Native Lipoprotein Research Articles

Isolation of antibodies to hypochlorite-modified low-density lipoproteins from human serum and study of their specificity

BACKGROUND: Modified low-density lipoproteins have immunogenic properties and induce the production of antibodies. In this case, HOCl promotes the formation of subsequent active halogen-containing compounds interacting with proteins and lipid parts of low-density lipoproteins, which leads to their modification and the production of antibodies to them. AIM: The aim of this work is to isolate antibodies to hypochlorite modified low-density lipoproteins from human blood sera and study their specificity. MATERIALS AND METHODS: Malondialdehyde, acetic anhydride and sodium hypochlorite were used to obtain modified low-density lipoproteins. IgG antibodies to hypochlorite modified low-density lipoproteins were isolated by affinity chromatography. The total IgG fraction antibodies was previously isolated from human blood serum using MabSelect Xtra. The specific antibodies to hypochlorite modified low-density lipoproteins were isolated from this IgG pool by affinity chromatography. CNBr-Sepharose 4B conjugated with human serum albumin modified with NaOCl was used as a sorbent. The specificity of antibodies against hypochlorite modified low-density lipoproteins was tested using a competitive enzyme-linked immunosorbent assay. The competitors were hypochlorite modified low-density lipoproteins, acetic anhydride modified low-density lipoproteins and malondialdehyde modified low-density lipoproteins in concentrations (1–250 μg/ml). RESULTS: IgG antibodies against hypochlorite-modified proteins that interact with hypochlorite modified low-density lipoproteins were detected in human blood. According to ELISA date the binding of the isolated antibodies to hypochlorite modified low-density lipoproteins was almost completely inhibited only by appropriately modified low-density lipoproteins, that is, hypochlorite modified low-density lipoproteins, but not native low-density lipoproteins or acetic anhydride modified low-density lipoproteins. Malondialdehyde modified low-density lipoproteins also showed some competitive activity, but much weaker than hypochlorite modified low-density lipoproteins. Hypochlorite modified low-density lipoproteins itself and, to a lesser extent, malondialdehyde modified low-density lipoproteins competed for binding with antibodies of human serum to hypochlorite modified low-density lipoproteins. Acetic anhydride modified low-density lipoproteins and native low-density lipoproteins did not reduce the efficiency of antibody binding to their antigen. CONCLUSIONS: Hypochlorite modified low-density lipoproteins forms epitopes independent of other low-density lipoproteins modifications studied. These epitopes are responsible for the formation of specific antibodies.

Antibodies to HIV-1 gp120 in an experimental model of atherosclerosis caused by immunization with native high-density lipoproteins

Antibodies to bacterial and viral antigens are detected in some systematic autoimmune diseases in the absence of infection. Autoimmune MRL mice and patients with mixed connective tissue disease produce antibodies to gp120 HIV-1, despite the fact that they had never been exposed to HIV-1. Conversely, viral infections may be accompanied by pathological autoimmune reactions. Reactivity to viral antigens in autoimmune diseases and autoimmune reactions in viral diseases are caused by the induction of antibodies via idiotype-anti-idiotype interactions between autoclones and lymphocyte clones against foreign antigens or homology of foreign antigens and autoantigens. Cardiovascular diseases caused by atherosclerosis are currently one of the main causes of mortality in HIV infected patients. It is assumed that HIV infection accelerates atherogenesis. The mechanisms of the association between HIV infection and atherosclerosis are not completely clear. Chronic activation of the immune system, disturbance of cytokine regulation caused by HIV infection, induction of autoantibodies against oxidized low-density lipoproteins (LDL) and antibodies to ApoB-D are considered as possible factors of atherogenesis in HIV infection. The aim of this research was to determine whether an atherogenic immune response against native human high-density lipoproteins (nHDL) could induce antibodies to gp120 HIV-1. Studies were conducted on model of autoimmune atherosclerosis caused by immunization with native human HDL in rabbits. Rabbits (n = 6) were one-time intradermally immunized with human nHDL at a dose of 200 μg of protein per animal in incomplete Freund’s adjuvant. Antibodies to gp120 HIV-1 were measured before immunization of rabbits and on the 28th and 42th day after immunization with HDL, and antibodies to HDL were weekly measured within 42 days after immunization. Histological analysis of the aorta was conducted after eight months after immunization. Transient anti-gp120 HIV-1 antibody production was detected in rabbits immunized with native HDL. The appearance of antibodies to gp120 HIV-1 in response to immunization with native HDL which causes atherosclerosis suggests that the immune response to gp120 HIV-1 during HIV infection may be accompanied by the production of atherogenic antibodies to HDL. Consequently, the antibodies to native lipoproteins caused by an immune response against gp120 HIV-1 may be a factor in atherogenesis during HIV-1 infection.

Platelets as an inter-player between hyperlipidaemia and atherosclerosis.

Platelet hyperreactivity and hyperlipidaemia contribute significantly to atherosclerosis. Thus, it is desirable to review the platelet-hyperlipidaemia interplay and its impact on atherogenesis. Native low-density lipoprotein (nLDL) and oxidized LDL (oxLDL) are the key proatherosclerotic components of hyperlipidaemia. nLDL binds to the platelet-specific LDL receptor (LDLR) ApoE-R2', whereas oxLDL binds to the platelet-expressed scavenger receptor CD36, lectin-type oxidized LDLR 1 and scavenger receptor class A 1. Ligation of nLDL/oxLDL induces mild platelet activation and may prime platelets for other platelet agonists. Platelets, in turn, can modulate lipoprotein metabolisms. Platelets contribute to LDL oxidation by enhancing the production of reactive oxygen species and LDLR degradation via proprotein convertase subtilisin/kexin type 9 release. Platelet-released platelet factor 4 and transforming growth factor β modulate LDL uptake and foam cell formation. Thus, platelet dysfunction and hyperlipidaemia work in concert to aggravate atherogenesis. Hypolipidemic drugs modulate platelet function, whereas antiplatelet drugs influence lipid metabolism. The research prospects of the platelet-hyperlipidaemia interplay in atherosclerosis are also discussed.

Influence of the rs4238001 Genetic Polymorphism of the SR-B1 Gene on Serum Lipid Levels and Response to Rosuvastatin in Myocardial Infarction Iraqi Patients.

Scavenger receptor type B (SR-BI) is a receptor that binds both native and altered lipoproteins. It was revealed to facilitate utilization of high-density lipoprotein HDL and significantly affect the reverse transport of cholesterol. Therefore, the objectives were to identify the possible role of the genetic variant rs4238001 in patients with myocardial infarction (MI) on serum lipid level, and how this variant could impact the response of rosuvastatin drug. The genotyping of the rs4238001 genetic polymorphism of the SR-B1 gene was performed in 300 participants, including 150 MI patients treated with 20mg/day/4 weeks of rosuvastatin and 150 healthy control using Taq man probes (FAM and VIC) by Real-time PCR technique. The concentrations of the lipid profile were evaluated. The significance of the anthropometric data was revealed in the ejection fraction and smoking status (p < 0.05) between groups. The lipid profile shows either significant differences between control and MI patients (pre-treatment) or between pre-and post-treatment of MI patients (p < 0.05), but not HDL-c (p > 0.05). The minor allele frequency MAF% of the T allele and TT genotype were more frequent in MI patients than in controls (P = 0.173; OR = 3.62; 95% CI = 0.74-17.64). CC genotype was found to be associated with response to rosuvastatin therapy with a change of % (29.08 ± 53.2; p = 0.021). In the Iraqi population, the rs4238001 polymorphism of the SR-B1 gene is associated with variations in serum lipids, and the CC genotype of the SNP is related to higher HDL-C in the lipid-lowering rosuvastatin response.

Native low-density lipoproteins are priming signals of the NLRP3 inflammasome/interleukin-1β pathway in human adipose tissue and macrophages

Elevated plasma numbers of atherogenic apoB-lipoproteins (apoB), mostly as low-density lipoproteins (LDL), predict diabetes risk by unclear mechanisms. Upregulation of the NLRP3 inflammasome/interleukin-1 beta (IL-1β) system in white adipose tissue (WAT) is implicated in type 2 diabetes (T2D); however, metabolic signals that stimulate it remain unexplored. We hypothesized that (1) subjects with high-apoB have higher WAT IL-1β-secretion than subjects with low-apoB, (2) WAT IL-1β-secretion is associated with T2D risk factors, and (3) LDL prime and/or activate the WAT NLRP3 inflammasome. Forty non-diabetic subjects were assessed for T2D risk factors related to systemic and WAT glucose and fat metabolism. Regulation of the NLRP3 inflammasome was explored using LDL without/with the inflammasome’s priming and activation controls (LPS and ATP). LDL induced IL1B-expression and IL-1β-secretion in the presence of ATP in WAT and macrophages. Subjects with high-apoB had higher WAT IL-1β-secretion independently of covariates. The direction of association of LDL-induced WAT IL-1β-secretion to T2D risk factors was consistently pathological in high-apoB subjects only. Adjustment for IL-1β-secretion eliminated the association of plasma apoB with T2D risk factors. In conclusion, subjects with high-apoB have higher WAT IL-1β-secretion that may explain their risk for T2D and may be related to LDL-induced priming of the NLRP3 inflammasome.ClinicalTrials.gov (NCT04496154): Omega-3 to Reduce Diabetes Risk in Subjects With High Number of Particles That Carry "Bad Cholesterol" in the Blood—Full Text View—ClinicalTrials.gov.

Construction and application of artificial lipoproteins using adiposomes

Lipoproteins are complex particles comprised of a neutral lipid core wrapped with a phospholipid monolayer membrane and apolipoproteins on the membrane, which is closely associated with metabolic diseases. To facilitate the elucidation of its formation and dynamics, as well as its applications, we developed an in vitro system in which adiposomes, consisting of a hydrophobic core encircled by a monolayer-phospholipid membrane, were engineered into artificial lipoproteins (ALPs) by recruiting one or more kinds of apolipoproteins, for example, apolipoprotein (Apo) A-I, ApoE, ApoA-IV, and ApoB. In vitro and in vivo studies demonstrated the stability and biological activity of ALPs derived from adiposomes, which resembles native lipoproteins. Of note, adiposomes bearing ApoE were internalized via clathrin-mediated endocytosis following LDLR binding and were delivered to lysosomes. On the other hand, adiposomes bearing ApoA-IV mimicked the existing form of endogenous ApoA-IV and exhibited significant improvement in glucose tolerance in mice. In addition, the construction process was simple, precise, reproducible, as well as easy to adjust for mass production. With this experimental system, different apolipoproteins can be recruited to build ALPs for some biological goals and potential applications in biomedicine.

Development and validation of a purification system for functional full-length human SR-B1 and CD36

Scavenger receptor class B type 1 (SR-B1) and CD36 are both members of the class B scavenger receptor family that play important roles in lipoprotein metabolism and atherosclerotic disease. SR-B1 is the primary receptor for high-density lipoproteins, while CD36 is the receptor responsible for the internalization of oxidized low-density lipoproteins. Despite their importance, class B scavenger receptor structure has only been studied by functional domain or peptide fragments-there are currently no reports of utilizing purified full-length protein. Here we report the successful expression and purification of full-length human SR-B1 and CD36 using an Spodoptera frugiperda insect cell system. We demonstrate that both SR-B1 and CD36 retained their normal functions in Spodoptera frugiperda cells, including lipoprotein binding, lipid transport, and the formation of higher order oligomers in the plasma membrane. Purification schemes for both scavenger receptors were optimized and their purity was confirmed by SDS-PAGE. Both purified scavenger receptors were assessed for stability by thermal shift assay and shown to maintain stable melting temperatures up to 6weeks post-purification. Microscale thermophoresis was used to demonstrate that purified SR-B1 and CD36 were able to bind their native lipoprotein ligands. Further, there was no difference in affinity of SR-B1 for high-density lipoprotein or CD36 for oxidized low-density lipoprotein, when comparing glycosylated and deglycosylated receptors. These studies mark a significant step forward in creating physiologically relevant tools to study scavenger receptor function and lay the groundwork for future functional studies and determination of receptor structure.

Investigation of Hyperlipidemia Associated with Increased Levels of Oxidized Low-Density Lipoproteins and 8-Hydroxy-2´-Deoxyguanosine.

Hyperlipidemia is a common risk factor for atherosclerosis, heart diseases, and other pathological conditions. The factors leading to the oxidation of native low-density lipoproteins remain of valuable importance for a better understanding of the mechanisms leading to these pathologies. The aim of the present study was to evaluate the association between lipid status and the levels of oxidized low-density lipoproteins and 8-hydroxy-2´-deoxyguanosine. One hundred and fourteen participants were enrolled. Lipid profile parameters were measured and used individually to categorize subjects into two groups of normal and hyperlipidemic cases according to the international reference ranges. Oxidized low-density lipoproteins and 8-hydroxy-2´-deoxyguanosine were then compared in normal and high lipid profile groups. The obtained results were then statistically analyzed. 8-Hydroxy-2´-deoxyguanosine was found to be positively correlated with hypercholesterolemia, hypertriglyceridemia, and high levels of low-density lipoproteins (r = 0.53, 0.41, and 0.60), respectively (p<0.001). A positive correlation was observed also between the levels of oxidized low-density lipoproteins and the same lipid profile parameters (r = 0.42, 0.31, and 0.45), respectively (p<0.001). The present study suggests that disturbance in lipid profile may result in increased levels of oxidized low-density lipoproteins and oxidative stress in the study group; however, a larger sample is needed to confirm the present findings.

Apolipoprotein E4 Effects a Distinct Transcriptomic Profile and Dendritic Arbor Characteristics in Hippocampal Neurons Cultured in vitro.

The APOE gene is diversified by three alleles ε2, ε3, and ε4 encoding corresponding apolipoprotein (apo) E isoforms. Possession of the ε4 allele is signified by increased risks of age-related cognitive decline, Alzheimer’s disease (AD), and the rate of AD dementia progression. ApoE is secreted by astrocytes as high-density lipoprotein-like particles and these are internalized by neurons upon binding to neuron-expressed apoE receptors. ApoE isoforms differentially engage neuronal plasticity through poorly understood mechanisms. We examined here the effects of native apoE lipoproteins produced by immortalized astrocytes homozygous for ε2, ε3, and ε4 alleles on the maturation and the transcriptomic profile of primary hippocampal neurons. Control neurons were grown in the presence of conditioned media from Apoe–/– astrocytes. ApoE2 and apoE3 significantly increase the dendritic arbor branching, the combined neurite length, and the total arbor surface of the hippocampal neurons, while apoE4 fails to produce similar effects and even significantly reduces the combined neurite length compared to the control. ApoE lipoproteins show no systemic effect on dendritic spine density, yet apoE2 and apoE3 increase the mature spines fraction, while apoE4 increases the immature spine fraction. This is associated with opposing effects of apoE2 or apoE3 and apoE4 on the expression of NR1 NMDA receptor subunit and PSD95. There are 1,062 genes differentially expressed across neurons cultured in the presence of apoE lipoproteins compared to the control. KEGG enrichment and gene ontology analyses show apoE2 and apoE3 commonly activate expression of genes involved in neurite branching, and synaptic signaling. In contrast, apoE4 cultured neurons show upregulation of genes related to the glycolipid metabolism, which are involved in dendritic spine turnover, and those which are usually silent in neurons and are related to cell cycle and DNA repair. In conclusion, our work reveals that lipoprotein particles comprised of various apoE isoforms differentially regulate various neuronal arbor characteristics through interaction with neuronal transcriptome. ApoE4 produces a functionally distinct transcriptomic profile, which is associated with attenuated neuronal development. Differential regulation of neuronal transcriptome by apoE isoforms is a newly identified biological mechanism, which has both implication in the development and aging of the CNS.

IDF21-0074 Role of methylglyoxal modified low density lipoprotein in type 2 diabetes mellitus and its role in periodontal disease

Background: Glycation, a physiological and pathological process mainly affects proteins, nucleic acids and lipids. Methylglyoxal (MGO) along with free radicals is known to contribute to the pathogenesis of several diseases, in disorders particularly having enhanced production of acetone and amino-acetone such as diabetes mellitus (DM). The accumulation of MGO induces β-cells dysfunction, insulin resistance as well as impairment of endothelial functions in DM patients. Also, the increased level of MGO intensifies ROS generation and AGEs formation which further accelerates the co-morbidities associated with type 2 diabetes mellitus (T2DM). There is an increased susceptibility and severity of periodontal diseases in case of patients with diabetes and vice-versa. Aim: The likely role of native low density lipoprotein (LDL) and glycated LDL in type 2 diabetes mellitus (T2DM) and periodontitis (PD) as well as in T2DM+PD patients has been evaluated. Method: Sera from T2DM+PD T2DM and PD patients (n=80), were screened for the presence of antibodies against MGO-LDL and native LDL. Besides, the conformational changes in LDL isolated from T2DM+PD, T2DM and PD patients (possibly due to glycoxidaton) were also studied by UV absorption, fluorescence and CD spectroscopy. ThT, Comet assay, SEM and TEM were also done. Cellular toxicity of the isolated LDL from both T2DM+PD, T2DM and PD patients was demonstrated by comet assay. Biochemical parameters like increase in carbonyl content, TBARS, TNF α and IL1β indicate LDL oxidation and increase in pro-inflammatory cytokines. Results: MGO-LDL (in comparison to parallel control) proved to be a potent antigen showing very high binding with T2DM+PD, T2DM and PD sera as observed in direct binding ELISA. The stronger binding of T2DM+PD, T2DM and PD auto-antibodies to MGO-LDL suggests the importance of this modification in the onset and pathogenesis of these diseases. Inhibition ELISA results showed very high percent inhibition of antibodies from T2DM+PD, T2DM and PD patients by MGO-LDL in comparison to native LDL pointing towards the possible role of glycoxidation of LDL in these patients. The modified LDL might have further generated high levels of circulating auto-antibodies in the sera of these patients. Further, to ascertain the specificity of auto-antibodies from T2DM+PD, T2DM and PD patients, competitive inhibition ELISA studies were done on IgG isolated from the sera of these patients. The samples of isolated IgG from T2DM+PD (T2DM+PD-IgG), T2DM (T2DM-IgG) and PD (PD-IgG) showed very strong recognition of MGO-LDL in comparison to the native counterpart. The specificity and affinity of T2DM+PD, T2DM and PD auto-antibodies towards MGO-LDL was further confirmed by gel retardation assay. The gel mobility results confirmed specificity of MGO-LDL towards auto-antibodies from T2DM and PD patients. Furthermore, high recognition of experimentally induced anti-MGO-LDL antibodies by T2DM+PD-LDL, T2DM-LDL and PD-LDL compared to NHS-LDL confirmed that the observed structural damage in T2DM+PD-LDL, T2DM-LDL and PD-LDL is due to glycoxidation. Discussion: The neo-epitopes on LDL created by its exposure to MGO appear to be responsible for induction and elevated levels of these antibodies, and thus MGO-LDL may be considered as potential antigenic candidate for auto-immune response in diabetes and periodontitis patients with a potential for biomarker development.

Lipoprotein-Inspired Nanoscavenger for the Three-Pronged Modulation of Microglia-Derived Neuroinflammation in Alzheimer’s Disease Therapy

The inflammatory dysfunction of microglia from excess amyloid-β peptide (Aβ) disposal is an overlooked but pathogenic event in Alzheimer's disease (AD). Here, we exploit a native high-density lipoprotein (HDL)-inspired nanoscavenger (pHDL/Cur-siBACE1) that combines the trinity of phosphatidic acid-functionalized HDL (pHDL), curcumin (Cur), and β-site APP cleavage enzyme 1 targeted siRNA (siBACE1) to modulate microglial dysfunction. By mimicking the natural lipoprotein transport route, pHDL can penetrate the blood-brain barrier and sequentially target Aβ plaque, where Aβ catabolism is accelerated without microglial dysfunction. The benefit results are from a three-pronged modulation strategy, including promoted Aβ clearance with an antibody-like Aβ binding affinity, normalized microglial dysfunction by blocking the NF-κB pathway, and reduced Aβ production by gene silence (44%). After treatment, the memory deficit and neuroinflammation of APPswe/PSEN 1dE9 mice are reversed. Collectively, this study highlights the double-edged sword role of microglia and provides a promising tactic for modulating microglial dysfunction in AD treatment.

Screening of the binding affinity of serum proteins to lipid nanoparticles in a cell free environment

Lipid nanoparticles (LNPs) are promising drug and gene carriers. Upon intravenous administration, LNPs’ experience different degree of cellular uptake depending on their formulation. Currently, in vitro and in vivo studies are the gold standard for assessing the fate of nano carriers once administered, but they are time consuming and expensive. In this work, we propose a time and cost-effective method to screen a wide range of LNP formulations and select the most promising candidates for in vitro and in vivo studies. Two different approaches were explored to investigate the binding affinity between LNPs and serum proteins using sensor functionalisation with either protein specific antibody or PEG specific antibody. The first approach allowed to identify the presence of a specific protein in the protein corona of lipid particles (reconstituted and native high-density lipoproteins (rHDL and HDL), and low-density lipoproteins LDL); while the second one provided a versatile platform for the immobilisation of pegylated-particles in order to follow the interaction with serum proteins and hence predict the composition of LNP protein corona. Sensing was done using Quartz Crystal Microbalance with Dissipation (QCM-D) but the approach is extendable to other surface sensing techniques such as Surface Plasmon Resonance (SPR) or ellipsometry.

AFM detects the effects of acidic condition on the size and biomechanical properties of native/oxidized low-density lipoprotein

Solution acidification exists under some physiological conditions (e.g. lysosomes in cells) and diseases (e.g. atherosclerosis, tumors, etc.). It is poorly understood whether and how acidification influences the size and biomechanical (stiffness and stickiness) properties of native Low-density lipoprotein (LDL) and its oxidized form (oxLDL) which plays a vital role in atherogenesis and tumorigenesis. Atomic force microscopy (AFM) evaluated that gradient acidification from pH 7.4 to pH 4.4 caused an expanding-first-and-then-shrinking decrease in size and a dramatic decrease in stiffness (but no statistically significant changes in stickiness) of LDL/oxLDL particles by influencing secondary/tertiary structures and lipid release detected by infrared spectral analysis and cholesterol detection, respectively. The smaller and softer characteristics of LDL/oxLDL at acidic conditions versus at the neutral pH partially explains the atherogenic role of acidification. The data may provide important information for a better understanding of LDL/oxLDL and some diseases (e.g. atherosclerosis and tumors).

Native Low-Density Lipoproteins Act in Synergy with Lipopolysaccharide to Alter the Balance of Human Monocyte Subsets and Their Ability to Produce IL-1 Beta, CCR2, and CX3CR1 In Vitro and In Vivo: Implications in Atherogenesis.

Increasing evidence has demonstrated that oxidized low-density lipoproteins (oxLDL) and lipopolysaccharide (LPS) enhance accumulation of interleukin (IL)-1 beta-producing macrophages in atherosclerotic lesions. However, the potential synergistic effect of native LDL (nLDL) and LPS on the inflammatory ability and migration pattern of monocyte subpopulations remains elusive and is examined here. In vitro, whole blood cells from healthy donors (n = 20) were incubated with 100 μg/mL nLDL, 10 ng/mL LPS, or nLDL + LPS for 9 h. Flow cytometry assays revealed that nLDL significantly decreases the classical monocyte (CM) percentage and increases the non-classical monocyte (NCM) subset. While nLDL + LPS significantly increased the number of NCMs expressing IL-1 beta and the C-C chemokine receptor type 2 (CCR2), the amount of NCMs expressing the CX3C chemokine receptor 1 (CX3CR1) decreased. In vivo, patients (n = 85) with serum LDL-cholesterol (LDL-C) >100 mg/dL showed an increase in NCM, IL-1 beta, LPS-binding protein (LBP), and Castelli’s atherogenic risk index as compared to controls (n = 65) with optimal LDL-C concentrations (≤100 mg/dL). This work demonstrates for the first time that nLDL acts in synergy with LPS to alter the balance of human monocyte subsets and their ability to produce inflammatory cytokines and chemokine receptors with prominent roles in atherogenesis.

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

CD36 Promotes Platelet Phosphatidylserine Exposure through MAP Kinase ERK5

Dyslipidemic conditions can promote atherosclerotic plaque instability and rupture, leading to thrombosis that can cause life threatening complications. Thrombosis in this setting is augmented by circulating oxidized lipids on oxidized low density lipoprotein particles (oxLDL) that are recognized by key scavenger receptors. CD36 is a major platelet scavenger receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype through multiple signaling pathways. We previously reported that recognition of oxLDL by CD36 generates specific reactive oxygen species through Src family kinases and NADPH oxidase. These species modulate redox sensitive signaling, such as the activation of the oxidant sensor MAP kinase ERK5, leading to platelet integrin activation, aggregation, and thrombus formation. Although ERK5 was a key effector for CD36 to promote thrombosis, the downstream signaling mechanisms are unclear. We hypothesize that CD36 promotes a procoagulant phenotype that requires ERK5. Platelets isolated and washed from healthy human donors were stimulated with buffer, native low-density lipoprotein (LDL), or oxLDL, and were stained with fluorophore-conjugated Annexin V or Lactadherin to detect exposure of procoagulant phosphatidylserine (PS). OxLDL-stimulated platelets showed dose-dependent 13.4 ± 2.8% and 18.5 ± 3.5% increases in Lactadherin binding at 50 and 100μg/mL oxLDL compared to control LDL (p=0.026; p<0.01), and a rapid binding within 5 min (11.0±0.35%, p<0.001). Furthermore, pre-treatment with oxLDL followed by addition of convulxin, a snake venom that activates platelets through the collagen signaling receptor GPVI, showed a 58.1 ± 4.62% increase in Lactadherin binding compared to oxLDL and convulxin alone (p<0.0001). Addition of ADP or thrombin, agonists that activates specific G-protein coupled receptors, following pre-treatment with oxLDL did not enhance Lactadherin binding. Platelets treated with FA6, a monoclonal CD36 antibody which prevents oxLDL binding, or BIX02188, an ERK5 inhibitor, showed an 80-90% decrease in Annexin V binding (p=0.039; p=0.02) after oxLDL exposure compared to a control non-immunizing IgG or DMSO. To link known pathways for PS exposure with oxLDL-induced PS exposure, platelets were stimulated with oxLDL followed by treatment with Z-VAD-FMK, a “pan” caspase inhibitor, or cyclosporin A, a cyclophilin D/mitochondrial permeability transition pore inhibitor. Z-VAD-FMK treated platelets showed 68% decrease in Lactadherin binding compared to control DMSO (p=0.0196), whereas cyclosporin A did not inhibit Lactadherin binding. Relevance of CD36/ERK5-mediated PS exposure was demonstrated using an in vivo arterial thrombosis model that relies on autologous transplantation of the epigastric artery, which has an adventitial surface that serves as a collagen-rich thrombogenic substrate for thrombosis, into the carotid artery. This model of thrombosis was chosen due to the potential impact of free radical generation in the standard ferric chloride or Rose Bengal-mediated arterial thrombosis models. Bone marrows from platelet ERK5-deficient mice (ERK5 flox/Pf4-cre+) or control mice (ERK5 flox/flox) were transplanted into irradiated atherogenic apoE null mice fed a high fat or control diet. Fibrin deposition, detected by a fluorophore-conjugated antibody to fibrin, was enhanced in hyperlipidemic conditions for mice with platelet ERK5. High fat diet-fed chimeric mice without ERK5 or animals on control diet do not show enhanced fibrin deposition. In conclusion, oxLDL promoted PS exposure through a CD36-Src family kinases-ERK5-caspase-dependent mechanism. PS exposure by this pathway potentially enhances fibrin deposition in an atherogenic arterial in vivo thrombosis model, which suggests a role for CD36 and ERK5 in thrombosis and coagulation in dyslipidemic conditions. DisclosuresNo relevant conflicts of interest to declare.

Native High-Density Lipoproteins (HDL) with Higher Paraoxonase Exerts a Potent Antiviral Effect against SARS-CoV-2 (COVID-19), While Glycated HDL Lost the Antiviral Activity.

Human high-density lipoproteins (HDL) show a broad spectrum of antiviral activity in terms of anti-infection. Although many reports have pointed out a correlation between a lower serum HDL-C and a higher risk of COVID-19 infection and progression, the in vitro antiviral activity of HDL against SARS-CoV-2 has not been reported. HDL functionality, such as antioxidant and anti-infection, can be impaired by oxidation and glycation and a change to pro-inflammatory properties. This study compared the antiviral activity of native HDL with glycated HDL via fructosylation and native low-density lipoproteins (LDL). After 72 h of fructosylation, glycated HDL showed a typical multimerized protein pattern with an elevation of yellowish fluorescence. Glycated HDL showed a smaller particle size with an ambiguous shape and a loss of paraoxonase activity up to 51% compared to native HDL. The phagocytosis of acetylated LDL was accelerated 1.3-fold by glycated HDL than native HDL. Native HDL showed 1.7 times higher cell viability and 3.6 times higher cytopathic effect (CPE) inhibition activity against SARS-CoV-2 than that of glycated HDL under 60 μg/mL (approximately final 2.2 μM) in a Vero E6 cell. Native HDL showed EC50 = 52.1 ± 1.1 μg/mL (approximately final 1.8 μM) for the CPE and CC50 = 79.4 ± 1.5 μg/mL (around 2.8 μM). The selective index (SI) of native HDL was calculated to be 1.52. In conclusion, native HDL shows potent antiviral activity against SARS-CoV-2 without cytotoxicity, while the glycation of HDL impairs its antiviral activity. These results may explain why patients with diabetes mellitus or hypertension are more sensitive to a COVID-19 infection and have a higher risk of mortality.

Low-density lipoprotein nanomedicines: mechanisms of targeting, biology, and theranostic potential

Native nanostructured lipoproteins such as low- and high-density lipoproteins (LDL and HDL) are powerful tools for the targeted delivery of drugs and imaging agents. While the cellular recognition of well-known HDL-based carriers occurs via interactions with an HDL receptor, the selective delivery and uptake of LDL particles by target cells are more complex. The most well-known mode of LDL-based delivery is via the interaction between apolipoprotein B (Apo-B) – the main protein of LDL – and the low-density lipoprotein receptor (LDLR). LDLR is expressed in the liver, adipocytes, and macrophages, and thus selectively delivers LDL carriers to these cells and tissues. Moreover, the elevated expression of LDLR in tumor cells indicates a role for LDL in the targeted delivery of chemotherapy drugs. In addition, chronic inflammation associated with hypercholesterolemia (i.e., high levels of endogenous LDL) can be abated by LDL carriers, which outcompete the deleterious oxidized LDL for uptake by macrophages. In this case, synthetic LDL nanocarriers act as ‘eat-me’ signals and exploit mechanisms of native LDL uptake for targeted drug delivery and imaging. Lastly, recent studies have shown that the delivery of LDL-based nanocarriers to macrophages via fluid-phase pinocytosis is a promising tool for atherosclerosis imaging. Hence, the present review summarizes the use of natural and synthetic LDL-based carriers for drug delivery and imaging and discusses various mechanisms of targeting.

REMODELING OF THE SMALL CALIBER CORONARY VESSELS IN EXPERIMENTAL ATHEROSCLEROSIS

Histological and morphometric studies of the small caliber coronary arteries’ structural rearrangement at different stages of atherosclerosis development in the experiment were performed. Atherosclerotic damage was modeled by administration of low-density human native lipoproteins to animals. Histological staining of the coronary arteries wall was carried out using hematoxylin-eosin, sudan III and the methods of Van Gieson and Malory. It was found that the degree of structural and morphometric changes in the small caliber coronary arteries depends on the term of the study. In the early stages after antigenic influence (6-11th week of the experiment), the initial signs of damage in the coronary arteries wall were observed. At the later stages (12–20th week of the experiment), the structural rearrangement of the small caliber coronary arteries was detected, which was characterized by a progressive statistically significant thickening of their wall by 38.7 %, due to narrowing of the lumen. The Vogenvort index increased by 2.3 times (p<0.05) and indicated the functional insufficiency of the arteries. Проведені гістологічні та морфометричні дослідження особливостей структурної перебудови вінцевих судин дрібного калібру на різних етапах розвитку атеросклерозу в експерименті. Атеросклеротичне пошкодження моделювали шляхом введення тваринам нативних ліпопротеїнів низької щільності людини. Гістологічне забарвлення стінки вінцевих судин проводили гемотоксілінеозином, за методами Ван-Гізон, Малорі і суданом ІІІ. Встановлено, що ступінь структурних та морфометричних змін вінцевих артерій залежить від терміну дослідження. В ранні терміни після антигенного навантаження (6-11-й тиждень експерименту) спостерігали початкові ознаки пошкодження судинної стінки. В пізні терміни (12-20-й тиждень експерименту) виявлена структурна реорганізація вінцевих артерій дрібного калібру, яка характеризувалась вираженим потовщенням їх стінки статистично вагомо на 38,7 %, за рахунок звуження просвіту. Індекс Вогенворта збільшувався в 2,3 рази (р<0,05), що свідчило про зниження пропускної здатності артерій.

Аtherosclerosis-like changes in the rabbit aortic wall induced by immunization with native high-density lipoproteins.

IntroductionA high level of total cholesterol or low‐density lipoprotein (LDL) cholesterol is considered the main cause of atherosclerosis and cardiovascular disease. For this reason, experimental atherosclerosis is induced by creating high blood cholesterol in animals. However, the hypothesis that atherosclerotic processes are mostly caused by immune (autoimmune) mechanisms has recently been gaining traction. At the same time, no experimental model has been developed that clearly demonstrates the autoimmune mechanism by which atherosclerosis develops and reproduces the full picture of atherosclerosis solely by means of an immune response, without resorting to additional interventions such as a high‐cholesterol diet or the use of genetic models of hyperlipidemia. Previously, we were able to induce atherosclerosis‐like lesions in the aorta and the development of pericardial fat in rats by immunizing them with human native lipoproteins. The purpose of this study was to test whether atherosclerosis can be induced in normocholesterolaemic rabbits by immunizing them with human native high‐density lipoproteins (hnHDL).MethodsRabbits were immunized with hnHDL. Aortic wall structure, plasma cholesterol level, and antibodies against HDL were studied.ResultsImmunization with hnHDL was found to cause atherosclerosis‐like lesions in the rabbit aorta such as adipocytic and chondrocytic metaplasia, proteoglycan deposits, leukocytic infiltration. Atherosclerosis‐like lesions developed in the aorta of hnHDL‐immunized rabbits against a background of normal blood LDL‐cholesterol level. Therefore, a high plasma cholesterol level is not the sole cause of atherosclerosis. The immune response against HDL is an independent cause of atherogenesis.ConclusionsA rabbit model of atherosclerosis caused by immunization with hnHDL can be widely used to examine the mechanisms occurring during atherogenesis.