Human Low Density Lipoprotein Research Articles

Malondialdehyde-Acetaldehyde Modified (MAA) Proteins Differentially Effect the Inflammatory Response in Macrophage, Endothelial Cells and Animal Models of Cardiovascular Disease.

Chronic inflammation plays a critical role in the pathogenesis of atherosclerosis. Currently, the mechanism(s) by which inflammation contributes to this disease are not entirely understood. Inflammation is known to induce oxidative stress, which can lead to lipid peroxidation. Lipid peroxidation can result in the production of reactive by-products that can oxidatively modify macromolecules including DNA, proteins, and lipoproteins. A major reactive by-product of lipid peroxidation is malondialdehyde (MDA). MDA can subsequently break down to form acetaldehyde (AA). These two aldehydes can covalently interact with the epsilon (ε)-amino group of lysines within proteins and lipoproteins leading to the formation of extremely stable, highly immunogenic malondialdehyde/acetaldehyde adducts (MAA-adducts). The aim of this study was to investigate the inflammatory response to MAA-modified human serum albumin (HSA-MAA) and low-density lipoprotein (LDL-MAA). We found that animals injected with LDL-MAA generate antibodies specific to MAA-adducts. The level of anti-MAA antibodies were further increased in an animal model of atherosclerosis fed a Western diet. An animal model that combined both high fat diet and immunization of MAA-modified protein resulted in a dramatic increase in antibodies to MAA-adducts and vascular fat accumulation compared with controls. In vitro exposure of endothelial cells and macrophages to MAA-modified proteins resulted in increased fat accumulation as well as increased expression of adhesion molecules and pro-inflammatory cytokines. The expression of cytokines varied between the different cell lines and was unique to the individual modified proteins. The results of these studies demonstrate that different MAA-modified proteins elicit unique responses in different cell types. Additionally, the presence of MAA-modified proteins appears to modulate cellular metabolism leading to increased accumulation of triglycerides and further progression of the inflammatory response.

Lipid Lowering Natural Products Targeting hPCSK9 May Prevent the Severity of COVID-19 Infection

Background: The deadly outbreak of COVID-19 disease caused by novel SARS CoV2 has created an unprecedented global health crisis affecting every sectors of human life and enormous damage to world’s economy. With >16.1 million infections and >650,000 deaths worldwide as of July 27, 2020, there is no treatment for this disease neither is there any available vaccine. Serious research efforts are ongoing on all fronts including treatment, prevention and diagnosis to combat the spread of this infection. A number of targets that include both viral and host proteins have been identified and became part of intense investigation. In this respect the viral surface spike (S) glycoprotein caught the attention most. It is cleaved by multiple host proteases to allow recognition by host receptor human Angiotensin Converting Enzyme2 (hACE2) leading to fusion and viral replication. Natural products, small compounds, antioxidants, peptides, proteins, oligonucleotides, antibodies and other compounds are under investigation for development of antiviral agents against COVID- 19. Objective: Recently cholesterol lowering phytocompounds Quercetin, Swertiamarin and Berberine which promote human Low Density Lipoprotein Receptor (hLDLR) via inhibition of human Proprotein Convertase Subtilisin Kexin9 (hPCSK9) have been shown to block viral infections caused by ebola, influenza, Respiratory Syncytial Virus (RSV), Hepatitis C virus (HCV) and other RNA type viruses. Since SARS CoV2 is a RNA virus with similar genetic structure and infection machinery, it is hypothesised that these phytocompounds may also exhibit antiviral property against COVID-19. Methods: Our above concept is based on recently published studies as well as our herein presented in silico modeling and computational data which suggested strong interactions of hPCSK9 with above phytocompounds and most importantly with hACE2 following its complexation with receptor binding domain (RBD) of SARS CoV2 S protein. Results: These results and a proposed schematic model showing association of hPCSK9 with SARS CoV2 infection are presented in this manuscript. It is proposed that hPCSK9 plays the role of a co-receptor in binding with hACE2:RBD complex and thereby facilitates viral fusion. Conclusion: Our studies suggest that PCSK9 inhibitors may provide beneficial effect against COVID-19 infection by retarding viral fusion through displacement of bound hPCSK9 from its complex with ACE2:RBD of SARS CoV2 S protein.

Human low-density lipoprotein receptor plays an important role in hepatitis B virus infection.

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, resulting in chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV vaccine is effective to prevent new HBV infection but does not offer therapeutic benefit to hepatitis B patients. Neither are current antiviral drugs curative of chronic hepatitis B. A more thorough understanding of HBV infection and replication holds a great promise for identification of novel antiviral drugs and design of optimal strategies towards the ultimate elimination of chronic hepatitis B. Recently, we have developed a robust HBV cell culture system and discovered that human apolipoprotein E (apoE) is enriched on the HBV envelope and promotes HBV infection and production. In the present study, we have determined the role of the low-density lipoprotein receptor (LDLR) in HBV infection. A LDLR-blocking monoclonal antibody potently inhibited HBV infection in HepG2 cells expressing the sodium taurocholate cotransporting polypeptide (NTCP) as well as in primary human hepatocytes. More importantly, small interfering RNAs (siRNAs)-mediated knockdown of LDLR expression and the CRISPR/Cas9-induced knockout of the LDLR gene markedly reduced HBV infection. A recombinant LDLR protein could block heparin-mediated apoE pulldown, suggesting that LDLR may act as an HBV cell attachment receptor via binding to the HBV-associated apoE. Collectively, these findings demonstrate that LDLR plays an important role in HBV infection probably by serving as a virus attachment receptor.

High-Throughput Screening Identifies MicroRNAs Regulating Human PCSK9 and Hepatic Low-Density Lipoprotein Receptor Expression.

Investigations into the regulatory mechanisms controlling cholesterol homeostasis have proven fruitful in identifying low-density lipoprotein (LDL)-lowering therapies to reduce the risk of atherosclerotic cardiovascular disease. A major advance was the discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9), a secreted protein that binds the LDL receptor (LDLR) on the cell surface and internalizes it for degradation, thereby blunting its ability to take up circulating LDL. The discovery that loss-of-function mutations in PCSK9 lead to lower plasma levels of LDL cholesterol and protection from cardiovascular disease led to the therapeutic development of PCSK9 inhibitors at an unprecedented pace. However, there remain many gaps in our understanding of PCSK9 regulation and biology, including its posttranscriptional control by microRNAs. Using a high-throughput region(3′-UTR) of human microRNA library screen, we identified microRNAs targeting the 3′ untranslated region of human PCSK9. The top 35 hits were confirmed by large-format PCSK9 3′-UTR luciferase assays, and 10 microRNAs were then selected for further validation in hepatic cells, including effects on PCSK9 secretion and LDLR cell surface expression. These studies identified seven novel microRNAs that reduce PCSK9 expression, including miR-221-5p, miR-342-5p, miR-363-5p, miR-609, miR-765, and miR-3165. Interestingly, several of these microRNAs were also found to target other genes involved in LDLR regulation and potently upregulate LDLR cell surface expression in hepatic cells. Together, these data enhance our understanding of post-transcriptional regulators of PCSK9 and their potential for therapeutic manipulation of hepatic LDLR expression.

Взаємозв’язок активності матриксної металопротеїнази-2 та -9 в гомогенаті міокарда зі змінами клітинного складу стінки вінцевих артерій в експерименті

Matrix metalloproteinases are involved in a complex multifactorial process of atherosclerotic plaque formation and play a leading role in the degradation of the extracellular matrix components and increase the migratory activity of cellular elements of the vascular wall. Despite a number of scientific studies, it is necessary to identify clear biochemical markers for the development of atherosclerotic lesions. The purpose of the study was to investigate the relationship between the activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 in myocardial homogenate with changes of the cells composition in the coronary arteries wall in experimental atherosclerosis. Materials and methods. The studies were performed on 76 nonlinear rats, which were divided into 3 groups: group Ia was the control (n=20) – animals, injected intracutaneously with 0.1 ml of 0.9 % sodium chloride solution; Ib – comparison group (n=20) – animals injected with incomplete Freund’s adjuvant at the dose of 0.1 ml intracutaneously and II – experimental group (n=36), which were immunized with native human low-density lipoprotein at a single dose of 200 μg, diluted in 0.1 ml of incomplete Freund’s adjuvant, regardless of the weight. The experiment lasted for 20 weeks. Material sampling was performed, starting from the 4th week after the drug administration. From the coronary arteries and the adjacent myocardium, microslides were made according to the generally accepted technique, which were stained with hematoxylin and eosin, according to the methods of Van Gizon, Mallory and Sudan III. The activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 was determined by enzyme-zimografic method. Results and discussion. The growth of matrix metalloproteinase-2 and matrix metalloproteinase-9 activity in atherosclerotic lesions showed stages of degradation of extracellular matrix components: the dynamics of matrix metalloproteinase-2 activity during the experiment increased from 109.1±1.23 % at the 12th week to 127.32±0.99 % at the 20th week. The increased activity of matrix metalloproteinase-2 was associated with an increase in the number of leukocytes and macrophages, including foam cells. The activity of matrix metalloproteinase-9 reached the highest values of 98.24±0.82% at the 8th period from the onset of changes to the final level of 86.26±0.54% at the maximum terms of the experiment. Conclusion. The growing activity of matrix metalloproteinase-9 indicated the development of early atherosclerotic lesions, while the high level of activity of matrix metalloproteinase-2 indicated significant structural changes in the wall of the coronary arteries

Application of enzymatic fluorometric assays to quantify phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in human plasma lipoproteins

Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) are important surface components of plasma lipoproteins, including very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). However, the pathophysiological roles of PC, PE and SM in lipoproteins have not been well characterized owing to the difficulties in quantifying phospholipid classes in lipoproteins. In this study, we assessed the precision and accuracy of the enzymatic fluorometric assays for measuring PC, PE and SM in VLDL, LDL and HDL, which were isolated from human plasma by ultracentrifugation. The within-run coefficients of variation (CV) for the measurements of PC, PE and SM in lipoproteins were 1.5–2.8 %, 1.1–2.4 % and 0.9–2.3 %, respectively, whereas the between-run CVs for the PC, PE and SM assays were 2.7–4.7 %, 2.1–4.5 % and 1.6–3.3 %, respectively. Excellent linearity and almost complete recovery were achieved for all assays measuring PC, PE and SM in VLDL, LDL and HDL. Our preliminary results using these enzymatic fluorometric assays suggested that the phospholipid compositions were different among VLDL, LDL and HDL. In conclusion, we established high-throughput enzymatic fluorometric assays to quantify PC, PE and SM in human plasma VLDL, LDL and HDL, which will be useful for further investigation of pathophysiological roles of phospholipids in lipoproteins.

Activation of pregnane X receptor induces atherogenic lipids and PCSK9 by a SREBP2-mediated mechanism.

Many drugs and environmental contaminants induce hypercholesterolemia and promote the risk of atherosclerotic cardiovascular disease. We tested the hypothesis that pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor, regulates the level of circulating atherogenic lipids in humans and utilized mouse experiments to identify the mechanisms involved. We performed serum NMR metabolomics in healthy volunteers administered rifampicin, a prototypical human PXR ligand or placebo in a crossover setting. We used high-fat diet fed wild-type and PXR knockout mice to investigate the mechanisms mediating the PXR-induced alterations in cholesterol homeostasis. Activation of PXR induced cholesterogenesis both in pre-clinical and clinical settings. In human volunteers, rifampicin increased intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) and total cholesterol and lathosterol-cholesterol ratio, a marker of cholesterol synthesis, suggesting increased cholesterol synthesis. Experiments in mice indicated that PXR activation causes widespread induction of the cholesterol synthesis genes including the rate-limiting Hmgcr and upregulates the intermediates in the Kandutsch-Russell cholesterol synthesis pathway in the liver. Additionally, PXR activation induced plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a negative regulator of hepatic LDL uptake, in both mice and humans. We propose that these effects were mediated through increased proteolytic activation of sterol regulatory element-binding protein 2 (SREBP2) in response to PXR activation. PXR activation induces cholesterol synthesis, elevating LDL and total cholesterol in humans. The PXR-SREBP2 pathway is a novel regulator of the cholesterol and PCSK9 synthesis and a molecular mechanism for drug- and chemical-induced hypercholesterolemia.

Autoimmune Rheumatic Diseases: An Update on the Role of Atherogenic Electronegative LDL and Potential Therapeutic Strategies.

Atherosclerosis has been linked with an increased risk of atherosclerotic cardiovascular disease (ASCVD). Autoimmune rheumatic diseases (AIRDs) are associated with accelerated atherosclerosis and ASCVD. However, the mechanisms underlying the high ASCVD burden in patients with AIRDs cannot be explained only by conventional risk factors despite disease-specific factors and chronic inflammation. Nevertheless, the normal levels of plasma low-density lipoprotein (LDL) cholesterol observed in most patients with AIRDs do not exclude the possibility of increased LDL atherogenicity. By using anion-exchange chromatography, human LDL can be divided into five increasingly electronegative subfractions, L1 to L5, or into electropositive and electronegative counterparts, LDL (+) and LDL (−). Electronegative L5 and LDL (−) have similar chemical compositions and can induce adverse inflammatory reactions in vascular cells. Notably, the percentage of L5 or LDL (−) in total LDL is increased in normolipidemic patients with AIRDs. Electronegative L5 and LDL (−) are not recognized by the normal LDL receptor but instead signal through the lectin-like oxidized LDL receptor 1 (LOX-1) to activate inflammasomes involving interleukin 1β (IL-1β). Here, we describe the detailed mechanisms of AIRD-related ASCVD mediated by L5 or LDL (−) and discuss the potential targeting of LOX-1 or IL-1β signaling as new therapeutic modalities for these diseases.

Clearance of Carbonyl-Modified Low-Density Lipoproteins in Rabbits

The elimination kinetics of carbonyl-modified low density lipoproteins (LDL) from rabbit bloodstream was studied using isolated LDL of rabbits and humans after preliminary biotinylation or labeling with FITC. Rabbit or human blood plasma LDL were isolated using differential ultracentrifugation in a density gradient; after labeling by biotinylation or by FITC, LDL were modified with various low molecular weight natural dicarbonyls: malondialdehyde (MDA), glyoxal or methylglyoxal. Native (control) and dicarbonyl-modified biotinylated or FITC-labeled LDL were injected into the ear vein of rabbits, and blood samples were taken at certain time intervals. The content of biotinylated LDL in blood plasma was determined by an enzyme immunoassay method; FITC-labeled LDL was determined from the fluorescence spectra. It has been found that glyoxal- and methylglyoxal-modified rabbit and human LDL circulate in the bloodstream of rabbits for almost the same period as native (unmodified) LDL. In contrast to this, MDA-modified rabbit and human LDL were very quickly eliminated from the rabbit bloodstream. Dicarbonyl-modified LDL from human blood plasma is not associated with red blood cells or endothelial cells. We found that using the Oxidized LDL ELISA kits (Mercodia, Sweden) it was possible to identify mainly MDA-modified LDL. The level of MDA-modified LDL in the blood plasma of CHD patients sharply decreased during therapy with evolocumab, the hypocholesterolemic inhibitor of PCSK9 (proprotein convertase of subtilisin/kexin type 9), which activates LDL reutilization in the liver cells. These results explain the extremely rapid clearance of MDA-modified LDL in our experiments by their increased utilization in hepatocytes. The results obtained indicate a high atherogenicity of glyoxal- and methylglyoxal-modified LDL, long-term circulating in the bloodstream.

Antioxidant Properties of Galanin and Its N-Terminal Fragments in in vitro and in vivo Oxidative Stress Modeling.

Antioxidant properties of rat galanin GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2 (Gal), N-terminal fragment of galanin (2-15 aa) WTLNSAGYLLGPHA (G1), and its modified analogue WTLNSAGYLLGPβAH (G2) were studied in vivo in the rat model of regional myocardial ischemia and reperfusion and in vitro in the process of Cu2+-induced free radical oxidation of human blood plasma low-density lipoproteins. Intravenous administration of G1, G2, and Gal to rats after ischemia induction reduced the infarction size and activities of the necrosis markers, creatine kinase-MB and lactate dehydrogenase, in blood plasma at the end of reperfusion. G1, G2, and Gal reduced formation of the spin adducts of hydroxyl radicals in the interstitium of the area at risk during reperfusion, moreover, G2 and Gal also reduced formation of the secondary products of lipid peroxidation in the reperfused myocardium. It was shown in the in vivo experiments and in the in vitro model system that the ability of galanin peptides to reduce formation of ROS and attenuate lipid peroxidation during myocardial reperfusion injury was not associated directly with their effects on activities of the antioxidant enzymes of the heart: Cu,Zn-superoxide dismutase, catalase, and glutathione peroxidase. The peptides G1, G2, and Gal at concentrations of 0.01 and 0.1 mM inhibited Cu2+-induced free radical oxidation of human low-density lipoproteins in vitro. The results of oxidative stress modeling demonstrated that the natural and synthetic agonists of galanin receptors reduced formation of the short-lived ROS in the reperfused myocardium, as well as of lipid radicals in blood plasma. Thus, galanin receptors could be a promising therapeutic target for cardiovascular diseases.

Riboflavin-Sensitized Photooxidation of Low-Density-Lipoprotein (LDL) Cholesterol: A Culprit in the Development of Cardiovascular Diseases (CVDs).

The oxidation of human low-density-lipoprotein (LDL) particles is responsible for the development of cardiovascular diseases (CVDs). In the present study, the occurrence of riboflavin-sensitized photooxidation of LDL particles was examined in an in vitro system. The presence of light, oxygen, and photosensitizer (50 μM riboflavin) caused the riboflavin-sensitized photooxidation of human LDL particles thereby increasing in the conjugated dienes (CDs) by 32.5 ± 4.8% (p < 0.05), indicating that this could serve as a major culprit in the development of CVDs. A 1 h radiation caused a 63.6 ± 0.3% degradation of the riboflavin content, and this indicates the extremely fast reaction of the riboflavin-sensitized photooxidation. The singlet oxygen quenching capacity of β-carotene was determined at three different concentrations (10, 50, and 100 μM), exhibiting both antioxidant and prooxidant effects, depending on the concentrations used. In addition, ascorbic acid displayed a high incorporation rate into the LDL particles, implying its potential in preventing riboflavin photosensitization of LDL particles. To the best of our knowledge, this is the first report on the riboflavin-sensitized photooxidation of LDL particles in an in vitro system, proposing a new possible mechanism in the development of CVDs.

Assessment of genetic diversity of Turkish maize landraces for phytic acid and total phenolic contents

Assessment of genetic diversity of Turkish maize landraces for phytic acid and total phenolic contents

Inhibition of low-density lipoprotein oxidation, antioxidative and bile acid-binding capacities of hydrolyzed proteins from carbohydrase-treated oat bran.

This study investigated the valorization of oat bran and the use of its proteins to generate polypeptides with antioxidant and bile acid-binding properties. Ten protein hydrolysates were prepared by treating cellulase (CPI) or Viscozyme (VPI) protein isolates with five proteases. VPI-pepsin was the best peroxyl radical scavenger (497±6-μM Trolox equivalents [TE]/g) while VPI-Flavourzyme quenched hydroxyl radicals (28±0.6) and VPI-pepsin superoxide anion radicals (45.3±6.6%). Hydrolysates, except those produced with pepsin, dose-dependently chelated iron whereas VPI-Protamex had the best copper-chelating capacity (59.83±1.40%). These antioxidative capacities were important in preventing by 50% in vitro copper-induced oxidation of human low-density lipoprotein. Furthermore, due to their aromatic amino acid contents and hydrophobicity, the hydrolysates bound up to 46.3% the bile acids taurodeoxycholate and taurocholate. PRACTICAL APPLICATIONS: The presence of oxidants in foods can damage food molecules and decrease their quality. They are also known to increase the risk of developing chronic conditions like cardiovascular disease. Finding new antioxidant molecules are therefore useful in the management of chronic diseases. Data from this work showed that hydrolyzed oat bran proteins can be useful in stabilizing commercial oil as they reduced the oxidation of peanut oil. Additionally, the protein hydrolysates not only prevented the oxidation of linoleic, a common component of both vegetable oils and biological cell membranes, they also inhibited the oxidation of human LDL cholesterol and chelated bile acids. These hydrolysates can then be further explored as multifunctional ingredients for the development of stable functional food products with potential beneficial effects on the cardiovascular system.

Optimization of microwave extraction method of phenolic compounds from red onion using response surface methodology and inhibition of lipoprotein low-density oxidation

In the present study, we have optimized by surface response methodology (BBD model) phenolic compounds recovery using microwave-assisted extraction (MAE) as an innovative technique. The extraction was carried out under different experimental conditions: ethanol/water (x1, 30–80 %), time (x2, 30–180 s), microwave power (x3, 500–900 W) and solvent to sample ratio (x4, 20:1 40:1, v/w). Total phenolics content (TPC) and antioxidant activities (AA) of onion extract were investigated on 2,2−9-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS+), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), oxygen radical absorbance capacity (ORAC) tests, and on biological model oxidation, human low-density lipoprotein (LDL). The maximum predicted TPC under the optimal MAE conditions (70 % ethanol), 700 W microwave power, 65 s extraction time and 25 mL g–1 solvent to solid ratio), was 10.90 ± 0.88 mg GAE g–1 DW. The AA of MAE or conventional extraction (CE) extract showed similar activities to neutralize DPPH, ABTS or AAPH derived radicals. The modified Gompertz model has been successfully used to describe CDs production kinetic in LDL Cu2+-mediated oxidation. This allowed the estimation of the lag time (Tlag) and the oxidation propagation rate (Rp) with precision. LDL oxidation assay showed that red onion extract increased significantly Tlag and giving an increased protection ratio at higher concentration (2.95 μmol L−1) of 180 % and 172 % for MAE and CE methods, respectively. However, Rp and CDs production remained constant. Results showed that microwave used for extraction does not affect the composition and antioxidant properties of onion sample. Red onion may be an important source of bioactive compounds and could promote health consumers.

Neuraminidases 1 and 3 Trigger Atherosclerosis by Desialylating Low-Density Lipoproteins and Increasing Their Uptake by Macrophages.

BackgroundChronic vascular disease atherosclerosis starts with an uptake of atherogenic modified low‐density lipoproteins (LDLs) by resident macrophages, resulting in formation of arterial fatty streaks and eventually atheromatous plaques. Increased plasma sialic acid levels, increased neuraminidase activity, and reduced sialic acid LDL content have been previously associated with atherosclerosis and coronary artery disease in human patients, but the mechanism underlying this association has not been explored.Methods and ResultsWe tested the hypothesis that neuraminidases contribute to development of atherosclerosis by removing sialic acid residues from glycan chains of the LDL glycoprotein and glycolipids. Atherosclerosis progression was investigated in apolipoprotein E and LDL receptor knockout mice with genetic deficiency of neuraminidases 1, 3, and 4 or those treated with specific neuraminidase inhibitors. We show that desialylation of the LDL glycoprotein, apolipoprotein B 100, by human neuraminidases 1 and 3 increases the uptake of human LDL by human cultured macrophages and by macrophages in aortic root lesions in Apoe−/− mice via asialoglycoprotein receptor 1. Genetic inactivation or pharmacological inhibition of neuraminidases 1 and 3 significantly delays formation of fatty streaks in the aortic root without affecting the plasma cholesterol and LDL levels in Apoe−/− and Ldlr−/− mouse models of atherosclerosis.ConclusionsTogether, our results suggest that neuraminidases 1 and 3 trigger the initial phase of atherosclerosis and formation of aortic fatty streaks by desialylating LDL and increasing their uptake by resident macrophages.

Discrimination between minimally modified LDL and fully oxidized LDL using monoclonal antibodies

Low density lipoprotein (LDL) can be oxidized in a stepwise process that leads to the production of minimally modified low density lipoprotein (mm-LDL), in which only the lipid component is oxidized, and then of fully oxidized LDL (oxLDL), in which both the lipids and the protein are oxidized. The thiobarbituric acid-reactive substances (TBARS) assay is a recognized method for determination of oxidized LDL, however this method is unable to distinguish between mm-LDL and oxLDL. In this study, seven specific monoclonal antibodies (mAbs) against human LDL were generated and selectively bound to the apolipoprotein B-100 (apoB-100) component of LDL. Oxidized LDL was produced by incubation of human LDL with 10 μM CuSO4 for various times. The TBARS assay revealed that the optimal incubation time to achieve maximal lipid oxidation was 9 h. Indirect ELISA using the newly generated mAbs was implemented to differentiate between mm-LDL and oxLDL and it was found that binding of the mAbs to oxLDL was significantly decreased after 48 h of incubation, reflecting the oxidative modification of apoB-100. Our results suggest that the optimal times for incubation of LDL with CuSO4 for generation of mm-LDL and oxLDL were 9 h and 48 h, respectively.

Radiation Impacts Early Atherosclerosis by Suppressing Intimal LDL Accumulation

Rationale: Bone marrow transplantation (BMT) is used frequently to study the role of hematopoietic cells in atherosclerosis, but aortic arch lesions are smaller in mice after BMT. Objective: To identify the earliest stage of atherosclerosis inhibited by BMT and elucidate potential mechanisms. Methods and Results: Ldlr-/- mice underwent total body γ-irradiation, bone marrow reconstitution and 6-week recovery. Atherosclerosis was studied in the ascending aortic arch and compared to mice without BMT. In BMT mice neutral lipid and myeloid cell topography were lower in lesions after feeding a cholesterol-rich diet (CRD) for 3, 6 and 12 weeks. Lesion coalescence and height were suppressed dramatically in mice post-BMT, whereas lateral growth was inhibited minimally. Targeted radiation to the upper thorax alone reproduced the BMT phenotype. Classical monocyte recruitment, intimal myeloid cell proliferation and apoptosis did not account for the post-BMT phenotype. Neutral lipid accumulation was reduced in 5-day lesions, thus we developed quantitative assays for LDL accumulation and paracellular leakage using DiI-labeled human LDL and rhodamine B-labeled 70kD dextran. LDL accumulation was dramatically higher in the intima of Ldlr-/- relative to Ldlr+/+ mice, and was inhibited by injection of HDL mimics, suggesting a regulated process. LDL, but not dextran, accumulation was lower in mice post-BMT both at baseline and in 5-day lesions. Since the transcript abundance of molecules implicated in LDL transcytosis was not significantly different in the post-BMT intima, transcriptomics from whole aortic arch intima, and at single cell resolution, was performed to give insights into pathways modulated by BMT. Conclusions: Radiation exposure inhibits LDL entry into the aortic intima at baseline and the earliest stages of atherosclerosis. Single cell transcriptomic analysis suggests that LDL uptake by endothelial cells is diverted to lysosomal degradation and reverse cholesterol transport pathways. This reduces intimal accumulation of lipid and impacts lesion initiation and growth.

Effect of Traditional Preparation Processing on the Total Phenol Content and Antioxidant Activity of Fallopia multiflora Thunb.

This study evaluates the effects of traditional preparation on the total phenol content and in vitro antioxidant activity of Fallopia multiflora Thunb.. The experimental results show that the total phenol content calculated with gallic acid (GAE) of Fallopia multiflora Thunb. increased during preparation processing. The Fallopia multiflora Thunb. after preparation had a total phenol content of 22.73 ± 0.21 mg GAE/g, about 3% higher than the raw sample (22.03 ± 0.40 mg GAE/g). The preparation processing also significantly increased the antioxidant activity of Fallopia multiflora Thunb.. The concentration of extract, which could neutralize 50% of the free radicals generated from 2.2-diphenyl-1-picrylhydrazyl of medicinal materials, after processing was 53.71 ± 0.44 µg/ml, about 2.3 times lower when compared to raw pharmaceutical materials (124,38 ± 0,56 µg/ml).&#x0D; Keywords&#x0D; Fallopia multilflora Thunb., processing, antioxidant, total phenol, neutralized free radicals.&#x0D; References&#x0D; [1] P.X. Sinh, Traditional pharmacology, Medical Publishing House, Ha Noi, 2014, pp. 352-353 (in Vietnamese).[2] L. Lin, C. Qu, J. Ni, A novel method to analyze hepatotoxic components in Polygonum multiflorum using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, Journal of hazardous materials 299 (2015) 249-259. https://doi.org/10.1016/j.jhazmat.2015.06.014[3] Y. Liu, Q. Wang, J. Yang, X. Guo, W. Liu, S. Ma, S. Li, Polygonum multiflorum Thunb.: a review on chemical analysis, processing mechanism, quality evaluation, and hepatotoxicity, Frontiers in pharmacology 9 (2018) 364. http://doi.org/10.3389 / fphar.2018.00364.[4] J. Huang, J.P. Zhang, J.Q. Bai, M.J. Wei, J. Zhang, Z.H. Huang, G.H. Qu, W. Xu, X.H. Qiu, Chemical profiles and metabolite study of raw and processed Polygoni Multiflori Radix in rats by UPLC-LTQ-Orbitrap MSn spectrometry, Chinese journal of natural medicines 16 (5) (2018) 375-400. http://doi.org/10.1016 / S1875-5364 (18) 30070-0.[5] L. Liang, J. Xu, W.W. Zhou, E. Brand, H.B. Chen, Z.Z. Zhao, Integrating targeted and untargeted metabolomics to investigate the processing chemistry of Polygoni Multiflori Radix, Frontiers in pharmacology 9 (2018) 934. http://doi.org/10.3389/fphar.2018.00934.[6] Ministry of Health, Vietnamese Pharmacopoeia V, Medical Publishing House, Ha Noi, part 2, 2018, pp 1180-1181 (in Vietnamese).[7] N.T.H. Ly, T.T. Thao, P.V. Truong, P.T. Thuong, Quality Evaluation of Fallopia multiflora in Vietnam Based on HPLC-FLD and Chemometrics, Natural Products Chemistry &amp; Research 6(6) (2018) 1-7. http://doi.org/10.4172/2329-6836.1000346.[8] L. Lin, B. Ni, H. Lin, M. Zhang, X. Li, Xi. Yin, C. Qu, J. Ni, Traditional usages, botany, phytochemistry, pharmacology and toxicology of Polygonum multiflorum Thunb.: a review, Journal of Ethnopharmacology 159 (2015) 158-183. https://doi.org/10.1016/j.jep.2014.11.009.[9] G.A. Bounda, Y.U. Feng, Review of clinical studies of Polygonum multiflorum Thunb. and its isolated bioactive compounds, Pharmacognosy research 7 (3) (2015) 225. http://doi.org/10.4103 / 0974-8490.157957.[10] M.P. Kähkönen, A.I. Hopia, H.J. Vuorela, J.P. Rauha, K. Pihlaja, T. S. Kujala, M.Heinonen, Antioxidant activity of plant extracts containing phenolic compounds, Journal of agricultural and food chemistry 47 (10) (1999) 3954-3962. http://doi.org/10.1021 / jf990146l.[11] H.H. Lin, A. L. Charles, C.W. Hsieh, Y. ChiLee, J.Y. Ciou, Antioxidant effects of 14 Chinese traditional medicinal herbs against human low-density lipoprotein oxidation, Journal of traditional and complementary medicine 5 (1) (2015) 51-55. https://doi.org/10.1016/j.jtcme.2014.10.001.&#x0D; &#x0D;

Testing lipid markers as predictors of all-cause morbidity, cardiac disease, and mortality risk in captive western lowland gorillas (Gorilla gorilla gorilla).

Great apes and humans develop many of the same health conditions, including cardiac disease as a leading cause of death. In humans, lipid markers are strong predictors of morbidity and mortality risk. To determine if they similarly predict risk in gorillas, we measured five serum lipid markers and calculated three lipoprotein ratios from zoo-housed western lowland gorillas (aged 6–52 years, , subset with routine immobilizations only: ): total cholesterol (TC), triglycerides (TGs), high-density lipoprotein (HDL), low-density lipoprotein (LDL), apolipoprotein A1 (apoA1), , , and . We examined each in relation to age and sex, then analyzed whether they predicted all-cause morbidity, cardiac disease, and mortality using generalized linear models (GLMs). Older age was significantly associated with higher TG, , , and , and lower HDL and apoA1. With all ages combined, compared to females, males had significantly lower TG, , , and , and higher HDL. Using GLMs, age, sex, and lower were significant predictors of all-cause morbidity; this is consistent with research demonstrating lower LDL in humans with arthritis, which was the second most prevalent condition in this sample. In contrast to humans, lipid markers were not better predictors of cardiac disease and mortality risk in gorillas, with cardiac disease best predicted by age and sex alone, and mortality risk only by age. Similar results were observed when multimodel inference was used as an alternative analysis strategy, suggesting it can be used in place of or in addition to traditional methods for predicting risk.

Berberis microphylla G. Forst (Calafate) Berry Extract Reduces Oxidative Stress and Lipid Peroxidation of Human LDL.

Calafate (Berberis microphylla G. Forst) is a Patagonian barberry very rich in phenolic compounds. Our aim was to demonstrate, through in vitro models, that a comprehensive characterized calafate extract has a protective role against oxidative processes associated to cardiovascular disease development. Fifty-three phenolic compounds (17 of them not previously reported in calafate), were tentatively identified by Ultra-Liquid Chromatography with Diode Array Detector, coupled to Quadrupole-Time of Fly Mass Spectrometry (UHPLC-DAD-QTOF). Fatty acids profile and metals content were studied for the first time, by Gas Chromatography Mass Spectrometry (GC-MS) and Total X-ray Fluorescence (TXRF), respectively. Linolenic and linoleic acid, and Cu, Zn, and Mn were the main relevant compounds from these groups. The bioactivity of calafate extract associated to the cardiovascular protection was evaluated using Human Umbilical Vein Endothelial Cells (HUVECs) and human low density lipoproteins (LDL) to measure oxidative stress and lipid peroxidation. The results showed that calafate extract reduced intracellular Reactive Oxygen Species (ROS) production (51%) and completely inhibited LDL oxidation and malondialdehyde (MDA) formation. These findings demonstrated the potential of the relevant mix of compounds found in calafate extract on lipoperoxidation and suggest a promising protective effect for reducing the incidence of cardiovascular disease.