High-density Lipoprotein Components Research Articles

Abstract 410: Binding of Apolipoprotein A-I to Inner and Outer Components of Bacterial Membranes

Human apolipoprotein A-I (apoA-I) is a 28 kDa protein and a major component of high-density lipoproteins, mediating several essential metabolic functions related to heart disease. The protein is able to suppress bacterial growth of gram-negative bacteria. To understand the basis of the bacterial growth suppression and its potential as an antimicrobial protein, the binding interaction between apoA-I and membrane components of gram-negative bacteria was characterized using wild-type, modified, and a deletion variant of apoA-I. To modify the protein, lysine residues were acetylated with acetic anhydride. The deletion variant was created by removing the C-terminal amino acid residues 190-143. The binding interaction of apoA-I with lipopolysaccharides (LPS), abundantly present in the outer bacterial membrane of E. coli , was investigated with non-denaturing PAGE and isothermal titration calorimetry. In addition, protein structural changes induced by LPS binding were monitored by circular dichroism and fluorescence spectroscopy. Binding analysis showed that wild-type and N-terminal apoA-I were able to associate with LPS, but the acetylated protein showed a strongly reduced binding interaction. The bacterial inner membrane is rich in negatively charged phosphatidylglycerol. ApoA-I showed a strong preference for bilayer vesicles made of phosphatidylglycerol compared to phosphatidylcholine. Acetylated apoA-I showed a marked decrease in phosphatidylglycerol binding, while the binding activity of N-terminal apoA-I was only moderately decreased. These results indicated that apoA-I interacts with components of the inner and outer membrane of gram-negative bacteria. In addition, lysine side-chains play a crucial role in the binding interaction, indicating the importance of ionic interactions. These results indicate the potential for apoA-I to function as an antimicrobial protein.

Disminución de los niveles plasmáticos de clusterina en pacientes con psoriasis

Introduction and objectivesPsoriasis is a chronic inflammatory disease that has been linked to increased cardiovascular risk. The glycoprotein clusterin (apolipoprotein J) is a component of high-density lipoproteins and has a protective role in atherosclerosis. The aim of the present study was to evaluate the plasma levels of clusterin and the proinflammatory cytokine macrophage migration inhibitory factor (MIF) in patients with severe psoriasis, comparing groups of patients with different risks of cardiovascular disease. Material and methodsTwenty-one patients with severe psoriasis (psoriasis area severity index and body surface area>10) and 11 healthy controls with no dermatologic disease were studied. Cardiovascular risk factors were assessed according to the Adult Treatment Panel (ATP) III criteria. Subclinical carotid atheromatosis was assessed by Doppler ultrasonography of the carotid arteries. Plasma clusterin and MIF levels were measured by enzyme-linked immunosorbent assay. ResultsATP-III criteria for metabolic syndrome were met by 47% of the patients, and 33% had carotid atheromatous plaque. Mean (SD) clusterin plasma levels were significantly lower in patients with psoriasis compared with controls (81.39 [27.30] μg/mL for the 21 patients vs 117 [21.6] μg/mL for the 11 controls; P=.0017). MIF plasma levels (ng/ml) were significantly higher in patients with atheromatous plaque compared with controls (53.22 [29.02] for the 6 patients with plaque vs 34.21 [9.65] for the 11 controls; P=.0394). ConclusionsThe decreased plasma levels of clusterin in psoriatic patients suggested an association with the disease and might be an indicator of systemic inflammatory activity. Increased levels of MIF appear to be associated with cardiovascular risk factors and carotid atheromatous plaque.

Secreted Progranulin Is a Homodimer and Is Not a Component of High Density Lipoproteins (HDL)

Progranulin is a secreted glycoprotein, and the GRN gene is mutated in some cases of frontotemporal dementia. Progranulin has also been implicated in cell growth, wound healing, inflammation, and cancer. We investigated the molecular nature of secreted progranulin and provide evidence that progranulin exists as a homodimer. Although recombinant progranulin has a molecular mass of ∼85 kDa by SDS-PAGE, it elutes in fractions corresponding to ∼170-180 kDa by gel-filtration chromatography. Additionally, recombinant progranulin can be intermolecularly cross-linked, yielding a complex corresponding to a dimer (∼180 kDa), and progranulins containing different epitope tags physically interact. In plasma, progranulin similarly forms complexes of ∼180-190 kDa. Although progranulin partially co-fractionated with high density lipoproteins (HDL) by gel-filtration chromatography, we found no evidence that progranulin in mouse or human plasma is a component of HDL either by ultracentrifugation or by lipid binding assays. We conclude that circulating progranulin exists as a dimer and is not likely a component of HDL.

Reducing plasma cholesterol is not the end of the quest

Despite the cholesterol lowering beneficial effects of statins, nearly 70% of cardiovascular disease remains unresolved. High density lipoproteins (HDL), apolipoprotein A-I (the major protein component of HDL), and apoA-I mimetic peptides exert antiinflammatory effects but have not been shown to reduce plasma cholesterol levels. Apolipoprotein E (apoE) is a protein component of chylomicron remnants, very low density lipoprotein (VLDL), and HDL. ApoE is ubiquitously expressed and secreted by many tissues in the body including liver, brain, skin, and tissue macrophages [1]. A common well-characterized polymorphism in human apoE gene accounts for the three alleles, e2, e 3a nde4 that are known to confer resistance or susceptibilitytomany diseasesincludingAlzheimer’sdisease[2]and atherosclerosis [3]. ApoE gene encodes for a 299 amino acid protein containing two domains: a receptor-binding domain at the N-ter

Liver Gene Transfer of Interkeukin-15 Constructs That Become Part of Circulating High Density Lipoproteins for Immunotherapy

Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα−/− mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.

Identification of sites in apolipoprotein A-I susceptible to chymase and carboxypeptidase A digestion

MCs (mast cells) adversely affect atherosclerosis by promoting the progression of lesions and plaque destabilization. MC chymase cleaves apoA-I (apolipoprotein A-I), the main protein component of HDL (high-density lipoprotein). We previously showed that C-terminally truncated apoA-I (cleaved at the carboxyl side of Phe225) is present in normal human serum using a newly developed specific mAb (monoclonal antibody). In the present study, we aimed to identify chymase-induced cleavage sites in both lipid-free and lipid-bound (HDL3) forms of apoA-I. Lipid-free apoA-I was preferentially digested by chymase, at the C-terminus rather than the N-terminus. Phe229 and Tyr192 residues were the main cleavage sites. Interestingly, the Phe225 residue was a minor cleavage site. In contrast, the same concentration of chymase failed to digest apoA-I in HDL3; however, a 100-fold higher concentration of chymase modestly digested apoA-I in HDL3 at only the N-terminus, especially at Phe33. CPA (carboxypeptidase A) is another MC protease, co-localized with chymase in severe atherosclerotic lesions. CPA, in vitro, further cleaved C-terminal Phe225 and Phe229 residues newly exposed by chymase, but did not cleave Tyr192. These results indicate that several forms of C-terminally and N-terminally truncated apoA-I could exist in the circulation. They may be useful as new biomarkers to assess the risk of CVD (cardiovascular disease).

Biomimetic Synthetic High Density Lipoprotein Nanostructures Target the SR-B1 Receptor and Differentially Manipulate Cellular Cholesterol Flux in Lymphoma Cells: A Novel Treatment Paradigm

Abstract 157We report a nanoparticle-enabled therapeutic approach to B cell lymphoma using synthetic, high-density lipoprotein nanoparticles (HDL-NP). Like natural HDLs, biomimetic HDL-NPs target scavenger receptor type B-1 (SR-B1), a high-affinity HDL receptor expressed by lymphoma cells. Functionally, and unlike natural HDL, a gold nanoparticle template used to control HDL-NP synthesis enables differential manipulation of cellular cholesterol flux through SR-B1. Recent evidence in lymphoblasts and myeloblasts from patients with acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) demonstrates enhanced uptake of cholesterol through high-density lipoprotein (HDL) carriers, which may result in increased cell proliferation. We therefore hypothesized that by targeting SR-B1, we could manipulate cholesterol flux in lymphoma cells thereby targeting cellular signaling pathways that would lead to cell death and offer an innovative approach to the treatment of lymphoma and other cancers. Methods:To accomplish this, we developed a biomimetic spherical nanoparticle (HDL-NP) with surface chemical properties similar to natural HDL, including the ability to sequester cholesterol. Biomimetic HDL-NPs are synthesized using a 5 nm diameter gold (Au) nanoparticle (NP) as a size- and shape-restrictive template on which to assemble the surface chemical components of natural HDLs, including phospholipids and the HDL-defining apolipoprotein A1 (Apo A1). Importantly, the core AuNP template occupies the real estate in natural cholesterol-rich HDLs reserved for esterified cholesterol, which inherently limits the ability of HDL-NPs to deliver cholesterol. We incubated the HDL-NPs with various lymphoma cell lines, and similarly tested the HDL-NPs in a xenograft model. Results:We first examined gene expression profiles of diffuse large B-cell lymphoma (DLBCL), Burkitt Lymphoma (BL) and normal B cells from patient samples in a database generated using Affymetrix U133plus 2.0 arrays in order to establish the prevalence of SR-B1 expression. We compared the expression of SR-B1 in BL cases (n=20), and DLBCL cases (n=40) that were further subdivided as activated B-cell (ABC)-like DLBCL (n=20), and germinal center (GC)-like DLBCL (n=20) to normal naive (n=3) and memory (n=3) B cells obtained from healthy donors. We found that SR-B1 was expressed at two to four-fold higher levels in the lymphomas (ABC and GC) compared with normal B cells. Next, we determined the expression of SR-B1 in lymphoma cell lines and normal peripheral lymphocytes by immunoblotting, and we found that SR-B1 is expressed in multiple B cell lymphoma cell lines, but not in Jurkat, a T-cell line, and is not expressed by normal human lymphocytes. Incubation of HDL-NP with Ramos, LY-3 and SUDHL-4 resulted in a dose-dependent decrease in cell viability and apoptosis (Figure 1) of the Ramos and SUDHL-4 cells, less so in LY-3 cells, and not in the Jurkat line. This required the nanoparticle construct and could not be duplicated by individual components of that construct, and was reversible with addition of acetylated low-density lipoprotein, indicating that the SR-B1 receptor was targeted. Xenograft experiments with SCID beige mice (C.B-Igh-1b/GbmsTac-Prkdcscid-Lystbg N7) bearing Ramos and Jurkat flank tumor xenografts confirmed the activity of the HDL-NP (Figure 2). Conclusion:We report a template-directed and bio-functional therapeutic nanostructure that could shift the paradigm for treating lymphoma and other cancers. A combination of SR-B1 binding and manipulation of cholesterol flux is responsible for selective induction of apoptosis in B cell lymphoma. [Display omitted] [Display omitted] Disclosures:Thaxton:Aurasense: Employment, Equity Ownership.

Cyclooxygenase-2 Expression, Prostacyclin Production and Endothelial Protection of High-density Lipoprotein

Atherosclerosis is a multi-factorial inflammatory disease with accumulation of lipids and recruitment of leukocytes into the subendothelial space. Due to the consistent inflammation in vessels, a subset of atherosclerotic plaques is even prone to physical disruption or producing thrombosis that triggers acute coronary syndromes (ACS). Cyclooxygenase (COX) and the downstream diverse prostanoids are involved in numerous physiological activities and inflammatory processes. Remarkably, prostanoids are a group of bioactive lipid mediators, and play a series of complicated and even contradictory roles during the progression of atherogenesis. Some, mainly prostacyclin (PGI2) have cardioprotective effects to inhibit the aggravation of atherosclerosis, however, others including prostaglandin E2 (PGE2) and thromboxane A2 (TXA2), increase the risk of cardiovascular thromboembolic events. Therefore, the controversial effects bring about a debate on the use of cyclooxygenase-2 (COX-2) specific and non-specific inhibitors in the prevention from cardiovascular diseases. In addition to reverse cholesterol transport (RCT), high-density lipoprotein (HDL) exerts several beneficial effects on endothelial protection. Moreover, HDL could also regulate the expression of COX-2 and the production of PGI2 in endothelial cells. While as a diverse complex of heterogeneous particle composed of various apolipoproteins, enzymes and lipids, the individual components of HDL, such as apolipoprotein A-I (apoA-I) and sphingosine-1-phosphate (S1P), have distinct effects on COX-2 expression and PGI2 production in endothelial cells. In the present review, we summarize the roles of COX-2 as well as PGI2 in atherosclerosis and atherothrombosis, the controversial vascular effects on prostanoid inhibition by COX-2 inhibitors, and the effects of HDL on the endothelial protection by the upregulation of COX-2 and the release of PGI2 during the progression of atherosclerosis.

Characterization of a Human Apolipoprotein A-I Construct Expressed in a Bacterial System

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins. This protein has key functions in lipoprotein metabolism and its plasma concentration is inversely correlated with the incidence of atherosclerosis and cardiovascular diseases. There is an increasing need to develop methods for efficient production of recombinant apoA-I for using it in basic research or pharmacological therapy. An apoA-I variant lacking two amino acid residues at the N-terminus can be easily produced by bacterial expression. We report here the characterization of this variant comparing its properties with those of the protein isolated from human serum. The results validate the use of this variant in future assays and investigations.

Enzymolysis of high density lipoprotein with a combination of membrane-immobilized esterase and trypsin

Apolipoprotein A-1 (apo A-1), a major component of high density lipoprotein (HDL), was efficiently digested by membrane-immobilized trypsin after HDL was treated with membrane-immobilized esterase. Compared to treatment with membrane-immobilized trypsin alone, the relative amounts of apo A-1 polypeptides, m/z 1723.78 and m/z 1568.82, increased by 2.7- and 3.9-fold, respectively, when HDL was treated with membrane-immobilized esterase and trypsin. Furthermore, the efficient digestion of apo A-1 by trypsin was inhibited when HDL was treated with membrane-immobilized esterase in the presence of an esterase inhibitor, 6,9-diamino-2-ethoxyacridine (acrinol). The data indicate that the lipid components of lipoproteins are released by membrane-immobilized esterase. This method can be used to investigate the structure and function of other apolipoproteins.

Harnessing a Physiologic Mechanism for siRNA Delivery With Mimetic Lipoprotein Particles

Therapeutics based on RNA interference (RNAi) have emerged as a potential new class of drugs for treating human disease by silencing the target messenger RNA (mRNA), thereby reducing levels of the corresponding pathogenic protein. The major challenge for RNAi therapeutics is the development of safe delivery vehicles for small interfering RNAs (siRNAs). We previously showed that cholesterol-conjugated siRNAs (chol-siRNA) associate with plasma lipoprotein particles and distribute primarily to the liver after systemic administration to mice. We further demonstrated enhancement of silencing by administration of chol-siRNA pre-associated with isolated high-density lipoprotein (HDL) or low-density lipoprotein (LDL). In this study, we investigated mimetic lipoprotein particle prepared from recombinant apolipoprotein A1 (apoA) and apolipoprotein E3 (apoE) as a delivery vehicle for chol-siRNAs. We show that apoE-containing particle (E-lip) is highly effective in functional delivery of chol-siRNA to mouse liver. E-lip delivery was found to be considerably more potent than apoA-containing particle (A-lip). Furthermore, E-lip–mediated delivery was not significantly affected by high endogenous levels of plasma LDL. These results demonstrate that E-lip has substantial potential as delivery vehicles for lipophilic conjugates of siRNAs.

Substituted Benzamides Containing Azaspiro Rings as Upregulators of Apolipoprotein A-I Transcription

Apolipoprotein A-I (Apo A-I) is the principal protein component of high density lipoprotein (HDL), which is generally considered as a potential therapeutic target against atherosclerosis. The understanding of the Apo A-I regulation mechanism has fuelled the development of novel HDL targeted therapeutic approaches. To identify novel agents that can upregulate Apo A-I expression, we performed a cell-based reporter assay to screen 25,600 small molecules. Based on the dataset obtained from screening, a series of novel analogs of substituted benzamides containing azaspiro rings were assessed for their ability to induce the transcription of the Apo A-I gene, and the structure-activity relationship (SAR) around these analogs was also proposed. The results indicated that the trifluoromethyl substituted benzamide containing an azaspiro ring is a promising backbone for designing Apo A-I transcriptional upregulator and could be viable leads for development of new drugs to prevent and treat atherosclerosis in the future.

OS041. Apolipoprotein A-I protects normal integration of the trophoblast into endothelial cellular networks in an in vitro model of preeclampsia

Failure of the trophoblast to appropriately invade uterine spiral arteries is thought to be an initiating event in preeclampsia, a disorder associated with endothelial dysfunction. A dyslipidemia characterised by low plasma levels of high density lipoproteins (HDL) and elevated triglycerides has also been described in preeclampsia. The pro-inflammatory cytokine TNF-α inhibits trophoblast invasion of uterine endothelial cells. Previous work using an in vitro JEG-3 cell/Uterine endothelial cell co-culture model investigated the effect of apoliopoprotein A-I, the main apolipoprotein component of HDL, on trophoblast incorporation into endothelial tubules in the presence and absence of TNF-α. These effects are now investigated using the human invasive trophoblast cell line HTR-8/SVneo. This study asks if apoA-I, which has established anti-inflammatory properties, can protect against the deleterious effect of TNF-α on trophoblast-endothelial cell interactions. The in vitro trophoblast-uterine endothelial cell co-culture model was used to investigate the effect of apoA-I on trophoblast incorporation into endothelial tubules in the presence and absence of TNF-α. Uterine endothelial cells were pre-incubated with lipid free apoA-I (final apoA-I concentration 1 mg/mL) for 16h prior to seeding on matrigel coated plates. Tubules formed within 4h. Fluorescence-labelled HTR-8/SVneo trophoblast cells were then co-cultured with the endothelial cells±TNF-α (final concentration of 0.2ng/mL). Bright field and fluorescent images were captured after 24h. The effect of TNF-α on HTR-8/SVneo cell invasion was quantified with Image J software. Integration of HTR-8/SVneo trophoblast cells into uterine endothelial tubular networks was also imaged using live cell imaging techniques (Zeiss Axiovert). TNF-α inhibited HTR-8/SVneo (trophoblast) cell integration into endothelial tubular structures by 24.1±3.7% p<0.001. This effect was reversed when the endothelial cells were pre-incubated for 16h with lipid free apoA-I (p<0.001 compared to non-incubated cells). Live cell images of the co-culture clearly demonstrate a disruption to the normal integration of trophoblast into endothelial tubular structures in the presence of TNF-α. The protective effect conferred by pre-incubation of endothelial cells with apoA-I against TNF-α is also clearly visible. Apolipoprotein A-I has been shown to enhance trophoblast-endothelial cell integration in the presence of a pro-inflammatory stimulus. A healthy lipid profile may affect pregnancy outcomes by priming endothelial cells in preparation for trophoblast invasion.

Extrahepatic High-Density Lipoprotein Receptor SR-BI and ApoA-I Protect Against Deep Vein Thrombosis in Mice

Deep vein thrombosis (DVT) and pulmonary embolism are frequent causes of morbidity and mortality. The goal of our study was to determine whether plasma high-density lipoprotein (HDL), which inversely correlates with the risk of cardiovascular events, affects DVT. Using a murine DVT model of inferior vena cava stenosis, we demonstrated that deficiency of the HDL receptor, scavenger receptor class B type I (SR-BI), promotes venous thrombosis. As SR-BI(-/-) mice have increased plasma cholesterol levels and abnormal HDL particles, we tested SR-BI(-/-) mice with an SR-BI liver transgene that normalizes both parameters. These mice also exhibited increased susceptibility to DVT, indicating a protective role of extrahepatic SR-BI. Mice lacking the major HDL apolipoprotein apoA-I or endothelial nitric oxide synthase (eNOS) (a downstream target of endothelial SR-BI signaling) also had a prothrombotic phenotype. Intravenous infusion of human apoA-I, an HDL component and SR-BI ligend, prevented DVT in wild-type but not SR-BI(-/-) or eNOS(-/-) mice, suggesting that its effect is mediated by SR-BI and eNOS. Intravenous apoA-I infusion abolished histamine-induced platelet-endothelial interactions, which are important for DVT initiation. An apoA-I (HDL)-SR-BI-eNOS axis is highly protective in DVT and may provide new targets for prophylaxis and treatment of venous thrombosis.

Paraoxonase 1 and homocysteine metabolism

Paraoxonase 1 (PON1), a component of high-density lipoprotein (HDL), is a calcium-dependent multifunctional enzyme that connects metabolisms of lipoproteins and homocysteine (Hcy). Both PON1 and Hcy have been implicated in human diseases, including atherosclerosis and neurodegeneration. The involvement of Hcy in disease could be mediated through its interactions with PON1. Due to its ability to reduce oxidative stress, PON1 contributes to atheroprotective functions of HDL in mice and humans. Although PON1 has the ability to hydrolyze a variety of substrates, only one of them-Hcy-thiolactone-is known to occur naturally. In humans and mice, Hcy-thiolactonase activity of PON1 protects against N-homocysteinylation, which is detrimental to protein structure and function. PON1 also protects against neurotoxicity associated with hyperhomocysteinemia in mouse models. The links between PON1 and Hcy in relation to pathological states such as coronary artery disease, stroke, diabetic mellitus, kidney failure and Alzheimer's disease that emerge from recent studies are the topics of this review.

Metabolism and Neurotoxicity of Homocysteine Thiolactone in Mice: Evidence for a Protective Role of Paraoxonase 1

Homocysteine (Hcy)-thiolactone is toxic, induces epileptic seizures in rodents, and has been implicated in Alzheimer's disease. Paraoxonase 1 (Pon1), a component of high-density lipoprotein, hydrolyzes Hcy-thiolactone in vitro. Whether this reflects a physiological function and whether Pon1 can protect against Hcy-thiolactone toxicity was unknown. Here we show that Hcy-thiolactone was elevated in brains of Pon1-/- mice (1.5-fold, p = 0.047) and that Pon1-/- mice excrete more Hcy-thiolactone than wild type animals (2.4-fold, p = 0.047). The frequency of seizures induced by intraperitoneal injections of L-Hcy-thiolactone was significantly higher in Pon1-/- mice compared with wild type animals (52.8% versus 29.5%, p = 0.042); the latency of seizures was lower in Pon1-/- mice than in wild type animals (31.8 min versus 41.2 min, p = 0.019). Using the Pon1 null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself is neurotoxic in vivo. Our findings indicate that Pon1 protects mice against Hcy-thiolactone neurotoxicity by hydrolyzing it in the brain, and suggest a mechanism by which Pon1 can protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer's disease.

Apolipoprotein A-I Attenuates Palmitate-Mediated NF-κB Activation by Reducing Toll-Like Receptor-4 Recruitment into Lipid Rafts

While high-density lipoprotein (HDL) is known to protect against a wide range of inflammatory stimuli, its anti-inflammatory mechanisms are not well understood. Furthermore, HDL's protective effects against saturated dietary fats have not been previously described. In this study, we used endothelial cells to demonstrate that while palmitic acid activates NF-κB signaling, apolipoprotein A–I, (apoA-I), the major protein component of HDL, attenuates palmitate-induced NF-κB activation. Further, vascular NF-κB signaling (IL-6, MCP-1, TNF-α) and macrophage markers (CD68, CD11c) induced by 24 weeks of a diabetogenic diet containing cholesterol (DDC) is reduced in human apoA-I overexpressing transgenic C57BL/6 mice compared to age-matched WT controls. Moreover, WT mice on DDC compared to a chow diet display increased gene expression of lipid raft markers such as Caveolin-1 and Flotillin-1, and inflammatory Toll-like receptors (TLRs) (TLR2, TLR4) in the vasculature. However apoA-I transgenic mice on DDC show markedly reduced expression of these genes. Finally, we show that in endothelial cells TLR4 is recruited into lipid rafts in response to palmitate, and that apoA-I prevents palmitate-induced TLR4 trafficking into lipid rafts, thereby blocking NF-κB activation. Thus, apoA-I overexpression might be a useful therapeutic tool against vascular inflammation.

Interplay Between SAFE and RISK Pathways in Sphingosine-1-Phosphate–Induced Cardioprotection

We studied the role of two powerful molecular signalling mechanisms involved in the cardioprotective effect of sphingosine-1-phosphate (S1P), a major component of high density lipoprotein (HDL) against myocardial ischaemic-reperfusion injury, namely the RISK pathway (Akt/Erk), including its downstream target FOXO-1 and, the SAFE pathway (TNF/STAT-3). Control hearts from wildtype, TNF deficient (TNF(-/-)) or cardiomyocyte STAT-3 deficient (STAT-3(-/-)) male mice were perfused on a Langendorff apparatus (35 min global ischaemia and 45 min reperfusion). S1P (10 nM) was given at the onset of reperfusion for the first 7 min, with/without STAT-3 or Akt inhibitors, AG490 and wortmannin (W), respectively. S1P reduced myocardial infarct size in wildtype hearts (39.3±4.4% in control vs 17.3±3.1% in S1P-treated hearts; n≥6; p<0.05) but not in STAT-3(-/-) or TNF(-/-) mice (34.2±4.3% in STAT-3(-/-) and 34.1±2.0% in TNF(-/-) mice; n≥6; p=ns vs. their respective control). Both STAT-3 and Akt inhibitors abolished the protective effects of S1P (33.7±3.3% in S1P + AG490 and 36.6±4.9% in S1P + W; n=6; p=ns vs. their respective control). Increased nuclear levels of phosphorylated STAT-3 (pSTAT-3), Akt and FOXO-1 were observed at 15 min reperfusion in wildtype mice with Western Blot analysis (53% STAT-3, 47% Akt, 41% FOXO-1; p<0.05 vs control) but not in STAT-3-/- mice or in wiltype hearts treated with the Akt inhibitor. Interestingly, an activation of pSTAT-3 was noticed in the mitochondria at 7 min but not 15 min of reperfusion. In conclusion, S1P activates both the SAFE and RISK pathways, therefore suggesting a dual protective signalling in S1P-induced cardioprotection.

Nonenzymatic glycation of high‐density lipoprotein impairs its anti‐inflammatory effects in innate immunity

In type 2 diabetes mellitus (T2DM), the abnormal protein and lipid composition of diabetic high-density lipoprotein (HDL) could impair its anti-inflammatory functions. Whether nonenzymatic glycation directly impaired the anti-inflammatory effects of HDL in innate immunity remained unclear. Human acute monocytic leukemia cell line (THP-1) cells, mouse RAW 264.7 macrophages and primary human monocytes derived macrophages were pre-incubated with native HDL, diabetic HDL isolated from T2DM patients or HDL glycated with different doses of d-glucose in vitro and then challenged with lipopolysaccharide (LPS). The release of tumor necrosis factor (TNF)-α and IL-1β was assayed by enzyme-linked immunosorbent assay (ELISA). Phosphorylation of Iκ-Bα in cytoplasm and nuclear translocation of NF-κB were detected by western blot. Glycation levels of native HDL, glycated HDL and diabetic HDL were determined using LC-MS/MS. The potency of diabetic HDL to inhibit the release of TNF-α (p < 0.05) and IL-1β (p < 0.001) was dramatically attenuated compared with that of native HDL. Similarly, glycation of HDL in vitro impaired its ability to inhibit TNF-α and IL-1β release in a glucose dose-dependent manner. Moreover, apoHDL still effectively inhibited the release of TNF-α and IL-1β induced by LPS, but glycated apoHDL partly lost such abilities. Nonenzymatic glycation levels of glycated HDL and diabetic HDL increased 28 fold (p < 0.001) and 4 fold (p < 0.001), respectively compared with that of native HDL. In this study, we observed that diabetic HDL and HDL glycated in vitro both partly lose their protective effects to inhibit cytokines release induced by LPS in macrophages, and nonenzymatic glycation of the protein components of HDL plays key roles in these impairments.

Surface-induced assembly of apolipoprotein A-I: Implications for symmetry-driven non-cooperative clustering

In condensed matter physics the geometry of a crystal is determined by the mechanism of condensation. In biology, the link between clustering mechanisms and the shape of a protein crystal is not well defined. To gain more insight into the problem, we studied clustering of apolipoprotein A-I (apo A-I) on a solid surface using AFM. The amyloidogenic protein apo A-I is the main protein component of high density lipoprotein and thus reduces the risk of atherosclerosis. We found that apo A-I clustered to form nano-scale, symmetrical clusters on mica. Statistical analysis of size distribution for several thousand clusters suggested that the clustering reaction followed a non-cooperative kinetic scheme characterized by a single equilibrium constant of 0.92·106M−1 and a change in free energy (ΔG) of −0.03kJmole−1/residue. This is close to ΔG of−0.04kJmole−1/residue for apo A-I binding to phospholipid membrane; and 30-fold smaller than ΔG for β-amyloid formation by apo A-I. The high symmetry of the clusters is consistent with an isotropic diffusion coefficient of protein monomers on the surface of the substrate. This previously unrecognized link between protein clustering mechanism and the symmetry of the growth pattern may have important implications in medicine, pharmaceutics and polymer science.