Lipoprotein Binding Research Articles

The effect of increased lipoproteins levels on the disposition of vincristine in rat.

BackgroundVincristine (VCR), an antineoplastic agent, is a key component in the treatment of acute lymphoblastic leukemia, lymphomas, rhabdomyosarcoma, neuroblastoma, and Wilms’ tumor diseases. Recently, high incidence of hyperlipidemia was reported to be associated with allogenic hematopoietic stem cell transplantation and VCR/L-asparaginase therapy.The aim of this study is to test the effects of incremental increase in lipoproteins levels on vincristine disposition in rat.MethodTo study VCR pharmacokinetics and protein binding, rats (n = 25) were assigned to three groups, normal lipidemic (NL), intermediate (IHL) and extreme hyperlipidemic (HL). Hyperlipidemia was induced by ip injection of (1 g/Kg) poloxamer 407 in rats. Serial blood samples were collected using the pre-inserted jugular vein cannula for 72 h post VCR (0.15 mg/Kg) i.v. dose. VCR unbound fractions in NL, IHL and HL plasma were determined using ultrafiltration kits.ResultsVCR demonstrated a rapid distribution phase (6–8 h) followed by a slower elimination phase with a mean elimination t½ of ~ 14 h. VCR exhibited moderate binding to plasma proteins ~ 83 %. It showed a relatively small Vc (~0.17 L/Kg) and a larger Vβ (1.53 L/Kg) indicating good tissue distribution. As the lipoproteins levels were increased, no significant changes were noted in VCR unbound fraction, plasma concentration, or volume of distribution indicating low affinity to lipoprotein binding. Induced HL also did not affect VCR elimination where similar VCR AUC0-∞, Cl and elimination phase t½ were reported along the different lipemic groups.ConclusionIncremental increase in lipoprotein levels resulted in no significant effect on VCR disposition as such ALL malignant lymphoma and allogenic hematopoietic stem cell transplantation patients need not to worry about HL-VCR interaction. Whether, HL can potentiate another drug-drug or drug-disease interaction involving VCR warrants further studying and monitoring to ensure therapeutic safety and efficiency.

Increased lipoprotein binding to arterial proteoglycans and normal macrophage cholesterol efflux capacity define the pro-atherogenic feature of CKD dyslipidemia

Increased lipoprotein binding to arterial proteoglycans and normal macrophage cholesterol efflux capacity define the pro-atherogenic feature of CKD dyslipidemia

Plasma anti-α-galactoside antibody mediates lipoprotein(a) binding to macrophages.

Lipoprotein (a) [Lp(a)] is the dominant lipid in atherosclerotic plaques though it is much less numerous than LDL or HDL in circulation. Molecular mechanism of selective uptake of Lp(a) into macrophages is unclear. Lp(a) was reported to form circulating immune complexes with the IgG-dominated plasma anti-α-galactoside antibody (anti-Gal) using the serine- and threonine-rich peptide sequences ( STPS) on its apo(a) subunit as surrogate ligand but left the other binding site of antibody free. We examined if these monovalent immune complexes could bind to smaller STPS-containing molecules on macrophage surface. Using placental membrane O-glycosylated proteins (PMOP) isolated by lectin affinity chromatography as model it was shown that human cell surface glycoproteins were small enough to occupy both binding sites of anti-Gal since they increased the fluorescence of FITC label at Fc part of anti-Gal and inhibited binding of anti-Gal and Griffonia simplicifolia lectin of similar specificity to immobilized ligands. Pre-incubation with anti-Gal facilitated Lp(a) attachment to macrophages unless anti-Gal-specific sugar was present. Anti-Gal-mediated attachment of apo(a) to macrophages increased with the number of apo(a) subunits. Further, anti-Gal-mediated binding of the same sample of apo(a) increased with the specific activity of anti-Gal sample. Finally binding of anti-Gal and anti-Gal-apo(a) complex to PMOP and macrophages respectively was mostly inhibited by LDL suggesting STPS as major anti-Gal epitopes on the cell surface. Results indicated that circulating Lp(a)-anti-Gal immune complexes anchor on macrophages using STPS-bearing cell surface glycoproteins as ligands and offer a pathway for Lp(a) sequestration into macrophages.

Circulation Enrichment of Functional Endothelial Progenitor Cells by Infantile Phototherapy.

Phototherapy is the most common therapy used for severe jaundice. There is increasing evidence that phototherapy can directly affect the expression and function of cell surface receptors including adhesion molecules, cytokines, and growth factor receptors. In this study, the effect of two infantile phototherapy regimens, including single and intensive phototherapy was investigated on biological features of circulation endothelial progenitor cells (cEPCs), as well as on serum secretion of two important chemotactic cytokines, SDF-1 and VEGF. Sixty infants diagnosed with severe hyperbilirubinemia and exposed to phototherapy were enrolled in this study. cEPCs were isolated before and after phototherapy and then migratory, proliferative, tubulogenic, and functional properties of these cells were analyzed. Our results revealed that intensive phototherapy markedly increased the release of EPCs into the circulation, and augmented the serum concentrations of both SDF-1 and VEGF cytokines. Cell proliferation, tubulogenic, and migratory properties of cEPCs isolated and expanded from infants with intensive phototherapy were significantly improved. cEPCs from infants with intensive phototherapy also showed greater levels of acetylated low-density lipoprotein and lectin binding. Overall, our results showed that the intensive phototherapy regimen can mobilize functional EPCs into the circulation through up-regulation of serum levels of VEGF and SDF-1, indicating phototherapy as an effective modality for improvement of stem cell mobilization in the therapeutic regenerative medicine. J. Cell. Biochem. 118: 330-340, 2017. © 2016 Wiley Periodicals, Inc.

The role of specific Smad linker region phosphorylation in TGF-β mediated expression of glycosaminoglycan synthesizing enzymes in vascular smooth muscle

Hyperelongation of glycosaminoglycan chains on proteoglycans facilitates increased lipoprotein binding in the blood vessel wall and the development of atherosclerosis. Increased mRNA expression of glycosaminoglycan chain synthesizing enzymes in vivo is associated with the development of atherosclerosis. In human vascular smooth muscle, transforming growth factor-β (TGF-β) regulates glycosaminoglycan chain hyperelongation via ERK and p38 as well as Smad2 linker region (Smad2L) phosphorylation. In this study, we identified the involvement of TGF-β receptor, intracellular serine/threonine kinases and specific residues on transcription factor Smad2L that regulate glycosaminoglycan synthesizing enzymes. Of six glycosaminoglycan synthesizing enzymes, xylosyltransferase-1, chondroitin sulfate synthase-1, and chondroitin sulfotransferase-1 were regulated by TGF-β. In addition ERK, p38, PI3K and CDK were found to differentially regulate mRNA expression of each enzyme. Four individual residues in the TGF-β receptor mediator Smad2L can be phosphorylated by these kinases and in turn regulate the synthesis and activity of glycosaminoglycan synthesizing enzymes. Smad2L Thr220 was phosphorylated by CDKs and Smad2L Ser250 by ERK. p38 selectively signalled via Smad2L Ser245. Phosphorylation of Smad2L serine residues induced glycosaminoglycan synthesizing enzymes associated with glycosaminoglycan chain elongation. Phosphorylation of Smad2L Thr220 was associated with XT-1 enzyme regulation, a critical enzyme in chain initiation. These findings provide a deeper understanding of the complex signalling pathways that contribute to glycosaminoglycan chain modification that could be targeted using pharmacological agents to inhibit the development of atherosclerosis.

Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a of Escherichia coli require the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b-LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.

Structure of serum amyloid A suggests a mechanism for selective lipoprotein binding and functions: SAA as a hub in macromolecular interaction networks.

Serum amyloid A is a major acute-phase plasma protein that modulates innate immunity and cholesterol homeostasis. We combine sequence analysis with x-ray crystal structures to postulate that SAA acts as an intrinsically disordered hub mediating interactions among proteins, lipids and proteoglycans. A structural model of lipoprotein-bound SAA monomer is proposed wherein two α-helices from the N-domain form a concave hydrophobic surface that binds lipoproteins. A C-domain, connected to the N-domain via a flexible linker, binds polar/charged ligands including cell receptors, bridging them with lipoproteins and rerouting cholesterol transport. Our model is supported by the SAA cleavage in the interdomain linker to generate the 1-76 fragment deposited in reactive amyloidosis. This model sheds new light on functions of this enigmatic protein.

Interfering lipoproteins in magnetic field-assisted agglutination of superparamagnetic particles immunoassay

ObjectiveThe technology of magnetic field-assisted immuno-agglutination of superparamagnetic particles allows sensitive detection of biomarkers in whole blood. However, we observed non-specific agglutination (NSA), due to interfering plasma proteins, that negatively affects C-reactive protein immunoassay. The objective of the study was to identify the plasma proteins involved and to eliminate these interferences. Design and methodsPlasma was fractionated by size exclusion HPLC and each fraction was tested for non-specific agglutination. In addition, plasma proteins bound to magnetic particles were analyzed by SDS-gel electrophoresis and identified by mass spectrometry. ResultsWe found that NSA was due to the binding of some lipoproteins to the particles. NSA was observed in the presence of purified LDL and VLDL but not HDL. NSA was mediated by the binding of ApoB100 to magnetic particles through its heparin binding sites. These interferences could be eliminated by addition of heparin or other polyanions like dextran sulfate to the assay buffer. ConclusionNSA results from the binding of some plasma lipoproteins to magnetic particles. The use of a polyanion to eliminate these interferences allows the formulation of a stable reagent.

Developmental Endothelial Locus-1 (Del-1) Inhibits Oxidized Low-Density Lipoprotein Activity by Direct Binding, and Its Overexpression Attenuates Atherogenesis in Mice.

Modified low-density lipoprotein (LDL) binding to scavenger receptors has been implicated in atherosclerosis. It is hypothesized that a third molecule may affect modified LDL binding, therefore, this study focuses on the soluble endogenous protein, developmental endothelial locus-1 (Del-1), as an inhibitor of oxidized LDL (oxLDL) interactions.Methods and Results:Del-1 preferentially bound oxLDL over native LDL in a cell-free binding assay. Del-1 also inhibited DiI-labeled oxLDL uptake by scavenger receptors irrespective of the receptor type (LOX-1, SR-AI, CD36, or SR-BI) expressed in COS-7 cells, and independent of cell type (human coronary artery endothelial cells (HCAECs) or THP-1-derived macrophages). Furthermore, Del-1 suppressed oxLDL-inducedMCP-1andICAM-1expression and endothelin-1 secretion in HCAECs. Then, male Del-1 transgenic (Del-1Tg) and wild-type mice (WT) mice were established and fed a Paigen diet for 20 weeks from the age of 24 weeks. While plasma lipid concentrations did not differ between WT and Del-1Tg mice, plasma LOX-1-ligand activity was significantly lower in Del-1Tg than in WT mice. Moreover, lipid accumulation in aortic roots was significantly less in the Del-1Tg mice, evaluated with Oil red-O. Taken together, Del-1 appears to block the activity of oxLDL pharmacologically by direct binding in vitro, and attenuates atherogenesis in vivo, although its role in physiological settings are yet to be resolved. Del-1 intercepted oxLDL before its receptor binding to reduce atherogenesis. (Circ J 2016; 80: 2541-2549).

Betaine prevented fructose-induced NAFLD by regulating LXRα/PPARα pathway and alleviating ER stress in rats

Betaine has been proven effective in treating nonalcoholic fatty liver disease (NAFLD) in animal models, however, its molecular mechanisms remain elusive. The aims of this study were to explore the mechanisms mediating the anti-inflammatory and anti-lipogenic actions of betaine in fructose-fed rats. In this study, betaine improved insulin resistance, reduced body weight gain and serum lipid levels, and prevented hepatic lipid accumulation in fructose-fed rats. It up-regulated hepatic expression of liver X receptor-alpha (LXRα) and peroxisome proliferator-activated receptor-alpha (PPARα), with the attenuation of the changes of their target genes, including hepatic carnitine palmitoyl transferase (CPT) 1α, glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1, apolipoprotein B, sterol regulatory element-binding protein 1c and adipocyte differentiation-related protein, involved in fatty acid oxidation and lipid storage in these model rats. Furthermore, betaine alleviated ER stress and inhibited acetyl-CoA carboxylase α, CPT II, stearoyl-CoA desaturase 1 and fatty acid synthase expression involved in fatty acid synthesis in the liver of fructose-fed rats. Betaine suppressed hepatic gluconeogenesis in fructose-fed rats by moderating protein kinase B -forkhead box protein O1 pathway, as well as p38 mitogen-activated protein kinase and mammalian target of rapamycin activity. Moreover, betaine inhibited hepatic nuclear factor kappa B /nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 inflammasome activation-mediated inflammation in this animal model. These results demonstrated that betaine ameliorated hepatic lipid accumulation, gluconeogenesis, and inflammation through restoring LXRα and PPARα expression and alleviating ER stress in fructose-fed rats. This study provides the potential mechanisms of betaine involved in the treatment of NAFLD.

Cardiomyocyte VEGF Regulates Endothelial Cell GPIHBP1 to Relocate Lipoprotein Lipase to the Coronary Lumen During Diabetes Mellitus.

Lipoprotein lipase (LPL)-mediated triglyceride hydrolysis is the major source of fatty acid for cardiac energy. LPL, synthesized in cardiomyocytes, is translocated across endothelial cells (EC) by its transporter glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1). Previously, we have reported an augmentation in coronary LPL, which was linked to an increased expression of GPIHBP1 following moderate diabetes mellitus. We examined the potential mechanism by which hyperglycemia amplifies GPIHBP1. Exposure of rat aortic EC to high glucose induced GPIHBP1 expression and amplified LPL shuttling across these cells. This effect coincided with an elevated secretion of heparanase. Incubation of EC with high glucose or latent heparanase resulted in secretion of vascular endothelial growth factor (VEGF). Primary cardiomyocytes, being a rich source of VEGF, when cocultured with EC, restored EC GPIHBP1 that is lost because of cell passaging. Furthermore, recombinant VEGF induced EC GPIHBP1 mRNA and protein expression within 24 hours, an effect that could be prevented by a VEGF neutralizing antibody. This VEGF-induced increase in GPIHBP1 was through Notch signaling that encompassed Delta-like ligand 4 augmentation and nuclear translocation of the Notch intracellular domain. Finally, cardiomyocytes from severely diabetic animals exhibiting attenuation of VEGF were unable to increase EC GPIHBP1 expression and had lower LPL activity at the vascular lumen in perfused hearts. EC, as the first responders to hyperglycemia, can release heparanase to liberate myocyte VEGF. This growth factor, by activating EC Notch signaling, is responsible for facilitating GPIHBP1-mediated translocation of LPL across EC and regulating LPL-derived fatty acid delivery to the cardiomyocytes.

Abstract 14167: Clinical Characteristics of Subjects With Different Levels of Systemic Erythrocyte-bound Apolipoprotein B

Introduction: The level of apolipoprotein (apo) B-containing lipoproteins in plasma is positively associated with cardiovascular risk. We have previously demonstrated that apo B bound to circulating erythrocytes (ery-apoB) may protect against atherosclerosis. We evaluated the clinical characteristics of subjects with different levels of ery-apoB. Methods: Ery-apoB was measured by flow cytometry in subjects with (n=177) and without (n=208) cardiovascular disease (CVD), and expressed as mean fluorescent intensity in arbitrary units (au). The number of criteria for metabolic syndrome as outlined by the NCEP was scored. Carotid intima media thickness (cIMT) was measured using B-mode ultrasound. Data are given as median (IQR). Results: Ery-apoB was lower in patients with CVD than in controls (0.80 (0.40-1.40) and 0.90 (0.46-1.79) au, respectively, p=0.039). Ery-apoB was inversely associated with cIMT, waist circumference, plasma triglycerides, C-reactive protein and complement C3, and was positively associated with HDL-cholesterol. Ery-apoB was similar in men and women. The number of criteria for the metabolic syndrome was closely associated with ery-apoB, in a dose-depending manner. Median ery-apoB decreased with increasing number of metabolic syndrome criteria (Kruskall Wallis test, p=0.02). Furthermore, ery-apoB was almost 3-fold higher in subjects with blood group O than in subjects with a non-O blood group (1.40 (0.75-2.15) and 0.50 (0.10-0.95) au, respectively, p<0.001). No correlation between ery-apoB and plasma apo B was found, and ery-apoB was unrelated to statin use. Discontinuation of statin use did not affect ery-apoB (n=54). Conclusion: Apo B bound to circulating erythrocytes is associated with several characteristics of the metabolic syndrome, inflammation and ABO blood group. These data provide novel insight into the mechanisms involved in the binding of atherogenic lipoproteins to erythrocytes.

Aquaporin-1 shifts the critical transmural pressure to compress the aortic intima and change transmural flow: theory and implications.

Transmural-pressure (ΔP)-driven plasma advection carries macromolecules into the vessel wall, the earliest prelesion atherosclerotic event. The wall's hydraulic conductivity, LP, the water flux-to-ΔP ratio, is high at low pressures, rapidly decreases, and remains flat to high pressures (Baldwin AL, Wilson LM. Am J Physiol Heart Circ Physiol 264: H26-H32, 1993; Nguyen T, Toussaint, Xue JD, Raval Y, Cancel CB, Russell LM, Shou S, Sedes Y, Sun O, Yakobov Y, Tarbell JM, Jan KM, Rumschitzki DS. Am J Physiol Heart Circ Physiol 308: H1051-H1064, 2015; Tedgui A, Lever MJ. Am J Physiol Heart Circ Physiol. 247: H784-H791, 1984. Shou Y, Jan KM, Rumschitzki DS. Am J Physiol Heart Circ Physiol 291: H2758-H2771, 2006) due to pressure-induced subendothelial intima (SI) compression that causes endothelial cells to partially block internal elastic laminar fenestrae. Nguyen et al. showed that rat and bovine aortic endothelial cells express the membrane protein aquaporin-1 (AQP1) and transmural water transport is both transcellular and paracellular. They found that LP lowering by AQP1 blocking was perplexingly ΔP dependent. We hypothesize that AQP1 blocking lowers average SI pressure; therefore, a lower ΔP achieves the critical force/area on the endothelium to partially block fenestrae. To test this hypothesis, we improve the approximate model of Huang et al. (Huang Y, Rumschitzki D, Chien S, Weinbaum SS. Am J Physiol Heart Circ Physiol 272: H2023-H2039, 1997) and extend it by including transcellular AQP1 water flow. Results confirm the observation by Nguyen et al.: wall LP and water transport decrease with AQP1 disabling. The model predicts 1) low-pressure LP experiments correctly; 2) AQP1s contribute 30-40% to both the phenomenological endothelial + SI and intrinsic endothelial LP; 3) the force on the endothelium for partial SI decompression with functioning AQP1s at 60 mmHg equals that on the endothelium at ∼43 mmHg with inactive AQP1s; and 4) increasing endothelial AQP1 expression increases wall LP and shifts the ΔP regime where LP drops to significantly higher ΔP than in Huang et al. Thus AQP1 upregulation (elevated wall LP) might dilute and slow low-density lipoprotein binding to SI extracellular matrix, which may be beneficial for early atherogenesis.

Abstract 1193: LSR transcript variant iota drives nuclear localization and altered transcriptome regulation in breast cancer

Abstract Breast cancer remains the second highest cause of cancer-related deaths among women despite continuous improvements in diagnosis and treatments. African-American (AA) women have higher mortality rates and shorter survival times then Caucasians. Higher prevalence of aggressive triple-negative tumors in young AA women, and increased mortality rates in AA women, at all ages, diagnosed with less aggressive Luminal A tumors denotes cancer disparities in multiple molecular phenotypes. Lipolysis stimulated lipoprotein receptor (LSR) was originally identified as a hepatocyte receptor involved in the dynamics of lipid distribution between the liver and peripheral tissue. Upon activation LSR mediates the binding, cellular uptake, and degradation of triacylglyceride-rich lipoproteins. LSR has also been highlighted as an important regulator of tricellular tight junctions (tTJs). tTJs are specialized junctions within tricellular contacts of epithelial sheets. LSR regulates tTJs through the recruitment and arrangement of several proteins including tricellulin, ILDR1 and ILDR2. While previous studies have described tTJ and hepatic functions of LSR, our lab was the first to directly show LSR enhances aggressive breast cancer cell behaviors including proliferation and migration, and promotes cancer-stem cell properties such as survival in anchorage-independent conditions. Herein, we have taken a closer examination of breast cancer samples, and found nuclear localization of LSR, a novel observation for a proposed membrane localized protein. Moreover, we show nuclear localization is significantly correlated with poor patient outcome. In vitro assays confirmed LSR localizes to the plasma membrane, cytoplasm, and the nucleus. From these results it is clear that LSR has an additional nuclear function that may alter gene expression and promote more severe cancer phenotypes. Of note, given the high breast cancer mortality rate in African-American women, we have looked for and identified LSR SNPs unique to African-Americans. Furthermore, we used in silico sequence analysis and determined putative functional motifs and post-translational modifications of the LSR protein as well as identified nine unique transcript variants of LSR. We then focused upon one variant (Iota), which lacks a transmembrane domain and features a nuclear localization sequence. We used in vitro assays to show cellular localization and behavioral consequence of LSR iota expression in breast cancer cell lines. Collectively, our novel data highlight the complex roles of LSR including alternate transcripts, nuclear localization, and possible enzymatic function, which may impact cancer progression and potentially provide insight into breast cancer health disparities. Citation Format: Katerina D. Fagan-Solis, David A. McDonald, Lynnelle W. Thorpe, Jodie M. Fleming. LSR transcript variant iota drives nuclear localization and altered transcriptome regulation in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1193. doi:10.1158/1538-7445.AM2015-1193

Lipoprotein lipase and atherosclerosis.

Lipoprotein lipase has long been known to hydrolyse triglycerides from triglycerides-rich lipoproteins. More recently, it has been shown to promote the binding of lipoproteins to various lipoprotein receptors. Evidence is also presented regarding the possible atherogenic role of lipoprotein lipase. In theory, lipoprotein lipase deficiency should help to clarify this question. However, the rarity of this condition means that it has not been possible to conduct epidemiological studies. An alternative approach is to investigate the correlation of lipoprotein lipase with onset of cardiovascular disease in prospective studies in large population-based cohorts. Complementary with this approach, animal models have been used to explore the atherogenicity of lipoprotein lipase expressed by macrophages.

Regulation of age-related macular degeneration-like pathology by complement factor H

Complement factor H (CFH) is a major susceptibility gene for age-related macular degeneration (AMD); however, its impact on AMD pathobiology is unresolved. Here, the role of CFH in the development of AMD pathology in vivo was interrogated by analyzing aged Cfh(+/-) and Cfh(-/-) mice fed a high-fat, cholesterol-enriched diet. Strikingly, decreased levels of CFH led to increased sub-retinal pigmented epithelium (sub-RPE) deposit formation, specifically basal laminar deposits, following high-fat diet. Mechanistically, our data show that deposits are due to CFH competition for lipoprotein binding sites in Bruch's membrane. Interestingly and despite sub-RPE deposit formation occurring in both Cfh(+/-) and Cfh(-/-) mice, RPE damage accompanied by loss of vision occurred only in old Cfh(+/-) mice. We demonstrate that such pathology is a function of excess complement activation in Cfh(+/-) mice versus complement deficiency in Cfh(-/-) animals. Due to the CFH-dependent increase in sub-RPE deposit height, we interrogated the potential of CFH as a previously unidentified regulator of Bruch's membrane lipoprotein binding and show, using human Bruch's membrane explants, that CFH removes endogenous human lipoproteins in aged donors. Thus, advanced age, high-fat diet, and decreased CFH induce sub-RPE deposit formation leading to complement activation, which contributes to RPE damage and visual function impairment. This new understanding of the complicated interactions of CFH in AMD-like pathology provides an improved foundation for the development of targeted therapies for AMD.

Simple Method Provides Resolution of Albumin, Lipoprotein, Free Fraction, and Chylomicron to Enhance the Utility of Protein Binding Assays

Medicinal chemists have been encouraged in recent years to embrace high speed protein binding assays. These methods employ dialysis membranes in 96 well format or in spin filters. Membrane‐based methods do not separate lipoprotein binding from albumin binding. While controls can account for membrane binding to an extent, membrane methods still introduce interference into the experiment. Ultracentrifugation methods, in contrast, do not introduce interference if density gradients can be avoided and they resolve lipoprotein from albumin. A new generation of compact, fast ultracentrifuges facilitate the rapid and fully‐informative separation of plasma into albumin, albumin/fatty‐acid complex, lipoprotein, protein‐free, and chylomicron fractions with no need of a salt or sugar density gradients. We present a simple and fast ultracentrifuge method here for two platinum compounds and a taxane that otherwise bound irreversibly to dialysis membranes and which exhibited distinctive lipoprotein binding behaviors. This new generation of ultracentrifugation methods underscores a need to further discuss protein binding assessments as they relate to medicinal chemistry efforts.

The tissue distribution of lipoprotein lipase determines where chylomicrons bind

To determine the role of LPL for binding of lipoproteins to the vascular endothelium, and for the distribution of lipids from lipoproteins, four lines of induced mutant mice were used. Rat chylomicrons labeled in vivo with [(14)C]oleic acid (primarily in TGs, providing a tracer for lipolysis) and [(3)H]retinol (primarily in ester form, providing a tracer for the core lipids) were injected. TG label was cleared more rapidly than core label. There were no differences between the mouse lines in the rate at which core label was cleared. Two minutes after injection, about 5% of the core label, and hence chylomicron particles, were in the heart of WT mice. In mice that expressed LPL only in skeletal muscle, and had much reduced levels of LPL in the heart, binding of chylomicrons was reduced to 1%, whereas in mice that expressed LPL only in the heart, the binding was increased to over 10%. The same patterns of distribution were evident at 20 min when most of the label had been cleared. Thus, the amount of LPL expressed in muscle and heart governed both the binding of chylomicron particles and the assimilation of chylomicron lipids in the tissue.

Simple method provides resolution of albumin, lipoprotein, free fraction, and chylomicron to enhance the utility of protein binding assays.

Medicinal chemists have been encouraged in recent years to embrace high speed protein binding assays. These methods employ dialysis membranes in 96-well format or spin filters. Membrane-based methods do not separate lipoprotein binding from albumin binding and introduce interference despite membrane binding controls. Ultracentrifugation methods, in contrast, do not introduce interference if density gradients can be avoided and they resolve lipoprotein from albumin. A new generation of compact, fast ultracentrifuges facilitates the rapid and fully informative separation of plasma into albumin, albumin/fatty acid complex, lipoprotein, protein-free, and chylomicron fractions with no need of salt or sugar density gradients. We present a simple and fast ultracentrifuge method here for two platinum compounds and a taxane that otherwise bound irreversibly to dialysis membranes and which exhibited distinctive lipoprotein binding behaviors. This new generation of ultracentrifugation methods underscores a need to further discuss protein binding assessments as they relate to medicinal chemistry efforts.

An integrated analysis of differential miRNA and mRNA expressions in human gallstones.

Gallstone disease, including cholesterol precipitation in bile, increased bile salt hydrophobicity and gallbladder inflammation. Here, we investigated miRNA and mRNA involved in the formation of gallstones, and explored the molecular mechanisms in the development of gallstones. Differentially expressed 17 miRNAs and 525 mRNA were identified based on Illumina sequencing from gallbladder mucosa of patients with or without gallstones, and were validated by randomly selected 6 miRNAs and 8 genes using quantitative RT-PCR. 114 miRNA target genes were identified, whose functions and regulating pathways were related to gallstones. The differentially expressed genes were enriched upon lipoprotein binding and some metabolic pathways, and differentially expressed miRNAs enriched upon ABC transportation and cancer related pathways. A molecular regulatory network consisting of 17 differentially expressed miRNAs, inclusive of their target genes, was constructed. miR-210 and its potential target gene ATP11A were found to be differentially expressed in both miRNA and mRNA profiles. ATP11A was a direct target of miR-210, which was predicted to regulate the ABC-transporters pathway. The expression levels of ATP11A in the gallstone showed inverse correlation with miR-210 expression, and up-regulation of miR-210 could reduce ATP11A expression in HGBEC. This is the first report that indicates the existence of differences in miRNA and mRNA expression in patients with or without gallstones. Our data shed light on further investigating the mechanisms of gallstone formation.