Low Density Lipoprotein Binding Research Articles

The lectin-like oxidized low-density-lipoprotein receptor: a pro-inflammatory factor in vascular disease

Scavenger receptors are membrane glycoproteins that bind diverse ligands including lipid particles, phospholipids, apoptotic cells and pathogens. LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is increasingly linked to atherosclerotic plaque formation. Transgenic mouse models for LOX-1 overexpression or gene knockout suggests that LOX-1 contributes to atherosclerotic plaque formation and progression. LOX-1 activation by oxidized LDL (low-density lipoprotein) binding stimulates intracellular signalling, gene expression and production of superoxide radicals. A key question is the role of leucocyte LOX-1 in pro-atherogenic lipid particle trafficking, accumulation and signalling leading to differentiation into foam cells, necrosis and plaque development. LOX-1 expression is elevated within vascular lesions and a serum soluble LOX-1 fragment appears diagnostic of patients with acute coronary syndromes. LOX-1 is increasingly viewed as a vascular disease biomarker and a potential therapeutic target in heart attack and stroke prevention.

Scavenger Receptor A and CD36 Are Implicated in Mediating Platelet Activation Induced by Oxidized Low- Density Lipoproteins

See ATVB 2007;2476–2483. CD36, previously known as GPIIIb or GPIV, is an 88-kDa membrane glycoprotein that was isolated from blood platelets soon after being identified on monocytes by monoclonal antibody OKM5. Cloning the CD36 gene showed this cell adhesion molecule to be a member of the scavenger receptor class B family. Moreover, it was observed that CD36 has the capacity to bind oxidized low-density lipoproteins (oxLDL) but not acetylated lipoproteins. Scavenger receptor class A (SRA),1 also expressed on platelets and monocytes/macrophages, is a trimer of 77 kDa, that binds to acetylated and oxidized lipoproteins. SRA is closely associated to macrophage CD36 in initiating atherosclerotic lesions, in some studies but not all.2,3,4 Very little was known, up to now, on the role played by oxLDL in activating platelets via CD36 and SRA and promoting a prothrombotic phenotype. The study, in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , by Suzanne Korporaal and coworkers,5 shows very elegantly that oxLDL, in tandem with CD36 and SRA, is activating platelets and potentially increasing the risk of thrombosis. This investigation brings some exciting data on a novel platelet-activating pathway triggered by partially oxidized (30% to 60%) LDL and mediated by the combined activity of CD36 and …

Liver X receptors inhibit human monocyte-derived macrophage foam cell formation by inhibiting fluid-phase pinocytosis of LDL

Liver X receptors (LXRs) are ligand-activated transcription factors involved in the control of lipid metabolism and inflammation. Several studies have recently shown that LXRs promote reverse cholesterol transport and inhibit atherosclerosis. Our study investigated whether LXRs affect macrophage uptake of LDL by human monocyte-derived macrophages. We have previously shown that human monocytes differentiated into macrophages with macrophage-colony-stimulating factor (M-CSF) constitutively take up large amounts of native LDL by receptor-independent, fluid-phase pinocytosis. In the research reported here, human monocytes were differentiated to macrophages in the presence of M-CSF with or without the LXR agonists T0901317 or 22(R)-hydroxycholesterol. Then, macrophages were incubated with native (125)I-LDL to determine LDL uptake. T0901317 and 22(R)-hydroxycholesterol inhibited (125)I-LDL uptake by 68 +/- 1% and 69 +/- 2%, respectively, and decreased pinocytotic vacuoles in the macrophages. (125)I-BSA uptake, a measure of fluid-phase pinocytosis, and (125)I-LDL uptake were the same, and T0901317 treatment inhibited uptake of both to the same degree. T0901317 did not affect receptor-mediated uptake of acetylated LDL, showing that the LXR effect is specific for fluid-phase pinocytosis of lipoproteins. Our results show that LXRs downregulate macrophage pinocytosis of LDL. The findings reveal an additional new mechanism by which LXR agonists may inhibit macrophage cholesterol accumulation and atherosclerosis, namely, by inhibiting macrophage uptake of LDL.

Fluoro-edenite fibers induce expression of Hsp70 and inflammatory response

The effect of the hypoglycemic biguanide drug Metformin was investigated after a 72 h pretreatment of human cultured fibroblasts. Metformin induced a moderate increase in low density lipoprotein binding, uptake and internalization (25% increase after treatment with 5 × 10−4 M of drug). A decrease in sterol, fatty acid and triacylglycerol synthesis from sodium acetate was observed after pretreatment with the drug, with a dose-dependent effect in the range of 5 × 10−5 to 5 × 10−4 M (50% reduction of sterol synthesis after treatment with Metformin 5 × 10−4 M). This effect was also observed in fibroblasts from a patient with homozygous familial hypercholesterolemia. Cholesterol esterification studied by incorporation of radiolabeled oleic acid was reduced by Metformin (40% of control after treatment with Metformin 5 × 10−4 M) whereas incorporation into triacylglycerols was less impaired. These effects of Metformin on cholesterol metabolism were observed either in the presence or in the absence of low density lipoproteins. Moreover, Metformin also reduced cholesterol esterification in J774 monocyte-macrophage cells. Metformin also induced a decrease of hydroxymethylglutaryl coenzyme A reductase activity in cultured fibroblasts and a reduction of acyl-coenzyme A :cholesterol-O-acyltransferase activity in cultured fibroblasts and J774 cells.

CdS Nanocrystal-Based Electrochemiluminescence Biosensor for the Detection of Low-Density Lipoprotein by Increasing Sensitivity with Gold Nanoparticle Amplification

Mercaptoacetic acid (RSH)-capped CdS nanocrystals (NCs) was demonstrated to be electrochemically reduced during potential scan and react with the coreactant S2O8(2-) to generate strong electrochemiluminescence (ECL) in aqueous solution. Based on the ECL of CdS NCs, a novel label-free ECL biosensor for the detection of low-density lipoprotein (LDL) has been developed by using self-assembly and gold nanoparticle amplification techniques. The biosensor was prepared as follows: The gold nanoparticles were first assembled onto a cysteamine monolayer on the gold electrode surface. This gold nanoparticle-covered electrode was next treated with cysteine and then reacted with CdS NCs to afford a CdS NC-electrode. Finally, apoB-100 (ligand of LDL receptor) was covalently conjugated to the CdS NC-electrode. The modification procedure was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy, respectively. The resulting modified electrode was tested as ECL biosensor for LDL detection. The LDL concentration was measured through the decrease in ECL intensity resulting from the specific binding of LDL to apoB-100. The ECL peak intensity of the biosensor decreased linearly with LDL concentration in the range of 0.025-16 ng mL-1 with a detection limit of 0.006 ng mL-1. The CdS NCs not only showed high ECL intensity and good biocompatibility but also could provide more binding sites for apoB-100 loading. In addition, the gold nanoparticle amplification for protein ECL analysis was applied to the improvement of the detection sensitivity. Thus, the biosensor exhibited high sensitivity, good reproducibility, rapid response, and long-term stability.

B-Type Natriuretic Peptide Inhibited Angiotensin II-Stimulated Cholesterol Biosynthesis, Cholesterol Transfer, and Steroidogenesis in Primary Human Adrenocortical Cells

In this study, we demonstrate that B-type natriuretic peptide (BNP) opposed angiotensin II (Ang II)-stimulated de novo cholesterol biosynthesis, cellular cholesterol uptake, cholesterol transfer to the inner mitochondrial membrane, and steroidogenesis, which are required for biosynthesis of steroid hormones such as aldosterone and cortisol in primary human adrenocortical cells. BNP dose-dependently stimulated intracellular cGMP production with an EC(50) of 11 nm, implying that human adrenocortical cells express the guanylyl cyclase A receptor. cDNA microarray and real-time RT-PCR analyses revealed that BNP inhibited Ang II-stimulated genes related to cholesterol biosynthesis (acetoacetyl coenzyme A thiolase, HMG coenzyme A synthase 1, HMG coenzyme A reductase, isopentenyl-diphosphate Delta-isomerase, lanosterol synthase, sterol-4C-methyl oxidase, and emopamil binding protein/sterol isomerase), cholesterol uptake from circulating lipoproteins (scavenger receptor class B type I and low-density lipoprotein receptor), cholesterol transfer to the inner mitochondrial membrane (steroidogenic acute regulatory protein), and steroidogenesis (ferredoxin 1,3beta-hydroxysteroid dehydrogenase, glutathione transferase A3, CYP19A1, CYP11B1, and CYP11B2). Consistent with the microarray and real-time PCR results, BNP also blocked Ang II-induced binding of (125)I-labeled low-density lipoprotein and (125)I-labeled high-density lipoprotein to human adrenocortical cells. Furthermore, BNP markedly inhibited Ang II-stimulated release of estradiol, aldosterone, and cortisol from cultured primary human adrenocortical cells. These findings demonstrate that BNP opposes Ang II-induced steroidogenesis via multiple steps from cholesterol supply and transfer to the final formation of steroid hormones. This study provides new insights into the cellular mechanisms by which BNP modulates Ang II-induced steroidogenesis in the adrenal gland.

Platelets express three different splice variants of ApoER2 that are all involved in signaling.

beta2-Glycoprotein I is the most relevant antigen in antiphospholipid syndrome. We have shown that binding of dimerized beta2-GPI to platelets via ApoER2' sensitizes platelets for second activating stimuli. Determine the region of ApoER2 involved in the binding of dimeric beta2-GPI. Cultured human megakaryocytes (MK) and three different human megakaryocytic cell lines were used for mRNA isolation to clone and express recombinant soluble platelet ApoER2. Domain deletion mutants of ApoER2 were constructed to identify the binding site for dimeric beta2-GPI. The presence of ApoER2 splice variants in platelets was demonstrated by immuno-blotting. Three different mRNA splice variants were isolated from all four types of megakaryocytic cells used. Sequence analysis identified the splice variants: (i) shApoER2Delta5 lacking low-density lipoprotein (LDL) binding domains 4, 5 and 6; (ii) shApoER2Delta4-5 lacking LDL binding domains 3, 4, 5, 6 and (iii) shApoER2Delta3-4-5 lacking LDL binding domains 3, 4, 5, 6 and 7. The presence of three splice variants of ApoER2 on platelets was confirmed by immuno-blotting, with ApoER2Delta4-5 being the most abundantly expressed splice variant. Upon stimulation with dimeric beta2-GPI, all three splice variants were translocated to the cytosol; however, ApoER2Delta4-5 translocation was most prominent. Dimeric beta2-GPI binds platelet ApoER2 variants via LDL-binding domain 1. Three different ApoER2 mRNA splice variants were isolated from MK and platelets express all three splice variants. All splice variants were shown to be functional by translocation upon stimulation with dimeric beta2-GPI. All three splice variants express LDL-binding domain 1.

Effects of Oxidation on the Structure and Stability of Human Low-Density Lipoprotein

Oxidation of low-density lipoprotein (LDL), the major cholesterol carrier in plasma, is thought to promote atherogenesis via several mechanisms. One proposed mechanism involves fusion of oxidized LDL in the arterial wall; another involves oxidation-induced amyloid formation by LDL apolipoprotein B. To test these mechanisms and to determine the effects of oxidation on the protein secondary structure and lipoprotein fusion in vitro, we analyzed LDL oxidized by nonenzymatic (Cu2+, H2O2, and HOCl) or enzymatic methods (myeloperoxidase/H2O2/Cl- and myeloperoxidase/H2O2/NO2-). Far-UV circular dichroism spectra showed that LDL oxidation induces partial unfolding of the secondary structure rather than folding into cross-beta amyloid conformation. This unfolding correlates with increased negative charge of oxidized LDL and with a moderate increase in thioflavin T fluorescence that may result from electrostatic attraction between the cationic dye and electronegative LDL rather than from dye binding to amyloid. These and other spectroscopic studies of low- and high-density lipoproteins, which encompass amyloid-promoting conditions (high protein concentrations, high temperatures, acidic pH), demonstrate that in vitro lipoprotein oxidation does not induce amyloid formation. Surprisingly, turbidity, near-UV circular dichroism, and electron microscopic data demonstrate that advanced oxidation inhibits heat-induced LDL fusion that is characteristic of native lipoproteins. Such fusion inhibition may result from the accumulation of anionic lipids and lysophospholipids on the particle surface and/or from protein cross-linking upon advanced lipoprotein oxidation. Consequently, oxidation alone may prevent rather than promote LDL fusion, suggesting that additional factors, such as albumin-mediated removal of lipid peroxidation products and/or LDL binding to arterial proteoglycans, facilitate fusion of oxidized LDL in vivo.

Estrogen induces endothelial progenitor cells proliferation and migration by estrogen receptors and PI3K-dependent pathways

Estrogen induces endothelial progenitor cells (EPCs) migration and proliferation, which may serve as a potential target for coronary artery disease, but the mechanisms are unclear. We hypothesized that estrogen receptors (ERs) and phosphatidylinositol 3-kinase (PI3K) signaling pathway, which represent particularly important roles of action for estrogen, may contribute to estrogen-induced EPCs migration and proliferation. Bone marrow mononuclear cells (MNCs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with growth factors as previously described. A total of 87.32 ± 5.13% of adherent cells showed uptake of acetylated low-density lipoprotein and lectin binding. Immunostaining and fluorescence activated cell sorting confirmed the endothelial progenitor phenotype. RT-PCR, immunocytochemistry staining and Western blot demonstrated expression of ERs. Exposure to 17β-estradiol significantly improved EPCs migration and proliferation. Those effects were blocked by pretreatment with the pharmacological PI3K blockers LY294002 (1 h, 10 umol/L) and ICI-182780 (1 h, 10 umol/L), a specific estrogen receptor antagonist, which show involvement of estrogen receptors and PI3K pathway. These results suggest that estrogen induces EPCs migration and proliferation via ERs and PI3K pathway which provided a novel insight and treatment strategy of vascular biology.

Suppression of endothelial or lipoprotein lipase in THP-1 macrophages attenuates proinflammatory cytokine secretion

LPL and endothelial lipase (EL) are associated with macrophages in human atherosclerotic lesions, and overexpression of LPL in mouse macrophages is associated with a greater extent of atherosclerosis. To investigate potential mechanisms by which macrophage-derived lipase expression may mediate proatherogenic effects, we used lentivirus-mediated RNA interference to suppress the expression of either LPL or EL within THP-1 macrophages. After suppression of either LPL or EL, significant decreases in the concentration of interleukin-1beta, interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha were observed. Incubation of THP-1 macrophages with either mildly or extensively oxidized LDL consistently decreased cytokine expression, which was additive to that contributed by lipase suppression. Decreased lipase expression was also associated with an altered lipid composition, with reduced percentages of cholesterol (unesterified and esterified), triglycerides, and lysophosphatidylcholine. Microarray data indicated a decreased expression of proinflammatory genes, growth factors, and antiapoptotic genes. By contrast, there was an increased expression of lipoprotein receptors (scavenger receptor 1, low density lipoprotein receptor, scavenger receptor class B type I, and CD36). Thus, we conclude that the suppression of either LPL or EL decreases proinflammatory cytokine expression and influences the lipid composition of THP-1 macrophages. These results provide further insight into the specific metabolic and potential pathological roles of LPL and EL in human macrophages.

Lipoprotein lipase-facilitated uptake of LDL is mediated by the LDL receptor

LPL mediates the uptake of lipoproteins into different cell types independent of its catalytic activity. The mechanism of this process and its physiological relevance are not clear. Taking into account the importance of the endothelial barrier for lipoprotein uptake, in vitro studies with primary aortic endothelial cells from wild-type and low density lipoprotein receptor (LDLR)-deficient (LDLR(-/-)) mice were performed. Addition of LPL almost doubled the uptake of LDL into wild-type cells. However, there was virtually no LPL-mediated change of LDL uptake into LDLR(-/-) cells. Upregulation of LDLR by lipoprotein-deficient serum/lovastatin in wild-type cells resulted in a 7-fold increase of LPL-mediated LDL uptake. Uptake of chylomicron remnants was not affected by LDLR expression. In proteoglycan-deficient cells, LPL did not increase the uptake of lipoproteins. The physiological relevance of this pathway was studied in mice that were both LDLR(-/-) and transgenic for catalytically inactive LPL in muscle. In the presence of LDLR, inactive LPL reduced LDL cholesterol significantly (13-24%). In the absence of LDLR, LDL cholesterol was not affected by transgenic LPL. Metabolic studies showed that in the presence of LDLR, LPL increased the muscular uptake of LDL by 77%. In the absence of LDLR, transgenic LPL did not augment LDL uptake. Chylomicron uptake was not affected by the LDLR genotype. We conclude that LPL-mediated cellular uptake of LDL, but not of chylomicrons, is dependent on the presence of both LDLR and proteoglycans.

Regulation of SR-BI-mediated selective lipid uptake in Chinese hamster ovary-derived cells by protein kinase signaling pathways

Scavenger receptor, class B, type I (SR-BI) mediates binding and internalization of a variety of lipoprotein and nonlipoprotein ligands, including HDL. Studies in genetically engineered mice revealed that SR-BI plays an important role in HDL reverse cholesterol transport and protection against atherosclerosis. Understanding how SR-BI's function is regulated may reveal new approaches to therapeutic intervention in atherosclerosis and heart disease. We utilized a model cell system to explore pathways involved in SR-BI-mediated lipid uptake from and signaling in response to distinct lipoprotein ligands: the physiological ligand, HDL, and a model ligand, acetyl LDL (AcLDL). In Chinese hamster ovary-derived cells, murine SR-BI (mSR-BI) mediates lipid uptake via distinct pathways that are dependent on the lipoprotein ligand. Furthermore, HDL and AcLDL activate distinct signaling pathways. Finally, mSR-BI-mediated selective lipid uptake versus endocytic uptake are differentially regulated by protein kinase signaling pathways. The protein kinase C (PKC) activator PMA and the phosphatidyl inositol 3-kinase inhibitor wortmannin increase the degree of mSR-BI-mediated selective lipid uptake, whereas a PKC inhibitor has the opposite effect. These data demonstrate that SR-BI's selective lipid uptake activity can be acutely regulated by intracellular signaling cascades, some of which can originate from HDL binding to murine SR-BI itself.

Low-density lipoproteins generated during an oral fat load in mild hypertriglyceridemic and healthy subjects are smaller, denser, and have an increased low-density lipoprotein receptor binding affinity

Triglyceride-rich lipoproteins generated during the postprandial phase are atherogenic. Large very low-density lipoproteins (LDLs) or chylomicrons (CMs) are not as atherogenic as their remnants (Rem). Small and dense LDLs are associated with cardiovascular disease. Low-density lipoprotein size is partly under genetic control and is considered as a relatively stable LDL feature. In this article, we present data on retinyl palmitate kinetics correlated with the modification of LDL features in terms of size, density, and in vitro receptor binding affinity after an oral fat load. Six nondiabetic, hypertriglyceridemic (HTG) patients and 6 healthy controls were examined. Low-density lipoprotein size was assessed by gradient gel electrophoresis, and LDL density by density gradient ultracentrifugation. Low-density lipoprotein binding affinity was tested by in vitro competition binding assay on normal human skin fibroblasts (HSFs) and hepatoma cells (HepG2). Kinetic parameters were estimated in CM and Rem fractions by compartmental modeling. Hypertriglyceridemic patients showed significantly higher triglyceride area and a slower CM fractional catabolic rate. Postprandial LDL density increased both in HTG patients and in the control group with a significant difference between groups at 6 hours. Fasting LDL size was lower in HTG patients vs controls but decreased similarly in the postprandial phase. Low-density lipoprotein size and density postprandial modifications were not correlated with any investigated parameter. Postprandial LDLs were internalized more efficiently by HSF than baseline LDL only in the HTG group. In conclusion, postprandial LDLs are smaller and denser compared with fasting LDLs after an oral fat load. Postprandial LDLs also slightly increased their affinity to the HSF cell receptors.

Synthetic nano-low density lipoprotein as targeted drug delivery vehicle for glioblastoma multiforme

The low density lipoprotein (LDL) receptor has been shown to be upregulated in GBM tumor cells and is therefore a potential molecular target for the delivery of therapeutic agents. A synthetic nano-LDL (nLDL) particle was developed and tested to determine its utility as a drug delivery vehicle targeted to GBM tumors. nLDL particles were constructed by combining a synthetic peptide containing a lipid binding motif and the LDL receptor (LDLR) binding domain of apolipoprotein B-100 with a lipid emulsion consisting of phosphatidyl choline, triolein, and cholesteryl oleate. Composition analysis, fast protein liquid chromatography, and electron microscopy revealed that nLDL was highly reproducible and intermediate in size between high density lipoprotein and LDL particles (10.5 ± 2.8 nm diameter). The binding and uptake of fluorescently labeled nLDL particles was assessed using fluorescence microscopy. Uptake of nLDL was time dependent, exhibiting saturation at approximately 3 h, and concentration dependent, exhibiting saturation at concentrations greater than 5 μM peptide. Using Lysotracker as a cellular marker, nLDL co-localized with lysosomes. nLDL binding was eliminated by blocking LDLRs with suramin and nLDL inhibited binding of plasma LDL to LDLRs. Collectively these data strongly suggest that the synthetic nano-LDLs described here are taken up by LDLR and can serve as a drug delivery vehicle for targeting GBM tumors via the LDLR.

Oxidized LDL binding to LOX-1 upregulates VEGF expression in cultured bovine chondrocytes through activation of PPAR-γ

It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in osteoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-γ was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-γ inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-γ and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1.

A Phage Display Screen and Binding Studies with Acetylated Low Density Lipoprotein Provide Evidence for the Importance of the Scavenger Receptor Cysteine-rich (SRCR) Domain in the Ligand-binding Function of MARCO

MARCO is a class A scavenger receptor capable of binding both gram-negative and -positive bacteria. Using the surface plasmon resonance technique, we show here that a recombinant, soluble form of MARCO, sMARCO, binds the major gram-negative and -positive bacterial surface components, lipopolysaccharide and lipoteichoic acid. Yet, the interaction of these two polyanions with sMARCO is of much lower affinity than that of polyinosinic acid, a polyanionic inhibitor of bacterial binding to MARCO. To further elucidate the ligand-binding functions of MARCO, we performed a phage display screen with sMARCO. The screening resulted in the enrichment of only a handful of phage clones. Contrary to expectations, no polyanionic peptides, but only those with a predominantly hydrophobic nature, were enriched. One peptide, VRWGSFAAWL, was displayed on two-thirds of the phages recovered after four rounds of screening. The VRWGSFAAWL phage-sMARCO interaction had significantly slower dissociation kinetics than that between sMARCO and lipopolysaccharide or lipoteichoic acid. Further work with this phage, and the second most enriched phage, displaying the peptide RLNWAWWLSY, demonstrated that both peptides bind to the SRCR domain of MARCO, and that they probably bind to the same site. Data base searches suggested that the VRWGSFAAWL peptide represents complement component C4, but we could not convincingly confirm this suggestion. A study with chimeric scavenger receptors indicated that even minor sequence changes in the MARCO scavenger receptor cysteine-rich (SRCR) domain can have profound effects on the binding of the prototypic scavenger receptor ligand, acetylated low density lipoprotein. As shown by differential binding of glutathione S-transferase-VR-WGSFAAWL, these differences were very likely due to conformational changes. These findings led to experiments that demonstrated a crucial role of the SRCR domain for acetylated low density lipoprotein binding in MARCO. Thus, our results strengthen the notion that the SRCR domain is the major ligand-binding domain in MARCO. Furthermore, they suggest that the domain may contain multiple ligand-binding sites.

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5% and 47.6% less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.

Potential role of a CD36-like class B scavenger receptor in the binding of modified low-density lipoprotein (acLDL) to the tegumental surface of Schistosoma mansoni sporocysts

Previous studies have documented the binding of low density lipoproteins (LDLs) to the tegumental surface of the mammalian stage of the human blood fluke Schistosoma mansoni, and that such binding may be functioning in the acquisition of host lipids for nutritional and/or immune evasion purposes. To determine if the intramolluscan mother sporocyst stage of S. mansoni also possess the ability to acquire exogenous LDL, live sporocysts, derived by in vitro transformation of isolated miracidia, were treated with DiI-labeled LDL (LDL-DiI) or acetylated LDL (acLDL-DiI). Sporocysts markedly differed in their binding, exhibiting strong labeling at the tegumental surface with acLDL-DiI, and only weak binding of LDL-DiI. As scavenger receptors (SRs) are known to selectively bind modified (acetylated or oxidized) LDL and other polyanionic molecules, various potential ligands of known SRs were used in an acLDL-DiI binding inhibition assay. Significant acLDL-DiI binding inhibition was observed for fucoidan, polyinosinic acid and dextran sulfate, but not for polycytidylic acid and dextran, a binding inhibition pattern consistent with SR class A or C activity. From a S. mansoni EST sequence, we cloned a scavenger receptor homologue from sporocyst cDNA that encoded a protein with 31% amino acid sequence identity and 50% similarity to a SR class B (SRB) molecule, belonging to the CD36 superfamily. Using an RNA interference assay, treatment of miracidia with a 517 bp double-stranded RNA of the S. mansoni SRB gene resulted in a significant and specific knockdown (60–70%) of SRB transcript levels in sporocysts after 6 days of dsRNA exposure and was associated with a significant reduction in acLDL-DiI binding to sporocysts at 8 and 10 days post-dsRNA incubation. There also was a time-dependent decrease in sporocyst length following dsRNA treatments. The functional linkage of acLDL binding to the cloned SRB-like S. mansoni gene using RNA interference (RNAi) suggests a possible role of the tegumental SRB-like protein as a receptor for modified LDL. Inhibition of sporocyst growth also indicates a potential involvement of this SR homologue in some aspect of larval growth and/or development.

Effects of Treatment for Diabetes Mellitus on Circulating Vascular Progenitor Cells

The circulating endothelial progenitor cells (EPCs) have an important role in angiogenesis, and the smooth muscle progenitor cells (SMPCs) participate in atherosclerosis. However, little is known about the effects of treatment of diabetes mellitus (DM) on EPCs and SMPCs. Therefore, we investigated the relations between the number of circulating vasucular progenitor cells before and after the treatment for DM. Ten previously untreated DM patients were enrolled in this study. Blood samples were collected before and after treatment. The peripheral mononuclear cells were purified and cultured to differentiate them into EPCs and SMPCs. After two weeks, the number of EPCs was determined by Dil-labeled acetylated low density lipoprotein and lectin binding. The number of SMPCs was evaluated by immunocytochemical staining of α-smooth muscle actin. Before treatment, the number of EPCs and SMPCs was significantly related to hemoglobin A1c and blood sugar. Serial examination revealed that improvement of glycemic control significantly increased the number of both EPCs and SMPCs. DM reduces the number of circulating EPCs and SMPCs according to its severity, and treatment of DM significantly increases the number of EPCs and SMPCs, which may be involved in angiogenesis and atherosclerosis in diabetes.

Synthesis andIn VitroSorption Properties of PAA-grafted Cellulose Beads for Selective Binding of LDL

Poly(acrylic acid) (PAA)-grafted cellulose copolymer beads were synthesized and tested in vitro as an adsorbent for selective removal of low-density lipoprotein (LDL) from human plasma. The copolymers were prepared by graft copolymerization of acrylic acid (AA) onto porous cellulose beads using cerium ammonium nitrate (CAN) as an initiator. The effect of initiator concentration, monomer amount and reaction time on the grafting was examined, and it revealed that the extent of grafting could be controlled by setting the appropriate reaction conditions. In vitro batch-wise adsorption tests were conducted to evaluate the lipoprotein sorption properties of the resulted copolymer beads, and the effect of grafting conditions on the adsorption performance was investigated. It was shown that the binding capacities of the best adsorbent derived from the appropriate reaction conditions could reach 4.96 mg/g total cholesterol (TC) and 4.46 mg/g LDL cholesterol (LDL-C) from human plasma, respectively, without significantly affecting the contents of beneficial constitutes such as high-density lipoprotein (HDL) and total proteins (TP). The influences of plasma amount and adsorption period on the adsorption properties were also determined and analyzed. It appears that this kind of copolymer is worthy of being developed as an alternative LDL adsorbent.