Sequential Ultracentrifugation Research Articles

Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) – a prominent extracellular matrix component – it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20–200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.

CBM-14GLIOBLASTOMA CELLS EXPOSED TO 5-ALA RELEASE PROTOPORPHYRIN IX CONTAINING EXTRACELLULAR VESICLES DETECTABLE BY HIGH-RESOLUTION FLOW CYTOMETRY

INTRODUCTION: Gliolan or 5-aminolevulinic acid (5-ALA) is used for fluorescence-guided neurosurgery in glioblastoma multiforme (GBM) patients. 5-ALA is part of the heme synthesis pathway. In GBM cells, accumulation of fluorescent protoporphyrin IX (PpIX) is observed after oral intake, providing the neurosurgeon with visual guidance during surgery. Extracellular vesicles (EVs) or exosomes are nano-sized (50-1000 nm) lipid membrane-enclosed vesicles released by cells. We hypothesized that EVs from fluorescent GBM cells can contain PpIX and are thus fluorescent as well, allowing for tumor derived extracellular vesicle detection by high-resolution flow cytometry. METHODS: U87-MG/EGFRvIII cells were cultured for 24-hours in presence of 500uM 5-ALA. Extracellular vesicles were isolated from cell culture supernatant by sequential (ultra)centrifugation and analysed using high-resolution flow cytometry. Fluorescent EVs were spiked into platelet-free plasma from healthy donors at different concentrations. Subsequently, plasma samples from GBM patients treated with 5-ALA were studied using high-resolution flow-cytometry. RESULTS: GBM cells treated with 5-ALA released fluorescent extracellular vesicles, which could be detected by high-resolution flow cytometry (488 nm excitation). These fluorescent EVs could be reliably quantified when spiked into healthy donor platelet-free plasma. In platelet-free plasma from GBM patients treated with 5-ALA, fluorescent extracellular vesicles could be observed. CONCLUSION: Here we provide a novel method to identify tumor-derived extracellular vesicles using high-resolution flow cytometry of PpIX positive particles. Current research focuses on detection of these particles in GBM patient derived bodily fluids and their potential relation to tumor volume and response to therapy.

Comparison of the effects of low-dose rosuvastatin on plasma levels of cholesterol and oxidized low-density lipoprotein in 3 ultracentrifugally separated low-density lipoprotein subfractions

Plasma-oxidized (ox) low-density lipoprotein (LDL) is an atherogenic lipoprotein. The distribution of ox-LDL in plasma LDL subfractions and the effect of statins on this distribution have not been investigated in detail. We examined the distribution of cholesterol and ox-LDL in 3 ultracentrifugally separated plasma LDL subfractions and investigated the effects of a statin, rosuvastatin, on the levels of these lipoproteins. Thirty-one polygenic hypercholesterolemic subjects were included in this study. Levels of cholesterol and ox-LDL in 3 plasma LDL subfractions and plasma levels of remnant-like particle cholesterol, ox-LDL, and adiponectin were measured after 0, 3, 6, and 12 months of treatment with rosuvastatin. Sequential ultracentrifugation was performed to subfractionate plasma lipoproteins. The mean daily dose of rosuvastatin over the 12 months of treatment was 2.9 ± 1.0 mg (mean ± standard deviation). The cholesterol subfraction distribution was 43 ± 10% as low-density LDL, 46 ± 8% as medium-density LDL, and 13 ± 5% as high-density LDL. Similarly, the distribution of ox-LDL was 31 ± 10% as low-density LDL, 48 ± 7% as medium-density LDL, and 22 ± 8% as high-density LDL. After 12 months of treatment with rosuvastatin, the level of cholesterol was significantly reduced in all 3 subfractions (P < .0001), as was the level of ox-LDL (P < .0001). Furthermore, the plasma cholesterol level in high-density lipoprotein2 increased significantly. The distribution of ox-LDL in plasma LDL subfractions was more skewed toward the denser subfractions, compared with cholesterol. Rosuvastatin treatment significantly reduced plasma levels of cholesterol and ox-LDL in all LDL subfractions.

214 Glycated LDL (glyc-LDL) Promotes Osteogenic Differentiation of Vascular Smooth Muscle Cells

Introduction It is well established that glyc-LDL is elevated in the serum of diabetic patients compared to healthy subjects, and that vascular calcification is common in diabetes. Furthermore, recent studies suggest that advanced glycation end products may play a pathogenic role in vascular calcification. We hypothesise that glyc-LDL promotes the osteogenic differentiation of vascular smooth muscle cells in vitro . Methods LDL was isolated from human serum by sequential density gradient ultracentrifugation and incubated at 37°C with a range of glucose concentrations at different time points to determine the optimum glycation conditions. The glyc-LDL was measured using an in-house ELISA method. Bovine Aortic Smooth Muscle Cells (BAoSMCs) were incubated with native or glycated LDL in the presence of osteogenic media and mineral deposition was determined using alizarin red staining and alkaline phosphatase (ALP) activity, which is an early marker of osteogenesis. Results The optimum conditions for LDL glycation was determined as 80mM glucose for 7 days, so these conditions were used for the production of glycated LDL throughout the study. BAoSMCs incubated in osteogenic media exhibited mineralisation after 7 days as determined by alizarin red staining. This calcification was significantly enhanced by treatment with glyc-LDL, but not by native LDL. Furthermore, we found that ALP activity was significantly elevated as early as day 4 in glyc-LDL treated cells, compared to those incubated in native LDL. The glyc-LDL-induced mineralisation was not attenuated in the presence of osteoprotegrin (OPG) nor in the presence of two novel small RAGE antagonist peptides, which were designed to prevent RAGE binding with several of its most important ligands, including HMGB-1, S100P and S1004A. Conclusion We have shown that human-derived glyc-LDL accelerates vascular calcification in vitro . This process does not appear to be attenuated by the addition of OPG, suggesting AGE-induced accelerated calcification occurs through a pathway independent of the RANKL/OPG signalling activity. Further studies with a range of RAGE inhibitors should allow the identification of the molecular pathways implicated in glyc-LDL induced calcification to enable the development of therapeutic peptides effective in blocking of RAGE-ligand interaction, which is prevalent in many pathologies.

Abstract 132: CETP Deficiency in Rabbits Protects High Fat High Cholesterol Diet Induced Atherosclerosis

CETP has been suggested as an effective and promising target for further reducing cardiovascular events through elevation of the HDL-C levels. The failure of Pfizer’s torcetrapib and Roche’s dalcetrapib raised the question whether increased HDL from pharmacological inhibition of CETP are cardioprotective. Because mice lack CETP naturally, we generated CETP knockout (KO) rabbits by Zinc-finger nucleases (ZFN) to gain insights onto CETP and HDL biology in CVD. The ZFN pairs target on exon 3 of rabbit CETP gene. By microinjection of ZFN mRNA into rabbit embryos, we generated 5 CETP knockout (CETPko) founder rabbits. The founder used for this study contained 4 nucleotides deletion in exon 3 that results in a premature stop codon. Western blot confirms that there is no detectable CETP protein in the homozygous CETPko rabbit plasma and liver. No CETP activity was detected in the CETPko rabbit plasma, consistent with their genotype. At the normal chow fed conditions, there is no significant difference between wild type and CETPko male rabbits in plasma HDLc and total cholesterol (TC) level; whereas female CETPko rabbits show 40 to 100% increase in both HDLc and TC levels compared to wild type ones. By sequential ultracentrifugation method to separate plasma lipoproteins, we found that there is much higher HDL3 component in female CETPko rabbits, but not in male CETPko ones, comparing to their WT age matched control. At the HFHC (3% soy bean oil and 0.3% cholesterol) diet fed conditions, both male and female CETPko rabbits show significantly lower level of TC and LDLc compared to wild type rabbits. At the same time, the HDLc levels are significantly higher in the CETPko rabbits. In addition, apoB depleted plasma from CETPko rabbit showed 2-fold increase in cholesterol efflux tested in human THP1 cells. Furthermore, we measured the atherosclerotic plaque in the rabbit aorta after 16 weeks HFHC diet treatment. The total gross lesion areas in CETPko rabbits were only one-third of those in the wild type rabbits. In conclusion, our study shows that genetic deficiency of CETP in rabbits has beneficial effects on enhancing HDL function and reducing atherosclerosis. Our work sheds light on CETP and HDL biology, and provides valuable information to the development of CETP drugs.

Extracellular vesicles derived from Gram-negative bacteria, such as Escherichia coli, induce emphysema mainly via IL-17A-mediated neutrophilic inflammation.

Recent evidence indicates that Gram-negative bacteria-derived extracellular vesicles (EVs) in indoor dust can evoke neutrophilic pulmonary inflammation, which is a key pathology of chronic obstructive pulmonary disease (COPD). Escherichia coli is a ubiquitous bacterium present in indoor dust and secretes nanometer-sized vesicles into the extracellular milieu. In the current study, we evaluated the role of E. coli-derived EVs on the development of COPD, such as emphysema. E. coli EVs were prepared by sequential ultrafiltration and ultracentrifugation. COPD phenotypes and immune responses were evaluated in C57BL/6 wild-type (WT), IFN-γ-deficient, or IL-17A-deficient mice after airway exposure to E. coli EVs. The present study showed that indoor dust from a bed mattress harbors E. coli EVs. Airway exposure to E. coli EVs increased the production of proinflammatory cytokines, such as TNF-α and IL-6. In addition, the repeated inhalation of E. coli EVs for 4 wk induced neutrophilic inflammation and emphysema, which are associated with enhanced elastase activity. Emphysema and elastase activity enhanced by E. coli EVs were reversed by the absence of IFN-γ or IL-17A genes. In addition, during the early period, lung inflammation is dependent on IL-17A and TNF-α, but not on IFN-γ, and also on TLR4. Moreover, the production of IFN-γ is eliminated by the absence of IL-17A, whereas IL-17A production is not abolished by IFN-γ absence. Taken together, the present data suggest that E. coli-derived EVs induce IL-17A-dependent neutrophilic inflammation and thereby emphysema, possibly via upregulation of elastase activity.

Study of frozen low density lipoprotein particles by using nanotechnology

ObjectivesTo investigate the impact of freezing in −80°C on the structure of isolated low density lipoproteins (LDLs), using nanotechnology, such as Atomic Force Microscopy (AFM). Design and methodsBlood EDTA plasma was obtained from healthy subject and used immediately to isolate LDL by sequential ultracentrifugation at 10°C in 55,000rpm for 3h, using a Beckmann XL-90 ultracentrifuge (75Ti rotor), in the presence of KBr in PBS. LDLs were then diluted with PBS until final concentrations of 5 and 15mg LDL/dl. After initial observation, samples were frozen in −80°C for two weeks and observed again after thawing. Experiments were performed in triplicate on two smooth and clean substrates of different hydrophobicity, glass (HOPG) and Si (c-Si). Statistical significance was set at 0.05. ResultsMacroscopically, LDL particles formed aggregations in a dendroid layout. There were no differences between images taken from both substrates (HOPG and c-Si). Frozen samples presented significantly smaller LDL particles, than fresh ones. In specific, mean diameter of LDL particle in the fresh LDL sample was 19.77nm, ranging from 13.34 to 28.76nm. The frozen LDL sample had a mean diameter of 5.2nm, ranging from 2.0 to 8.0nm, which was significantly different from the unfrozen. ConclusionsAtomic Force Microscopy showed that freezing of LDL causes alterations in their size.

Crosstalk Between Glycoxidative Modification of Low-Density Lipoprotein, Angiotensin II-Sensitization, and Adrenocortical Aldosterone Release

Low-density lipoprotein (LDL) is considered to be a risk factor for atherosclerosis. In the presence of hyperglycemia, LDL undergoes glycoxidative modification and this glycoxidized (glycox) LDL promotes atherosclerosis in type 2 diabetic (T2D) individuals. Moreover, because of its cholesterol content, LDL contributes to aldosterone biosynthesis, which is modulated by angiotensin II (AngII) and has been implicated in cardiovascular complications of T2D. However, the molecular mechanism of the crosstalk between glycoxLDL, AngII, and aldosterone has not been explained clearly. Therefore, this study has been aimed to investigate the impact of in vitro modified glycoxLDL on aldosterone release in an AngII-sensitized adrenocortical carcinoma cell line (NCI H295R). Native LDL (natLDL), isolated from healthy volunteers by sequential density gradient ultracentrifugation, was subjected to d-glucose (200 mmol/l), for glycoxidative modification, at 37 °C for 6 days. The AngII-sensitized H295R cells were treated with natLDL and glycoxLDL for 24 h and the supernatant was used for aldosterone measurement. The treated cells were utilized for protein isolation and mRNA quantification. Compared to natLDL, glycoxLDL produced a significantly greater effect on aldosterone release from AngII-sensitized cells. The treatment with specific pharmacological inhibitors suggests that modified LDL recruits ERK1/2 and janus kinase-2 for transcriptional regulation of aldosterone synthase. Moreover, glycoxLDL modulates aldosterone release via cAMP-dependent protein kinase A (PKA) pathway. However, glycoxLDL induces ERK phosphorylation independent of PKA activation and this novel mechanism could be targeted for therapeutic trials. In conclusion, this in vitro study emphasizes a possible causal relationship between LDL glycoxidative modification, AngII-sensitization, and adrenocortical steroid hormone release.

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Objectives Preeclampsia (PE) and IUGR share common pathomechanisms. Recently, we reported on serum lipid concentrations in both conditions in the early-onset form of PE before 34 weeks of gestation (Pecks et al., 2012). Subtle changes of triglyceride concentrations were noticed in PE while a significant decrease in LDL levels was found in IUGR subjects when compared to a control group (CTRL). We now aimed to extend our lipid analysis within our cohort to gain a deeper understanding of lipid metabolism and its role in the pathogenesis of both diseases. Methods Serum of (i) PE, (ii) PE with IUGR, and (iii) normotensive IUGR each n = 10 was matched to a CTRL group (iv, n = 30). HDL, IDL, LDL, VLDL were isolated by sequential preparative ultracentrifugation. Esterified and non-esterified cholesterol, phospholipid, and triglyceride concentrations were determined in each fraction. Statistical analysis was done using 2-way ANOVA. Results No significant difference between the case groups and CTRL could be observed in the HDL fraction. In the IDL fraction triglyceride levels were higher in PE as compared to CTRL. Within the LDL fraction esterified and non-esterified cholesterol and phospholipid levels were lower in IUGR and IUGR + PE. Within the VLDL fraction triglycerides were higher in all three case groups as compared to CTRL (table). Conclusions Our results suggest a disturbed conversion of VLDL to LDL is involved in both IUGR and preeclampsia. Triglyceride metabolism appears to be most affected in preeclampsia whereas cholesterol metabolism is disturbed in IUGR irrespectively of additional preeclampsia. One has to carefully separate all three entities to avoid misinterpretation. Disclosures U. Pecks: None. B. Winkler: None. T. Goecke: None. N. Maass: None. C. Contini: None. K. Winkler: None.

Changes in ultracentrifugally separated plasma lipoprotein subfractions in patients with polygenic hypercholesterolemia, familial combined hyperlipoproteinemia, and familial hypercholesterolemia after treatment with atorvastatin

Plasma levels of low-density lipoproteins (LDLs) are decreased through stimulation of their hepatic uptake by statins via an LDL receptor. However, it is unclear whether statins equally stimulate the hepatic uptake of all LDL subfractions. We compared the effects of atorvastatin on 3 LDL subfractions, and their associations with LDL-receptor activities, in Japanese patients with polygenic hypercholesterolemia (PHC), familial combined hyperlipoproteinemia (FCHL), and familial hypercholesterolemia (FH). Atorvastatin was administered to patients with PHC (n=11), FCHL (n=16), and FH (n=13). We measured plasma levels of lipids, remnant-like particle cholesterol, apoproteins, and cholesterol in lipoprotein fractions. Sequential ultracentrifugation was performed to subfractionate the plasma lipoproteins, and lymphocyte LDL-receptor activities were estimated using flow cytometry. The average daily dosage of atorvastatin was 10, 27, and 40mg in patients with PHC, FCHL, and FH, respectively; after 12months of atorvastatin treatment, LDL cholesterol (LDL-C) plasma levels decreased by 44%, 50%, and 53%, respectively (all, P<.0001). Atorvastatin reduced low-density LDL-C plasma levels in patients with PHC (48% reduction), FCHL (53%), and FH (46%) (all, P<.0001). Plasma levels of medium-density and high-density LDL-C were also significantly reduced in the 3 patient groups (all, P≤.0147). LDL-receptor activity was negatively correlated with baseline levels of medium-density LDL-C and with the decreases in plasma md-LDL-C levels. Atorvastatin decreased the levels of the 3 LDL fractions. The md-LDL decrease appeared to be mainly because of stimulation of LDL-receptor activity.

Low concentrations of phospholipids and plasma HDL cholesterol subclasses in asymptomatic subjects with high coronary calcium scores

Objective: To determine whether HDL size distribution and HDL subclasses are associated with coronary calcium scores. Methods: We screened 677 apparently healthy individuals by coronary tomography. One hundred twenty subjects were then recruited for the study and grouped by coronary artery calcification scores (CAC). Forty asymptomatic patients with atherosclerosis with CAC scores ≥75th percentile for gender and age were placed in the first group. Forty patients with CAC scores ≤25th percentile and 40 matched controls (CAC = 0) made up the two remaining groups. HDL samples were separated via sequential ultracentrifugation, followed by electrophoresis: they were then enzymatically stained and densitometrically analyzed to determine the triglycerides (Tg), phospholipids (Ph), and plasma cholesterol (C) concentrations corresponding to each HDL subclass. Results: HDL size distribution, lipid and non-lipid risk factors for atherosclerosis were similar among the three groups: HDL-cholesterol and HDL-phospholipids were significantly lower in the CAC ≥75th percentile group, whereas HDL-lipids in the CAC ≤25th group were comparable to the controls. HDL2b- and HDL2a-cholesterol were decreased, whereas phospholipids were lower in patients constituting 4 of the 5 HDL subclasses. The Ph-to-Tg ratios of small HDL were higher in both experimental groups compared with the controls, suggesting that these lipoproteins had abnormal structures. In spite of the significant differences between the high-CAC score subjects and the controls, statistical analyses demonstrated no substantial relationship between CAC scores and HDL parameters: other lipid and non-lipid risk factors for atherosclerosis were not statistically linked to CAC scores. Only male gender and age contributed to CAC scores in our study population. Conclusions: Our results suggest that CAC scores and traditional lipid profiles are independent aspects of atherosclerosis and that only lipids may be biomarkers of coronary calcification during the asymptomatic stages of the disease; however, HDL subclasses do not contribute to CAC scores.

In vitro evaluation of endothelial exosomes as carriers for small interfering ribonucleic acid delivery.

Exosomes, one subpopulation of nanosize extracellular vesicles derived from multivesicular bodies, ranging from 30 to 150 nm in size, emerged as promising carriers for small interfering ribonucleic acid (siRNA) delivery, as they are capable of transmitting molecular messages between cells through carried small noncoding RNAs, messenger RNAs, deoxyribonucleic acids, and proteins. Endothelial cells are involved in a number of important biological processes, and are a major source of circulating exosomes. In this study, we prepared exosomes from endothelial cells and evaluated their capacity to deliver siRNA into primary endothelial cells. Exosomes were isolated and purified by sequential centrifugation and ultracentrifugation from cultured mouse aortic endothelial cells. Similar to exosome particles from other cell sources, endothelial exosomes are nanometer-size vesicles, examined by both the NanoSight instrument and transmission electron microscopy. Enzyme-linked immunosorbent assay analysis confirmed the expression of two exosome markers: CD9 and CD63. Flow cytometry and fluorescence microscopy studies demonstrated that endothelial exosomes were heterogeneously distributed within cells. In a gene-silencing study with luciferase-expressing endothelial cells, exosomes loaded with siRNA inhibited luciferase expression by more than 40%. In contrast, siRNA alone and control siRNA only suppressed luciferase expression by less than 15%. In conclusion, we demonstrated that endothelial exosomes have the capability to accommodate and deliver short foreign nucleic acids into endothelial cells.

Chylomicrons produced by Caco-2 cells contained ApoB-48 with diameter of 80-200 nm.

The small intestine generally transports dietary fats to circulation in triglyceride (TG)‐rich lipoproteins. The two main intestinal lipoproteins are chylomicron (CM) and very low‐density lipoprotein (VLDL). Unfortunately, studies on the CM biogenesis and intestinal transport of dietary fats have been hampered by the lack of an adequate in vitro model. In this study, we investigated the possible factors that might increase the efficiency of CM production by Caco‐2 cells. We utilized sequential NaCl gradient ultracentrifugation to isolate the CMs that were secreted by the Caco‐2 cells. To confirm the successful isolation of the CMs, we performed Fat Red 7B staining, TG reading, apolipoprotein B (ApoB) measurement, and transmission electron microcopy (TEM) analysis. We then tested the effects of cell differentiation, oleic acid, mono‐olein, egg lecithin, incubation time, and collagen matrix on CM secretion. We found that cell differentiation, oleic acid, and lecithin were critical for CM secretion. Using the Transwell system, we further confirmed that the CMs produced by our Caco‐2 cells contained significant amount of TGs and ApoB‐48 such that they could be detected without the use of isotope labeling. In conclusion, when fully differentiated Caco‐2 were challenged with oleic acid, lecithin, and sodium taurocholate, 21% of their total number of lipoproteins were CMs with the diameter of 80–200 nm.

Inhibitory effects of LDL-associated tissue factor pathway inhibitor

IntroductionTissue factor pathway inhibitor (TFPI) is present in plasma as full-length free TFPI (TFPIα) and as C-terminally degraded forms mainly associated with low-density lipoprotein particles (LDL-TFPI). Addition of TFPIα to plasma induces a prolongation of the clotting time when tested in a diluted prothrombin time (dPT) assay, whereas no prolongation is observed with LDL-TFPI or truncated recombinant TFPI (TFPI1–161). The aim was to further characterize kinetic properties of purified LDL-TFPI in thrombin generation and chromogenic activity assays. Materials and MethodsLDL-TFPI was purified from human plasma by sequential flotation ultracentrifugation. Thrombin generation was measured in human plasma or in FVIII-immunodepleted plasma using either 1 pM tissue factor and 4μM phospholipids or 0.5nM factor Xa (FXa) and 4μM phospholipids, respectively. ResultsTFPIα prolonged the lag-phase and decreased the thrombin peak in tissue factor-induced thrombin generation, whereas LDL-TFPI exclusively decreased the peak height of thrombin without effecting the lag phase. Steady-state and transient kinetics showed that LDL-TFPI was a more potent inhibitor of FXa than TFPIα and TFPI1–161, indicating that FXa inhibition was not rate determining for the lag phase, whereas it appeared to affect thrombin generation during the propagation phase. This was supported by FXa-induced thrombin generation showing that LDL-TFPI, compared with TFPIα, more actively decreased the peak height. ConclusionsOur results suggest that LDL-TFPI affects thrombin generation during the propagation phase, and is kinetically different from TFPI1–161. It may therefore play a more prominent physiological role in vivo than hereto anticipated from dPT measurements.

High density lipoproteins improve insulin sensitivity in high-fat diet-fed mice by suppressing hepatic inflammation

Obesity-induced liver inflammation can drive insulin resistance. HDL has anti-inflammatory properties, so we hypothesized that low levels of HDL would perpetuate inflammatory responses in the liver and that HDL treatment would suppress liver inflammation and insulin resistance. The aim of this study was to investigate the effects of lipid-free apoAI on hepatic inflammation and insulin resistance in mice. We also investigated apoAI as a component of reconstituted HDLs (rHDLs) in hepatocytes to confirm results we observed in vivo. To test our hypothesis, C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks and administered either saline or lipid-free apoAI. Injections of lipid-free apoAI twice a week for 2 or 4 weeks with lipid-free apoAI resulted in: i) improved insulin sensitivity associated with decreased systemic and hepatic inflammation; ii) suppression of hepatic mRNA expression for key transcriptional regulators of lipogenic gene expression; and iii) suppression of nuclear factor κB (NF-κB) activation. Human hepatoma HuH-7 cells exposed to rHDLs showed suppressed TNFα-induced NF-κB activation, correlating with decreased NF-κB target gene expression. We conclude that apoAI suppresses liver inflammation in HFD mice and improves insulin resistance via a mechanism that involves a downregulation of NF-κB activation.

Myeloid Derived Suppressor Cells (MDSCs) Are Increased and Exert Immunosuppressive Activity In CML Patients At Diagnosis

IntroductionIn some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase 1 (arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. The aim of this study was to analyze MDSCs and investigate their activity in CML patients. MethodsMDSCs were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis. In 21 patients MDSCs were also measured during TKI treatment. Granulocytic MDSCs (G-MDSCs) were identified as CD11b+CD33+CD14-HLADR- cells, while the monocytic MDSCs (Mo-MDSCs) as CD14+HLADR by cytofluorimetric analysis. Arg1 expression was assessed using real time PCR and Western Blot. Arg activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. Microvesicles (MV) were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation. ResultsCML patients showed high levels of Mo- and G-MDSCs at diagnosis in comparison to HD (41±8 and 82,5±12,2% respectively for CML vs 9±2,1 and 55±5,3% for HD; p<0.001), while after TKIs therapy both subpopulations decreased, returning to normal values. T-reg (CD4+ CD25high Foxp3+ cells) were also significantly increased in CML patients at diagnosis in respect to HD (9±2% vs 6,1±0,8%, p<0.001) with a significant correlation with the percentage of Gr-MDSCs (r=0,6254; p<0.001). Both in PB and purified granulocytic cells, Arg1 expression showed a 30 fold increase in CML at diagnosis compared to HD (p<0.001) and decreased after therapy. The same data were confirmed by Western Blot analysis. Arg enzymatic activity in granulocytes resulted also increased in CML (n=10) compared to HD (n=10) (p<0.001). The suppressive function of CML G-MDSCs was demonstrated by their ability to inhibit the proliferation of CFSE+ HD T cells (p<0.001). In addition, an increase of Mo-MDSCs in vitro was observed after incubation of HD monocytes with CML sera (29±13%; p<0.0001) or MV (8±2,8%; p<0.05). ConclusionsMDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. CML granulocytes have high arg1 activity and immunosuppressive activity. Moreover, CML serum as well as CML microvesicles increase the percentage of HD Mo-MDSCs. Disclosures:No relevant conflicts of interest to declare.

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.

Scavenger receptor-BI is a receptor for lipoprotein(a)

Scavenger receptor class B type I (SR-BI) is a multi-ligand receptor that binds a variety of lipoproteins, including high density lipoprotein (HDL) and low density lipoprotein (LDL), but lipoprotein(a) [Lp(a)] has not been investigated as a possible ligand. Stable cell lines (HEK293 and HeLa) expressing human SR-BI were incubated with protein- or lipid-labeled Lp(a) to investigate SR-BI-dependent Lp(a) cell association. SR-BI expression enhanced the association of both (125)I- and Alexa Fluor-labeled protein from Lp(a). By confocal microscopy, SR-BI was also found to promote the internalization of fluorescent lipids (BODIPY-cholesteryl ester (CE)- and DiI-labeled) from Lp(a), and by immunocytochemistry the cellular internalization of apolipoprotein(a) and apolipoprotein B. When dual-labeled ((3)H-cholesteryl ether,(125)I-protein) Lp(a) was added to cells expressing SR-BI, there was a greater relative increase in lipid uptake over protein, indicating that SR-BI mediates selective lipid uptake from Lp(a). Compared with C57BL/6 control mice, transgenic mice overexpressing human SR-BI in liver were found to have increased plasma clearance of (3)H-CE-Lp(a), whereas mouse scavenger receptor class B type I knockout (Sr-b1-KO) mice had decreased plasma clearance (fractional catabolic rate: 0.63 ± 0.08/day, 1.64 ± 0.62/day, and 4.64 ± 0.40/day for Sr-b1-KO, C57BL/6, and human scavenger receptor class B type I transgenic mice, respectively). We conclude that Lp(a) is a novel ligand for SR-BI and that SR-BI mediates selective uptake of Lp(a)-associated lipids.

Carbamylated LDL induces a pro-thrombotic state via the LOX-1 receptor and arterial thrombus formation: a novel mechanism of cardiovascular events in end-stage renal disease

Background: Carbamylation of low-density lipoproteins (cLDL) alters protein structure and is thought to promote vascular inflammation and dysfunction during end-stage renal disease (ESRD). However, it remains unknown whether cLDL exerts prothrombotic effects in vascular cells and platelets and whether cLDL affects arterial thrombus formation in vivo. Methods: LDL was isolated from healthy subjects or patients with ESRD by sequential ultra-centrifugation. Ex vivo carbamylation of LDL (cLDL) from healthy subjects was induced with potassium cyanate. Arterial thrombus formation was analysed in a murine carotid artery photochemical injury model. Analysis of tissue factor (TF) and plasminogen activator inhibitor type-1 (PAI-1) expression was performed by Western blot and real-time PCR analysis. Platelet aggregation was analysed by impedance aggregometry. Results: Intravenous administration of cLDL (2mg/kg body weight) accelerated arterial thrombus formation in a murine photochemical carotid artery injury model as compared to treatment with native LDL (2mg/kg body weight) or vehicle (PBS) (n=8; p<0.05 versus nLDL and control). Analyses of tissue lysates demonstrated that cLDL induced the expression of TF and PAI-1 mRNA levels in carotid arteries (n=5, p<0.01). In human aortic smooth muscle cells (AoSMC) cLDL (10-300 μg/ml) induced TF and PAI-1 expression by 3.5- and 3-fold, as compared with nLDL (n=4; p<0.01). cLDL also enhanced basal and TNF-α induced expression of TF and PAI-1 in human aortic endothelial cells (n=4; p<0.02). In contrast, native LDL (nLDL) had no effect on endothelial expression or activity of TF and PAI-1. In both cell types, cLDL activated the mitogen-activated protein kinases p38 and ERK (n=3; p<0.05) as well as the transcription factor NFkB (n=4, p<0.05). Silencing of lectin-like oxidized LDL receptor-1 (LOX-1) using specific siRNA prevented the induction of TF and PAI-1 expression in vascular cells. In platelets cLDL activated the p38 MAP kinase and induced platelet aggregation. Finally, LDL isolated from patients with ESRD mimicked the prothrombotic effects of cLDL on vascular cells and platelets. Conclusions: These data demonstrate that cLDL at physiologically relevant concentrations,via activation of the LOX-1 receptor induces potent pro-thrombotic effects in vascular cells and platelets leading to enhanced thrombus formation in vivo. This observation may explain the markedly increased incidence of fatal acute thrombotic events in patients with end-stage renal disease and lead to the development of novel LDL targeting strategies in this population.

Torcetrapib therapy in patients with coronary artery disease does not lead to further impairment of endothelial effects of High-Density Lipoprotein (HDL)

Introduction: CETP inhibition leading to increased HDL cholesterol and reduced LDL cholesterol is currently examined as a potential strategy for prevention of cardiovascular events in patients with coronary disease. However, vascular effects of HDL are recently found to be highly heterogeneous in patients with coronary artery disease (CAD). We therefore studied in patients with CAD the effect of the CETP inhibitor by torcetrapib, that resulted in increased cardiovascular events due to side effects, to understand whether intense CETP inhibition impairs endothelial effects of HDL. Methods: HDL was isolated by sequential ultracentrifugation from patients randomized to receive either a combination of atorvastatin plus torcetrapib (60 mg/day; n=31) or atorvastation alone (n=36) at baseline and 6 months, and from healthy subjects. The effect of HDL on endothelial nitric oxide (NO) production was measured by ESR spectroscopy and on endothelial pro-inflammatory and pro-apoptotic activation determined by VCAM-1 and active caspase-3 expression assays, respectively. Results: There was no significant improvement in the capacity of HDL to stimulate endothelial NO production, reduce VCAM-1 activation or inhibit active caspase-3 expression following torcetrapib/atorvastatin treatment or atorvastatin alone in patients with CAD. These vascular effects of HDL remained largely impaired as compared to HDL from healthy subjects. Importantly, however, there was no further impairment in the capacity of HDL to stimulate endothelial NO production (10.6±52.1% vs 20.0±72.8% at baseline, p=0.33), reduce VCAM-1 activation (11.0±7.3% vs 9.0±8.1% at baseline, p=0.12) or inhibit caspase-3 activation (5.5±14.9% vs 10.0±7.3% at baseline, p=0.061) following 6-month treatment with torcetrapib. Conclusions: Despite a significant increase in HDL-C levels, torcetrapib treatment did not restore the potential anti-atherosclerotic properties of HDL on the endothelium as found in the healthy controls. Importantly, torcetrapib treatment did not further impair the endothelial effects of HDL. These data suggest that the adverse effects of torcetrapib on cardiovascular events were not caused by worsening of vascular effects of HDL.