Human high-density lipoprotein particles prevent activation of the JNK pathway induced by human oxidised low-density lipoprotein particles in pancreatic beta cells.

AIMS/HYPOTHESIS: We explored the potential adverse effects of pro-atherogenic oxidised LDL-cholesterol particles on beta cell function. MATERIALS AND METHODS: Isolated human and rat islets and different insulin-secreting cell lines were incubated with human oxidised LDL with or without HDL particles. The insulin level was monitored by ELISA, real-time PCR and a rat insulin promoter construct linked to luciferase gene reporter. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Prolonged incubation with human oxidised LDL particles led to a reduction in preproinsulin expression levels, whereas the insulin level was preserved in the presence of native LDL-cholesterol. The loss of insulin production occurred at the transcriptional levels and was associated with an increase in activator protein-1 transcriptional activity. The rise in activator protein-1 activity resulted from activation of c-Jun N-terminal kinases (JNK, now known as mitogen-activated protein kinase 8 [MAPK8]) due to a subsequent decrease in islet-brain 1 (IB1; now known as MAPK8 interacting protein 1) levels. Consistent with the pro-apoptotic role of the JNK pathway, oxidised LDL also induced a twofold increase in the rate of beta cell apoptosis. Treatment of the cells with JNK inhibitor peptides or HDL countered the effects mediated by oxidised LDL. CONCLUSIONS/INTERPRETATION: These data provide strong evidence that oxidised LDL particles exert deleterious effects in the progression of beta cell failure in diabetes and that these effects can be countered by HDL particles.

Cu and Fe metallic ions-mediated oxidation of low-density lipoproteins studied by NMR, TEM and Z-scan technique

In this work we report on a study of the morphological changes of LDL induced in vitro by metallic ions (Cu(2+) and Fe(3+)). These modifications were characterized by transmission electron microscopy, nuclear magnetic resonance and the Z-scan technique. The degree of oxidative modification of LDL was determined by the TBARS and lipid hydroperoxides assays. It is shown that distinct pathways for modifying lipoproteins lead to different morphological transformations of the particles characterized by changes in size and/or shape of the resulting particles, and by the tendency to induce aggregation of the particles. There were no evidence of melting of particles promoted by oxidative processes with Cu and Fe. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Impedimetric immunosensor for electronegative low density lipoprotein (LDL(-)) based on monoclonal antibody adsorbed on (polyvinyl formal)-gold nanoparticles matrix

Monoclonal antibodies (MAb) have been commonly applied to measure LDL in vivo and to characterize modifications of the lipids and apoprotein of the LDL particles. The electronegative low density lipoprotein (LDL(-)) has an apolipoprotein B-100 modified at oxidized events in vivo. In this work, a novel LDL-electrochemical biosensor was developed by adsorption of anti-LDL(-) MAb on an (polyvinyl formal)-gold nanoparticles (PVF-AuNPs)-modified gold electrode. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the recognition of LDL-. The interaction between MAb-LDL(-) leads to a blockage in the electron transfer of the [Fe(CN)(6)](4-)/K(4)[Fe(CN)(6)](3-) redox couple, which may could result in high change in the electron transfer resistance (R(CT)) and decrease in the amperometric responses in CV analysis. The compact antibody-antigen complex introduces the insulating layer on the assembled surface, which increases the diameter of the semicircle, resulting in a high R(CT), and the charge transferring rate constant k(0) decreases from 18.2 x 10(-6) m/s to 4.6 x 10(-6) m/s. Our results suggest that the interaction between MAb and lipoprotein can be quantitatively assessed by the modified electrode. The PVF-AuNPs-MAb system exhibited a sensitive response to LDL(-), which could be used as a biosensor to quantify plasmatic levels of LDL(-). (C) 2011 Elsevier B.V. All rights reserved.

Low density lipoprotein signaling and pancreatic beta cells protection

SummaryLow-density lipoproteins (LDLs) have an important physiological role in organism transporting cholesterol and other fatty substances to target tissues. However, elevated LDL levels in the blood are associated with the formation of arterial plaques and consequently atherosclerosis. It is therefore important to characterize the intracellular pathways induced upon LDL stimulation as they might be involved in the pathological properties of these lipoproteins. It has been previously found that LDL stimulation of mouse embryonic fibroblasts activates p38 mitogen activated protein kinases (MAPKs). This leads to cell spreading and increase in the wound healing capabilities of the cells. These two responses might occur within atherosclerotic plaques.The aim of this project is to reveal the missing links between LDL particle and activation of p38 MAPK kinase. As previously shown in our lab activation of p38 MAPK kinase by the LDL particles occur independently of classical LDL receptor (LDLR). In this study we have shown that scavenger receptor type Β class I (SR-BI) is responsible for the signal transduction from the LDLs to the p38 MAPK. We have also shown that Mitogen activated kinase kinases (MKKs) that can directly activate ρ 38 MAPK in these conditions are MKK3 and MKK6 but not MKK4. We have also tested some of the intermediate components of the pathway like Ras and PI3 kinase but found that they do not play a role.The data obtained in this study showed a part of molecular mechanism responsible for p38 MAPK activation and subsequent wound healing and can contribute to our knowledge on function of the fibroblasts in the development of the atherosclerotic plaques.Diabetes Mellitus is a condition caused by disordered metabolism of blood glucose level. It is one of the most commonly spread disease in the western world, with the incidence reaching 8% of population in United States. Two most common types of diabetes are type 1 and 2 that differs slightly in the mechanism of the development. However in the basis of both types lies the cell death of pancreatic beta cells. The aim of this work is to improve beta cells survival in different pathophysiological settings. This could be extrapolated to the conditions in which Diabetes develops in humans. We decided to use RasGAP- derived fragment Ν with its strong antiapoptotic effect in beta cells. In our lab we have demonstrated that in the mild stress conditions RasGAP can be cleaved by caspases at the position 455 producing two fragments, fragment Ν and fragment C. Fragment Ν exerts

Modification of Linear Low Density Polyethylene for Improved Photo and Biodegradation

LLDPE was blended with poly (vinyl alcohol) and mechanical, thermal, spectroscopic properties and biodegradability were investigated. The biodegradability of LLDPE/PVA blends has been studied in two environments, viz. (1) a culture medium containing Vibrio sp. and (2) a soil environment over a period of 15 weeks. Nanoanatase having photo catalytic activity was synthesized by hydrothermal method using titanium-iso-propoxide. The synthesized TiO2 was characterized by X-Ray diffraction (XRD), BET studies, FTIR studies and scanning electron microscopy (SEM). The crystallite size of titania was calculated to be ≈ 6nm from the XRD results and the surface area was found to be about 310m2/g by BET method. SEM shows that nanoanatase particles prepared by this method are spherical in shape. Linear low density polyethylene films containing polyvinyl alcohol and a pro-oxidant (TiO2 or cobalt stearate with or without vegetable oil) were prepared. The films were then subjected to natural weathering and UV exposure followed by biodegradation in culture medium as well as in soil environment. The degradation was monitored by mechanical property measurements, thermal studies, rate of weight loss, FTIR and SEM studies. Higher weight loss, texture change and greater increments in carbonyl index values were observed in samples containing cobalt stearate and vegetable oil. The present study demonstrates that the combination of LLDPE/PVA blends with (I) nanoanatase/vegetable oil and (ii) cobalt stearate/vegetable oil leads to extensive photodegradation. These samples show substantial degradation when subsequent exposure to Vibrio sp. is made. Thus a combined photodegradation and biodegradation process is a promising step towards obtaining a biodegradable grade of LLDPE.

Antioxidant activity and protective effects of green and dark coffee components against human low density lipoprotein oxidation

The in vitro antioxidant activity and the protective effect against human low density lipoprotein oxidation of coffees prepared using different degrees of roasting was evaluated. Coffees with the highest amount of brown pigments (dark coffee) showed the highest peroxyl radical scavenging activity. These coffees also protected human low-density lipoprotein (LDL) against oxidation, although green coffee extracts showed more protection. In a different experiment, coffee extracts were incubated with human plasma prior to isolation of LDL particles. This showed, for the first time, that incubation of plasma with dark, but not green coffee extracts protected the LDL against oxidation by copper or by the thermolabile azo compound AAPH. Antioxidants in the dark coffee extracts must therefore have become associated with the LDL particles. Brown compounds, especially those derived from the Maillard reaction, are the compounds most likely to be responsible for this activity.

Mathematical model for low density lipoprotein (LDL) endocytosis by hepatocytes

Individuals with elevated levels of plasma low density lipoprotein (LDL) cholesterol (LDL-C) are considered to be at risk of developing coronary heart disease. LDL particles are removed from the blood by a process known as receptor-mediated endocytosis, which occurs mainly in the liver. A series of classical experiments delineated the major steps in the endocytotic process; apolipoprotein B-100 present on LDL particles binds to a specific receptor (LDL receptor, LDL-R) in specialized areas of the cell surface called clathrin-coated pits. The pit comprising the LDL-LDL-R complex is internalized forming a cytoplasmic endosome. Fusion of the endosome with a lysosome leads to degradation of the LDL into its constituent parts (that is, cholesterol, fatty acids, and amino acids), which are released for reuse by the cell, or are excreted. In this paper, we formulate a mathematical model of LDL endocytosis, consisting of a system of ordinary differential equations. We validate our model against existing in vitro experimental data, and we use it to explore differences in system behavior when a single bolus of extracellular LDL is supplied to cells, compared to when a continuous supply of LDL particles is available. Whereas the former situation is common to in vitro experimental systems, the latter better reflects the in vivo situation. We use asymptotic analysis and numerical simulations to study the longtime behavior of model solutions. The implications of model-derived insights for experimental design are discussed.

Inter-relationships between small, dense low-density lipoprotein (LDL), plasma triacylglycerol and LDL apoprotein B in an atherogenic lipoprotein phenotype in free-living subjects

A predominance of small, dense low-density lipoprotein (LDL) is a major component of an atherogenic lipoprotein phenotype, and a common, but modifiable, source of increased risk for coronary heart disease in the free-living population. While much of the atherogenicity of small, dense LDL is known to arise from its structural properties, the extent to which an increase in the number of small, dense LDL particles (hyper-apoprotein B) contributes to this risk of coronary heart disease is currently unknown. This study reports a method for the recruitment of free-living individuals with an atherogenic lipoprotein phenotype for a fish-oil intervention trial, and critically evaluates the relationship between LDL particle number and the predominance of small, dense LDL. In this group, volunteers were selected through local general practices on the basis of a moderately raised plasma triacylglycerol (triglyceride) level (>1.5 mmol/l) and a low concentration of high-density-lipoprotein cholesterol (<1.1 mmol/l). The screening of LDL subclasses revealed a predominance of small, dense LDL (LDL subclass pattern B) in 62% of the cohort. As expected, subjects with LDL subclass pattern B were characterized by higher plasma triacylglycerol and lower high-density lipoprotein cholesterol (<1.1 mmol/l) levels and, less predictably, by lower LDL cholesterol and apoprotein B levels (P<0.05; LDL subclass A compared with subclass B). While hyper-apoprotein B was detected in only five subjects, the relative percentage of small, dense LDL-III in subjects with subclass B showed an inverse relationship with LDL apoprotein B (r=-0.57; P<0.001), identifying a subset of individuals with plasma triacylglycerol above 2.5 mmol/l and a low concentration of LDL almost exclusively in a small and dense form. These findings indicate that a predominance of small, dense LDL and hyper-apoprotein B do not always co-exist in free-living groups. Moreover, if coronary risk increases with increasing LDL particle number, these results imply that the risk arising from a predominance of small, dense LDL may actually be reduced in certain cases when plasma triacylglycerol exceeds 2.5 mmol/l.

Investigation of the Europium Emission Spectra of the Europium-Oxytetracycline Complex in the Presence of Human Low-Density Lipoproteins

Low-Density Lipoprotein (LDL), often known as ""bad cholesterol"" is one of the responsible to increase the risk of coronary arterial diseases. For this reason, the cholesterol present in the LDL particle has become one of the main parameters to be quantified in routine clinical diagnosis. A number of tools are available to assess LDL particles and estimate the cholesterol concentration in the blood. The most common methods to quantify the LDL in the plasma are the density gradient ultracentrifugation and nuclear magnetic resonance (NMR). However, these techniques require special equipments and can take a long time to provide the results. In this paper, we report on the increase of the Europium emission in Europium-oxytetracycline complex aqueous solutions in the presence of LDL. This increase is proportional to the LDL concentration in the solution. This phenomenum can be used to develop a method to quantify the number of LDL particles in a sample. A comparison between the performances of the oxytetracycline and the tetracycline in the complexes is also made.

Measurement of the nonlinear optical response of low-density lipoprotein solutions from patients with periodontitis before and after periodontal treatment: evaluation of cardiovascular risk markers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phenol degradation in an anaerobic fluidized bed reactor packed with low density support materials

The objective of this research was to study phenol degradation in anaerobic fluidized bed reactors (AFBR) packed with polymeric particulate supports (polystyrene - PS, polyethylene terephthalate - PET, and polyvinyl chloride - PVC). The reactors were operated with a hydraulic retention time (HRT) of 24 h. The influent phenol concentration in the AFBR varied from 100 to 400 mg L-1, resulting in phenol removal efficiencies of similar to 100%. The formation of extracellular polymeric substances yielded better results with the PVC particles; however, deformations in these particles proved detrimental to reactor operation. PS was found to be the best support for biomass attachment in an AFBR for phenol removal. The AFBR loaded with PS was operated to analyze the performance and stability for phenol removal at feed concentrations ranging from 50 to 500 mg L-1. The phenol removal efficiency ranged from 90-100%.

Behavior of the thermal diffusivity of native and oxidized human low-density lipoprotein solutions studied by the Z-scan technique

Modifications in low-density lipoprotein (LDL) have emerged as a major pathogenic factor of atherosclerosis, which is the main cause of morbidity and mortality in the western world. Measurements of the heat diffusivity of human LDL solutions in their native and in vitro oxidized states are presented by using the Z-Scan (ZS) technique. Other complementary techniques were used to obtain the physical parameters necessary to interpret the optical results, e. g., pycnometry, refractometry, calorimetry, and spectrophotometry, and to understand the oxidation phase of LDL particles. To determine the sample's thermal diffusivity using the thermal lens model, an iterative one-parameter fitting method is proposed which takes into account several characteristic ZS time-dependent and the position-dependent transmittance measurements. Results show that the thermal diffusivity increases as a function of the LDL oxidation degree, which can be explained by the increase of the hydroperoxides production due to the oxidation process. The oxidation products go from one LDL to another, disseminating the oxidation process and caring the heat across the sample. This phenomenon leads to a quick thermal homogenization of the sample, avoiding the formation of the thermal lens in highly oxidized LDL solutions. (C) 2012 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.JBO.17.10.105003]

Atherogenic lipoprotein phenotype and low-density lipoprotein size and subclasses in patients with growth hormone deficiency before and after short-term replacement therapy

Patients with growth hormone deficiency (GHD) have increased cardiovascular risk and may show elevated triglyceride and reduced high density lipoprotein (HDL) cholesterol concentrations, two lipid abnormalities usually accompanied by increased small dense LDL in the 'atherogenic lipoprotein phenotype' (ALP). In the present study, we directly investigated (1) whether hypopituitary patients with GHD have increased small dense LDL; (2) whether growth hormone replacement therapy (GHRT) beneficially impact on such particles; (3) the prevalence of ALP in GHD and GHRT patients.

Effects of growth hormone (GH) replacement therapy on low-density lipoprotein apolipoprotein B100 kinetics in adult patients with GH deficiency: a stable isotope study

GH replacement therapy has been shown to improve the dyslipidemic condition in a substantial proportion of patients with adult GH deficiency. The mechanisms are not yet fully elucidated. Low-density lipoprotein (LDL) apolipoprotein B100 (apoB) formation and catabolism are important determinants of plasma cholesterol concentrations. This study examined the effect of GH replacement therapy on LDL apoB metabolism using a stable isotope turnover technique. LDL apoB kinetics was determined in 13 adult patients with GH deficiency before and after 3 months GH/placebo treatment in a randomized, double-blind, placebo-controlled study. LDL apoB (13)C-leucine enrichment was determined by isotope-ratio mass spectrometry. Plasma volume was assessed by standardized radionuclide dilution technique. GH replacement therapy significantly decreased LDL cholesterol, LDL apoB concentrations, and LDL apoB pool size compared with placebo. Compared with baseline, GH replacement therapy resulted in a significant increase in plasma volume and fractional catabolic rate, whereas LDL formation rate remained unchanged. LDL lipid content did not significantly change after GH and placebo. This study suggests that short-term GH replacement therapy decreases the LDL apoB pool by increasing removal of LDL particles without changing LDL composition or LDL apoB production rate. In addition, it is possible that the beneficial effects of GH on the cardiovascular system contribute to these findings.

Interactions between cyclosporin A, low-density lipoprotein and the low-density lipoprotein receptor

Cyclosporine A (CSA) is a cyclic eleven amino acid, lipophilic molecule used therapeutically as an immunosuppressive agent. Cyclosporine can specifically inhibit the transcription of a number of different genes. It is known that CSA is bound almost exclusively to lipoproteins in plasma, however, the relationship between the low density lipoprotein (LDL), the LDL receptor, and CSA has not been fully elucidated. The exact mechanism of cellular uptake of CSA is unknown, but it is believed to be by simple passive diffusion across the cell membrane. In addition, it has been recently shown that the frequent finding of hypercholesterolemia seen in patients treated with CSA can be explained by a CSA-induced effect. The mechanism by which CSA induces hypercholesterolemia is not known. We have used an LDL receptor-deficient animal model, the Watanabe Heritable Hyperlipidemic (WHHL) rabbit to investigate the role of LDL and the LDL receptor in the cellular uptake of CSA. Using this animal model, we have shown that CSA uptake by lymphocytes is predominantly LDL receptor-mediated. Chemical modification of apoB-100 on LDL particles abolishes their ability to bind to the LDL receptor. When CSA is incubated with modified LDL much less is taken-up than when native LDL is incubated with CSA. Treatment of two human cell lines with CSA results in a dose-dependent decrease in LDL receptor mRNA levels. Using a novel transfection system involving the 5$\sp\prime$-flanking region of the LDL receptor gene, we have found that CSA decreases the number of transcripts, but is dependent on whether or not cholesterol is present and the stage of growth of the cells. ^