Decreases TLR4-Target Gene Expression in Cultured Macrophages Exposed to T. brucei Ghosts.

Trypanosoma brucei causes African trypanosomiasis in humans. Infection with T. brucei elicits a potent pro-inflammatory immune response within infected human hosts, and this response is thought to at least be partially due to Toll-like receptor (TLR) activation. In response to stimulation by lipopolysaccharide and other pathogen antigens, TLR4 translocates to lipid rafts, which induces the expression of pro-inflammatory genes. However, cholesterol efflux is acknowledged as anti-inflammatory due to promoting lipid raft disruption. In this study, we wanted to assess the impact of T. brucei "ghosts", which are non-viable T. brucei essentially devoid of intracellular contents, in stimulating macrophage TLR4 translocation to lipid rafts, and whether promoting cholesterol efflux in macrophages incubated with T. brucei ghosts attenuates TLR4-target gene expression. When cultured macrophages were exposed to T. brucei ghosts, we observed an increase in lipid raft TLR4 protein content, which suggests certain surface molecules of T. brucei serve as ligands for TLR4. However, pretreating macrophages with cholesterol acceptors before T. brucei ghost exposure decreased lipid raft TLR4 protein content and the expression of pro-inflammatory TLR4-target genes. Taken together, these results imply that macrophage cholesterol efflux weakens pro-inflammatory responses which occur from T. brucei infection via increasing macrophage lipid raft disruption.

SCD1 activity in muscle increases triglyceride PUFA content, exercise capacity, and PPARδ expression in mice.

Stearoyl-CoA desaturase (SCD)1 converts saturated fatty acids into monounsaturated fatty acids. Using muscle overexpression, we sought to determine the role of SCD1 expression in glucose and lipid metabolism and its effects on exercise capacity in mice. Wild-type C57Bl/6 (WT) and SCD1 muscle transgenic (SCD1-Tg) mice were generated, and expression of the SCD1 transgene was restricted to skeletal muscle. SCD1 overexpression was associated with increased triglyceride (TG) content. The fatty acid composition of the muscle revealed a significant increase in polyunsaturated fatty acid (PUFA) content of TG, including linoleate (18:2n6). Untrained SCD1-Tg mice also displayed significantly increased treadmill exercise capacity (WT = 6.6 ± 3 min, Tg = 71.9 ± 9.5 min; P = 0.0009). SCD1-Tg mice had decreased fasting plasma glucose, glucose transporter (GLUT)1 mRNA, fatty acid oxidation, mitochondrial content, and increased peroxisome proliferator-activated receptor (PPAR)δ and Pgc-1 protein expression in skeletal muscle. In vitro studies in C2C12 myocytes revealed that linoleate (18:2n6) and not oleate (18:1n9) caused a 3-fold increase in PPARδ and a 9-fold increase in CPT-1b with a subsequent increase in fat oxidation. The present model suggests that increasing delta-9 desaturase activity of muscle increases metabolic function, exercise capacity, and lipid oxidation likely through increased PUFA content, which increases PPARδ expression and activity. However, the mechanism of action that results in increased PUFA content of SCD1-Tg mice remains to be elucidated.

Role of stearoyl-CoA desaturase-1 in skeletal muscle function and metabolism.

Stearoyl-CoA desaturase-1 (SCD1) converts saturated fatty acids (SFA) into monounsaturated fatty acids and is necessary for proper liver, adipose tissue, and skeletal muscle lipid metabolism. While there is a wealth of information regarding SCD1 expression in the liver, research on its effect in skeletal muscle is scarce. Furthermore, the majority of information about its role is derived from global knockout mice, which are known to be hypermetabolic and fail to accumulate SCD1's substrate, SFA. We now know that SCD1 expression is important in regulating lipid bilayer fluidity, increasing triglyceride formation, and enabling lipogenesis and may protect against SFA-induced lipotoxicity. Exercise has been shown to increase SCD1 expression, which may contribute to an increase in intramyocellular triglyceride at the expense of free fatty acids and diacylglycerol. This review is intended to define the role of SCD1 in skeletal muscle and discuss the potential benefits of its activity in the context of lipid metabolism, insulin sensitivity, exercise training, and obesity.

Dissecting the Impact of Vascular Smooth Muscle Cell ABCA1 versus ABCG1 Expression on Cholesterol Efflux and Macrophage-like Cell Transdifferentiation: The Role of SR-BI.

Cholesterol-laden macrophages are recognized as a major contributor to atherosclerosis. However, recent evidence indicates that vascular smooth muscle cells (VSMC) that accumulate cholesterol and transdifferentiate into a macrophage-like cell (MLC) phenotype also play a role in atherosclerosis. Therefore, removing cholesterol from MLC may be a potential atheroprotective strategy. The two transporters which remove cholesterol from cells are ABCA1 and ABCG1, as they efflux cholesterol to apoAI and HDL, respectively. In this study, the well-characterized immortalized VSMC line MOVAS cells were edited to generate ABCA1- and ABCG1-knockout (KO) MOVAS cell lines. We cholesterol-loaded ABCA1-KO MOVAS cells, ABCG1-KO MOVAS cells, and wild-type MOVAS cells to convert cells into a MLC phenotype. When we measured apoAI- and HDL-mediated cholesterol efflux in these cells, we observed a drastic decrease in apoAI-mediated cholesterol efflux within ABCA1-KO MOVAS MLC, but HDL-mediated cholesterol efflux was only partially reduced in ABCG1-KO MOVAS cells. Since SR-BI also participates in HDL-mediated cholesterol efflux, we assessed SR-BI protein expression in ABCG1-KO MOVAS MLC and observed SR-BI upregulation, which offered a possible mechanism explaining why HDL-mediated cholesterol efflux remains maintained in ABCG1-KO MOVAS MLC. When we used lentivirus for shRNA-mediated knockdown of SR-BI in ABCG1-KO MOVAS MLC, this decreased HDL-mediated cholesterol efflux when compared to ABCG1-KO MOVAS MLC with unmanipulated SR-BI expression. Taken together, these major findings suggest that SR-BI expression in MLC of a VSMC origin plays a compensatory role in HDL-mediated cholesterol efflux when ABCG1 expression becomes impaired and provides insight on SR-BI demonstrating anti-atherogenic properties within VSMC/MLC.

miR-33a Expression Attenuates ABCA1-Dependent Cholesterol Efflux and Promotes Macrophage-Like Cell Transdifferentiation in Cultured Vascular Smooth Muscle Cells.

Recent evidence suggests that the majority of cholesterol-laden cells found in atherosclerotic lesions are vascular smooth muscle cells (VSMC) that have transdifferentiated into macrophage-like cells (MLC). Furthermore, cholesterol-laden MLC of VSMC origin have demonstrated impaired ABCA1-dependent cholesterol efflux, but it is poorly understood why this occurs. A possible mechanism which may at least partially be attributed to cholesterol-laden MLC demonstrating attenuated ABCA1-dependent cholesterol efflux is a miR-33a expression, as a primary function of this microRNA is to silence ABCA1 expression, but this has yet to be rigorously investigated. Therefore, the VSMC line MOVAS cells were used to generate miR-33a knockout (KO) MOVAS cells, and we used KO and wild-type (WT) MOVAS cells to delineate any possible proatherogenic role of miR-33a expression in VSMC. When WT and KO MOVAS cells were cholesterol-loaded to convert into MLC, this resulted in the WT MOVAS cells to exhibit impaired ABCA1-dependent cholesterol efflux. In the cholesterol-loaded WT MOVAS MLC, we also observed a delayed restoration of the VSMC phenotype when these cells were exposed to the ABCA1 cholesterol acceptor, apoAI. These results imply that miR-33a expression in VSMC drives atherosclerosis by triggering MLC transdifferentiation via attenuated ABCA1-dependent cholesterol efflux.

The Impact of MiR-33a-5p Inhibition in Pro-Inflammatory Endothelial Cells.

Evidence suggests cholesterol accumulation in pro-inflammatory endothelial cells (EC) contributes to triggering atherogenesis and driving atherosclerosis progression. Therefore, inhibiting miR-33a-5p within inflamed endothelium may prevent and treat atherosclerosis by enhancing apoAI-mediated cholesterol efflux by upregulating ABCA1. However, it is not entirely elucidated whether inhibition of miR-33a-5p in pro-inflammatory EC is capable of increasing ABCA1-dependent cholesterol efflux. In our study, we initially transfected LPS-challenged, immortalized mouse aortic EC (iMAEC) with either pAntimiR33a5p plasmid DNA or the control plasmid, pScr. We detected significant increases in both ABCA1 protein expression and apoAI-mediated cholesterol efflux in iMAEC transfected with pAntimiR33a5p when compared to iMAEC transfected with pScr. We subsequently used polymersomes targeting inflamed endothelium to deliver either pAntimiR33a5p or pScr to cultured iMAEC and showed that the polymersomes were selective in targeting pro-inflammatory iMAEC. Moreover, when we exposed LPS-challenged iMAEC to these polymersomes, we observed a significant decrease in miR-33a-5p expression in iMAEC incubated with polymersomes containing pAntimR33a5p versus control iMAEC. We also detected non-significant increases in both ABCA1 protein and apoAI-mediated cholesterol in iMAEC exposed to polymersomes containing pAntimR33a5p when compared to control iMAEC. Based on our results, inhibiting miR-33a-5p in pro-inflammatory EC exhibits atheroprotective effects, and so precisely delivering anti-miR-33a-5p to these cells is a promising anti-atherogenic strategy.

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.

Utilizing the LoxP-Stop-LoxP System to Control Transgenic ABC-Transporter Expression In Vitro.

ABCA1 and ABCG1 are two ABC-transporters well-recognized to promote the efflux of cholesterol to apoAI and HDL, respectively. As these two ABC-transporters are critical to cholesterol metabolism, several studies have assessed the impact of ABCA1 and ABCG1 expression on cellular cholesterol homeostasis through ABC-transporter ablation or overexpressing ABCA1/ABCG1. However, for the latter, there are currently no well-established in vitro models to effectively induce long-term ABC-transporter expression in a variety of cultured cells. Therefore, we performed proof-of-principle in vitro studies to determine whether a LoxP-Stop-LoxP (LSL) system would provide Cre-inducible ABC-transporter expression. In our studies, we transfected HEK293 cells and the HEK293-derived cell line 293-Cre cells with ABCA1-LSL and ABCG1-LSL-based plasmids. Our results showed that while the ABCA1/ABCG1 protein expression was absent in the transfected HEK293 cells, the ABCA1 and ABCG1 protein expression was detected in the 293-Cre cells transfected with ABCA1-LSL and ABCG1-LSL, respectively. When we measured cholesterol efflux in transfected 293-Cre cells, we observed an enhanced apoAI-mediated cholesterol efflux in 293-Cre cells overexpressing ABCA1, and an HDL2-mediated cholesterol efflux in 293-Cre cells constitutively expressing ABCG1. We also observed an appreciable increase in HDL3-mediated cholesterol efflux in ABCA1-overexpressing 293-Cre cells, which suggests that ABCA1 is capable of effluxing cholesterol to small HDL particles. Our proof-of-concept experiments demonstrate that the LSL-system can be used to effectively regulate ABC-transporter expression in vitro, which, in turn, allows ABCA1/ABCG1-overexpression to be extensively studied at the cellular level.

Comparison of Human Urinary Exosomes Isolated via Ultracentrifugation Alone versus Ultracentrifugation Followed by SEC Column-Purification.

Chronic kidney disease is a progressive, incurable condition that involves a gradual loss of kidney function. While there are no non-invasive biomarkers available to determine whether individuals are susceptible to developing chronic kidney disease, small RNAs within urinary exosomes have recently emerged as a potential candidate to use for assessing renal function. Ultracentrifugation is the gold standard for urinary exosome isolation. However, extravesicular small RNA contamination can occur when isolating exosomes from biological fluids using ultracentrifugation, which may lead to misidentifying the presence of certain small RNA species in human urinary exosomes. Therefore, we characterized human urinary exosomal preparations isolated by ultracentrifugation alone, or via ultracentrifugation followed by size exclusion chromatography (SEC) column-purification. Using nanoparticle tracking analysis, we identified SEC fractions containing robust amounts of exosome-sized particles, that we further characterized using immunoblotting. When compared to exosomal preparations isolated by ultracentrifugation only, SEC fractionated exosomal preparations showed higher levels of the exosome-positive marker CD81. Moreover, while the exosome-negative marker calnexin was undetectable in SEC fractionated exosomal preparations, we did observe calnexin detection in the exosomal preparations isolated by ultracentrifugation alone, which implies contamination in these preparations. Lastly, we imaged SEC fractionated exosomal preparations using transmission electron microscopy to confirm these preparations contained human urinary exosomes. Our results indicate that combining ultracentrifugation and SEC column-purification exosome isolation strategies is a powerful approach for collecting contaminant-free human urinary exosomes and should be considered when exosomes devoid of contamination are needed for downstream applications.

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.

Urinary Exosomal MicroRNAs as Biomarkers for Obesity-Associated Chronic Kidney Disease.

The early detection of chronic kidney disease (CKD) is key to reducing the burden of disease and rising costs of care. This need has spurred interest in finding new biomarkers for CKD. Ideal bi-omarkers for CKD should be: easy to measure; stable; reliably detected, even when interfering substances are present; site-specific based on the type of injury (tubules vs. glomeruli); and its changes in concentration should correlate with disease risk or outcome. Currently, no single can-didate biomarker fulfills these criteria effectively, and the mechanisms underlying kidney fibrosis are not fully understood; however, there is growing evidence in support of microRNA-mediated pro-cesses. Specifically, urinary exosomal microRNAs may serve as biomarkers for kidney fibrosis. In-creasing incidences of obesity and the recognition of obesity-associated CKD have increased interest in the interplay of obesity and CKD. In this review, we provide: (1) an overview of the current scope of CKD biomarkers within obese individuals to elucidate the genetic pathways unique to obesi-ty-related CKD; (2) a review of microRNA expression in obese individuals with kidney fibrosis in the presence of comorbidities, such as diabetes mellitus and hypertension; (3) a review of thera-peutic processes, such as diet and exercise, that may influence miR-expression in obesity-associated CKD; (4) a review of the technical aspects of urinary exosome isolation; and (5) future areas of research.

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.

Combined LXR and RXR Agonist Therapy Increases ABCA1 Protein Expression and Enhances ApoAI-Mediated Cholesterol Efflux in Cultured Endothelial Cells.

Endothelial ABCA1 expression protects against atherosclerosis and this atheroprotective effect is partially attributed to enhancing apoAI-mediated cholesterol efflux. ABCA1 is a target gene for LXR and RXR; therefore, treating endothelial cells with LXR and/or RXR agonists may increase ABCA1 expression. We tested whether treating cultured immortalized mouse aortic endothelial cells (iMAEC) with the endogenous LXR agonist 22(R)-hydroxycholesterol, synthetic LXR agonist GW3965, endogenous RXR agonist 9-cis-retinoic acid, or synthetic RXR agonist SR11237 increases ABCA1 protein expression. We observed a significant increase in ABCA1 protein expression in iMAEC treated with either GW3965 or SR11237 alone, but no significant increase in ABCA1 protein was observed in iMAEC treated with either 22(R)-hydroxycholesterol or 9-cis-retionic acid alone. However, we observed significant increases in both ABCA1 protein expression and apoAI-mediated cholesterol efflux when iMAEC were treated with a combination of either 22(R)-hydroxycholesterol and 9-cis-retinoic acid or GW3965 and SR11237. Furthermore, treating iMAEC with either 22(R)-hydroxycholesterol and 9-cis-retinoic acid or GW3965 and SR11237 did not trigger an inflammatory response, based on VCAM-1, ICAM-1, CCL2, and IL-6 mRNA expression. Based on our findings, delivering LXR and RXR agonists precisely to endothelial cells may be a promising atheroprotective approach.

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.