Recombinant apoA-I Research Articles

Abstract 227: Measuring HDL-apolipoprotein A-I Exchange By Subclass In Reconstituted And Human HDL

Objective: Levels of high density lipoprotein (HDL) are inversely associated with cardiovascular disease risk. HDL particles contain a diverse array of lipids, cholesterol, and proteins, and exist over a range of sizes ranging from about 8 nm to about 17 nm diameter, but it is not well understood how HDL size affects its function. In this study we examine the effect of HDL particle size on the rate of HDL-apolipoprotein A-I (apoA-I) exchange in reconstituted and human HDL particles. Method and Results: Reconstituted HDL particles were synthesized from 16:0-18:1n-9 PC, cholesterol, and recombinant apoA-I. Human HDL was isolated from plasma from healthy volunteers by density gradient ultracentrifugation. In both cases, the HDL was further separated by subclass using size exclusion chromatography. ApoA-I exchange was measured by incubating fluorescently-labeled, lipid-free apoA-I with HDL and quantifying the fluorescent signal appearing in the HDL region versus the lipid-free region of a nondenaturing gel over time. ApoA-I exchange rates increased inversely with particle size in both reconstituted and human HDL, with medium-sized HDL particles exhibiting twofold faster exchange rates compared to the largest HDL particles. Conclusions: Smaller HDL particles exhibit faster rates of HDL-apoA-I exchange in both reconstituted and human HDL particles. Reconstituted HDL particles provide an accurate approximation of apoA-I exchange in human HDL particles.

Human apoA-I[Lys107del] mutation affects lipid surface behavior of apoA-I and its ability to form large nascent HDL

Population studies have found that a natural human apoA-I variant, apoA-I[K107del], is strongly associated with low HDL-C but normal plasma apoA-I levels. We aimed to reveal properties of this variant that contribute to its unusual phenotype associated with atherosclerosis. Our oil-drop tensiometry studies revealed that compared to WT, recombinant apoA-I[K107del] adsorbed to surfaces of POPC-coated triolein drops at faster rates, remodeled the surfaces to a greater extent, and was ejected from the surfaces at higher surface pressures on compression of the lipid drops. These properties may drive increased binding of apoA-I[K107del] to and its better retention on large triglyceride-rich lipoproteins, thereby increasing the variant’s content on these lipoproteins. While K107del did not affect apoA-I capacity to promote ABCA1-mediated cholesterol efflux from J774 cells, it impaired the biogenesis of large nascent HDL particles resulting in the formation of predominantly smaller nascent HDL. Size-exclusion chromatography of spontaneously reconstituted 1,2-dimyristoylphosphatidylcholine-apoA-I complexes showed that apoA-I[K107del] had a hampered ability to form larger complexes but formed efficiently smaller-sized complexes. CD analysis revealed a reduced ability of apoA-I[K107del] to increase α-helical structure on binding to 1,2-dimyristoylphosphatidylcholine or in the presence of trifluoroethanol. This property may hinder the formation of large apoA-I[K107del]-containing discoidal and spherical HDL but not smaller HDL. Both factors, the increased content of apoA-I[K107del] on triglyceride-rich lipoproteins and the impaired ability of the variant to stabilize large HDL particles resulting in reduced lipid:protein ratios in HDL, may contribute to normal plasma apoA-I levels along with low HDL-C and increased risk for CVD.

Development of an Apolipoprotein AI Chimera for Targeted Nanodisc Drug Delivery to Breast Cancer Cells

A current challenge in cancer treatment is the ability to selectively target diseased cells with chemotherapeutics. Several promising treatments suffer this limitation and have toxic off target effects on healthy cells. Our overall objective is to generate a platform for targeted drug delivery employing self‐assembling nanodiscs comprised of phospholipids and apolipoprotein AI (apoAI). ApoAI is an exchangeable apolipoprotein located on high density lipoproteins (HDL), which promotes cholesterol efflux from macrophages and facilitates selective uptake of cholesterol by binding scavenger receptor, class B type 1 (SR‐B1), also known as the HDL receptor. We hypothesize that a chimeric protein of apoAI with MT1‐AF7p (apoAI‐AF7p), a peptide ligand that binds membrane metalloproteinase 14 (MMP14), will enhance the targeting capability of the nanodiscs to MDA‐MB‐231 breast cancer cells. MMP14 is a cell surface protein that is overexpressed in many types of cancers responsible for metastasis and linked to poor prognosis in patients. We designed a construct bearing the coding sequence for human apoAI with a hexa‐His‐tag at the N‐terminal end to facilitate protein purification by affinity chromatography, and MT1‐AF7p (HWKHLHNTKTFL) at the C‐terminal end with an intervening linker segment ([G4S]3). The construct was designed and custom‐generated with optimized codon sequence in a pCR2.1 vector. We successfully sub‐cloned the chimeric insert into a pET20b(+) expression vector employing standard molecular biology protocols and verified the resulting plasmid through Sanger sequencing. Recombinant apoAI and apoAI‐AF7p proteins were over‐expressed in E. coli and purified using a nickel affinity chromatography. The purified proteins were visualized by 15% acrylamide SDS‐PAGE, which revealed major bands at ~ 29 kDa band for apoAI and 32 kDa band for apoAI‐AF7p consistent with their expected size difference. We obtained ~ 10 mg purified protein/L culture medium, which is sufficient to carry out structural and functional characterization. Chimeric apoAI‐AF7p nanodiscs have potential not only for targeting, but also for drug delivery since MDA‐MB‐231 cells are reported to have high expression levels of both SR‐B1 and MMP14 which mediates homing and selective uptake of the hydrophobic core of HDL respectively. Preliminary data from fluorescence microscopy indicates the presence of both these proteins as well as the LDLr. Additionally, preliminary circular dichroism data shows similar helical content in the chimera as apoA1. Our next steps are to characterize the expression of MMP14, SR‐B1, and LDLr on MDA‐MB‐231 cells via FACS, qPCR and immunoblotting as well prepare nanodiscs with the chimera. Once particle formation and protein surface expression is established, cells will be incubated with particles and we’ll assess cell binding characteristics. The chimeric nanodiscs bears potential not only for targeting breast cancer cells, but also for drug delivery since these cells are reported to have high expression levels of SRB1, which mediate uptake of the contents within the hydrophobic core.Support or Funding InformationThis project is supported by the National Institute of Health (NIH) under the award numbers GM105561 (VN), R25GM071638 (DK) and T34GM008074 (RM)ApoA1‐AF7p chimera design: hexa‐His tag at the N‐terminus of ApoA1 and the affinity peptide MT1‐AF7p at the C terminus.Figure 1

Structural-functional characterization of recombinant Apolipoprotein A-I fromLabeo rohitademonstrates heat-resistant antimicrobial activity.

Apolipoprotein A-I is an anti-inflammatory, antioxidative, cardioprotective, anti-tumorigenic, and anti-diabetic in mammals. Apolipoprotein A-I also regulates innate immune defense mechanisms in vertebrates and invertebrates.Apolipoproteins A-I from mammals and several teleosts display antibacterial activities against Gram negative and Gram positive bacteria. The present study describes strategies to obtain high amounts of soluble purified recombinant Apolipoprotein A-I of Labeo rohita, an Indian major carp (rLrApoA-I). The study also reports its detailed structural and functional characterization i.e. antimicrobial activity against a number of important marine and fresh water bacterial pathogens. The rLrApoA-I was expressed inEscherichia coliBL21(DE3) pLysS expression hostas a soluble protein under optimized conditions. Theyield of purified rLrApoA-I was ~ 75 mg/L from soluble fraction using metal ion affinity chromatography. The authenticity of the rLrApoA-I was confirmed by MALDI-TOF-MS analysis. The secondary structure analysis showed rLrApoA-I to be predominantly alpha helical, an evolutionary conservedcharacteristic acrossmammals and teleosts. The purified rLrApoA-I exhibited antimicrobial activity as evident from inhibition of growth of a number of bacteria namelyAeromonas hydrophila, A. liquefaciens, A. culicicola, A. sobria, Vibrio harveyi, V. parahaemolyticus and Edwardsiella tardain a dose-dependent manner. Minimum bactericidal concentration forA. liquefaciens, A. culicicola, and A. sobria, was determined to be 25 μg/ml or 0.81 μM whereas for A. hydrophila, E. tarda,V. parahaemolyticus andV. harveyi, it was determined to be 100 μg/ml or 3.23 μM. These data strongly suggest that recombinant ApoA-I fromLabeo rohitacould play a role in primary defense against fish pathogen. Further, at temperature ≥ 55 °C, though a loss in secondary structure was observed, no effect on its antibacterial activity was observed. This is of significance as the antibacterial activity is not likely to be lost even if the protein is subjected to high temperatures during transport.

Abstract 201: Generation of Photoactivatable apoA I to Study HDL Transport in vivo Reveals Impaired HDL Recirculation in a Murine Model of Psoriasis

HDL is cardioprotective, but plasma HDL levels do not necessarily predict cardiovascular outcomes. The major HDL-associated protein apoA-I picks up its cholesterol from cells within extravascular compartments to return it to plasma and then bile. Yet, tools are lacking to quantify the important step of HDL transit through extravascular spaces. Here, we developed recombinant photoactivatable apoA-I to quantify endogenous HDL recirculation. Using the tool, we studied HDL passage through skin in healthy mice versus those with experimental psoriasis, wherein collagen density increased in the skin in a CD4 + T cell-dependent manner. In control mice, photoactivated HDL mobilized to plasma within 2 h but was retained in collagen-enriched skin of mice with psoriasis. These data suggest that cardiovascular comorbidity in psoriasis might be linked to T cell-mediated structural changes in skin that impedes systemic recirculation of HDL. This new tool is likely to find wide application in HDL research.

Abstract 387: HDL-apolipoprotein A-I Exchange Occurs in the Absence of HDL Particle Remodeling

Objective: Apolipoprotein A-I (apoA-I) spontaneously exchanges between high-density lipoprotein (HDL)-bound and lipid-free states. This is a key event in reverse cholesterol transport, necessary for de novo HDL biogenesis. However, the mechanism of HDL-apoA-I exchange (HAE) is poorly understood. In this study, we test whether HDL remodeling is obligatory to HAE or an independent process. Method and Results: Recombinant human apoA-I modified with single cysteine mutations were expressed in bacteria and labeled with Alexa 488 and Alexa 647 fluorophores. ApoA-I Alexa647 was used to synthesize rHDL particles of defined sizes with phosphatidyl choline (POPC) and cholesterol. Reconstituted HDL of 17.0, 12.2, 9.6, 8.4 and 7.8 nm were obtained. These particles were incubated with excess lipid-free apoA-I Alexa488 to initiate HAE, which was quantified using non-denaturing gradient gel electrophoresis to separate HDL particles from lipid-free apoA-I. Gels were imaged using FITC and Cy5 filters. The extent of apoA-I Alexa488 binding and apoA-I Alexa647 released from HDL was determined by densitometry. HAE occurred without significant changes in rHDL particle size for all subclasses of rHDL. HAE in the forward (lipid-free to bound) direction did not exhibit size dependence, but HAE in the reverse (lipid-bound to free) direction appeared to be size-dependent, with the 8.4 nm rHDL displaying 2-fold higher apoA-I Alexa647 dissociation compared to the 17.0 nm particle. Conclusions: The bidirectional rate of HAE can be measured simultaneously using fluorescent apoA-I probes. Reconstituted HDL particles predominantly retained their size, indicating that apoA-I exchange occurs without remodeling of HDL particles. This result has significant implications as to the possible molecular processes driving apoA-I exchange with HDL.

Human macrophage cathepsin B-mediated C-terminal cleavage of apolipoprotein A-I at Ser228 severely impairs antiatherogenic capacity.

Apolipoprotein A-I (apoA-I) is the major component of HDL and central to the ability of HDL to stimulate ATP-binding cassette transporter A1 (ABCA1)-dependent, antiatherogenic export of cholesterol from macrophage foam cells, a key player in the pathology of atherosclerosis. Cell-mediated modifications of apoA-I, such as chlorination, nitration, oxidation, and proteolysis, can impair its antiatherogenic function, although it is unknown whether macrophages themselves contribute to such modifications. To investigate this, human monocyte-derived macrophages (HMDMs) were incubated with human apoA-I under conditions used to induce cholesterol export. Two-dimensional gel electrophoresis and Western blot analysis identified that apoA-I is cleaved (∼20-80%) by HMDMs in a time-dependent manner, generating apoA-I of lower MW and isoelectric point. Mass spectrometry analysis identified a novel C-terminal cleavage site of apoA-I between Ser228-Phe229 Recombinant apoA-I truncated at Ser228 demonstrated profound loss of capacity to solubilize lipid and to promote ABCA1-dependent cholesterol efflux. Protease inhibitors, small interfering RNA knockdown in HMDMs, mass spectrometry analysis, and cathepsin B activity assays identified secreted cathepsin B as responsible for apoA-I cleavage at Ser228 Importantly, C-terminal cleavage of apoA-I was also detected in human carotid plaque. Cleavage at Ser228 is a novel, functionally important post-translational modification of apoA-I mediated by HMDMs that limits the antiatherogenic properties of apoA-I.-Dinnes, D. L. M., White, M. Y., Kockx, M., Traini, M., Hsieh, V., Kim, M.-J., Hou, L., Jessup, W., Rye, K.-A., Thaysen-Andersen, M., Cordwell, S. J., Kritharides, L. Human macrophage cathepsin B-mediated C-terminal cleavage of apolipoprotein A-I at Ser228 severely impairs antiatherogenic capacity.

Production and Analysis of Biological Properties of Recombinant Human Apolipoprotein A-I.

Production of recombinant human apolipoprotein A-I (apoA-I) in E. coli cells is described and its biological properties are compared with those of natural protein. Recombinant apoA-I was isolated as a chimeric polypeptide and then processed to a mature form apoA-I (rapo-I). We studied the ability of the resulting protein to penetrate into hepatocyte nuclei and regulate the rate of DNA biosynthesis in complex with estriol. Penetration of rapoA-I conjugated with FITC into hepatocyte nuclei was demonstrated. rapoA-I-estriol and apoA-I-estriol complexes induced similar increase in DNA biosynthesis rate in isolated hepatocytes, which confi rms functional similarity of the obtained recombinant mature protein (rapoA-I) and native human apoA-I.

Why do we fail to achieve Glagovian atheroregression in lipid-lowering trials?

• Reduction of total atheroma volume provides cardiology with a hope to reverse atherosclerosis in hands of the modern-day lipidlowering medications: • ASTEROID and SATURN trials demonstrated the potential of statins which reduce the total atheroma volume up to 6.38 mm3 in coronary arteries and 30% decrease in outcomes; • Recombinant ApoA-I Milano demonstrated a 14.1 mm3 reduction in total atheroma volume with unproven effect on clinical outcomes; • ZEUS trial with ezetimibe revealed an 8.2 mm3 atheroregression. • The plaque burden with a Glagovian threshold of 40% (PAV [percent atheroma volume] above 40% at the baseline, and below 40% at the follow-up) is the ultimate criterion of the clinically valuable lesion we are able to examine in order to judge the genuine Glagovian atheroregression. • Some methodological flaws of the modern-day interventional imaging approaches including improper interpretation of the vessel contours, misunderstanding of such parameters as total atheroma volume and PAV, absence of the unified CoreLab expert methodology for assessment of both intravascular and noninvasive coronary imaging significantly impact results and further progress in this field: • There is a difference in methodology how to assess plaque burden between histology of Glagov and IVUS-imaging of Nissen; • Neither intravascular intravascular ultrasound (IVUS) or optical coherence tomography nor noninvasive computed tomography angiography allow us to comprehensively distinguish all the artery layers; • External elastic membrane and adventitia could be quantitatively assessed by IVUS only; • Some new medications as well as progress in the development of the bioresorbable scaffolds and nanomedicine promise to revolutionize the cardiovascular biomedicine with the main goal to achieve Glagovian atheroregression below 40% threshold of the PB: • The bioresorbable scaffold Absorb BVS (Abbott Vascular, CA, USA) is the first coronary device which has shown phenomena such as late lumen enlargement and wall thinning with at least 12% reduction of plaque burden; • NANOM-FIM trial of plasmonic photothermal therapy with silica-gold nanoparticles showed truly unprecedented 60.3 mm3 plaque volume reduction. • The modern-day statin trials were conducted in patients with relatively small lesions when PAV never exceeded 40% and without proper methodology, which means that the revealed phenomenon of atheroregression is essentially the pseudo reduction of the atheroma volume. • The PAV or plaque burden is the only parameter to describe changes in the vessel geometry from the Glagov phenomenon point of view because it mathematically reflects patterns between both vessel and lumen size. • IVUS with assessment of plaque burden remains the golden standard to evaluate atheroregressive patterns of medications or medical devices in clinical trials: • Trials with the baseline PAV above 40% demonstrated higher potential of the plaque burden reduction; • Further comprehensive analysis is required in order to validate the genuine threshold of the artery enlargement between a 20 and 55% PAV when positive correlation between lumen area and PB gets replaced by the negative correlation with the progressive narrowing of the lumen.

Abstract 223: The Structural Organization of Apolipoprotein A-I in Reconstituted High-Density Lipoprotein Discs Using Stable Isotope-Assisted Cross-Linking

X-ray crystallography studies of lipid-free apolipoprotein A-I (apoA-I) and related apoA-IV have shown that the proteins form a reciprocating dimer which could be directly related to the structural organization of lipid-bound apoA-I. Previous work has demonstrated the success of chemical cross-linking combined with mass spectrometry to derive distance constraints for molecular modeling of apoA-I in reconstituted high density lipoprotein (HDL) particles. However, these studies have been limited by i) the relative paucity of distance constraint data, and ii) ambiguities in determining the molecular span of the cross-links. Here, we have addressed the first issue by using an affinity-tagged cross-linker to purify cross-linked peptides away from non-modified peptides prior to sampling with mass spectrometry. This essentially boosts the signal from cross-linked peptides of interest that would have otherwise been suppressed by detection of non-modified peptides. Using this method, we identified >25 cross-linked peptides; the most reported to date for these particles. To address the issue of molecular span, we used a stable isotopic-labeling approach in which wild-type human recombinant apoA-I ( 14 N) was mixed 1:1 with an isotopically labeled ( 15 N) apoA-I. Analysis of the isotopic patterns of the resulting mass spectra allowed the unambiguous determination of the molecular span of the cross-linked peptides; i.e. whether the links were within the same molecule of apoA-I (intramolecular) or between the dimers of apoA-I (intermolecular). This information will be used to derive a detailed novel model of lipid-bound apoA-I in reconstituted HDL, an important step in our overall understanding of how native HDL species are organized and how they interact with plasma remodeling factors and cell surface proteins.

Site-specific Nitration of Apolipoprotein A-I at Tyrosine 166 Is Both Abundant within Human Atherosclerotic Plaque and Dysfunctional

We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr(166)), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr(166) nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO2-Tyr(166)-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNACUA pair for functional studies. Studies with mAb 4G11.2 showed that NO2-Tyr(166)-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for ∼ 8% of total apoA-I within the artery wall but was nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO2-Tyr(166)-apoA-I existed as a lipid-poor lipoprotein with <3% recovered within the HDL-like fraction (d = 1.063-1.21). NO2-Tyr(166)-apoA-I in plasma showed a similar distribution. Recovery of NO2-Tyr(166)-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 ± 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr(166) is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall.

Recent progress in the development of apoA–I and apoE mimetic therapies

Historical perspectives Epidemiological studies demonstrate a clear inverse association of HDL with cardiovascular disease. Results from the Framingham Heart Study demonstrate that low levels of HDL are an independent risk factor for coronary heart disease (CHD). Statins, which can reduce levels of apoB-containing lipoproteins and plasma cholesterol levels, have been only partially successful at reducing the risk of CHD, while 70% of the problem remains to be solved. In addition, with the increasing prevalence of metabolic syndrome, in order to successfully address the prevailing CHD problem, there is a need for an alternate mode of therapy than simply to reduce plasma cholesterol levels. Thus, much recent attention has focused on studying HDL as an alternate therapy. Badimon et al. showed that administering human HDL to cholesterol-fed rabbits regressed atherosclerotic plaque formation [1]. Since apoA-I is the major protein component of HDL, studies with isolated apoA-I in the presence of lipid have been performed [2], termed recombinant HDL therapy. Transgenic expression of human apoA-I in athero sclerosis-sensitive mouse models also demonstrated significant inhibition of atherosclerosis [3]. The field got a boost when apoA-I milano –lipid complexes (40 mg/kg of protein) were infused in humans and the results were followed by intravenous ultrasound. When apoA-I milano –POPC complexes were infused in 40 patients, there was a significant decrease in the atheroma volume (between 3.5 and 5%) compared with baseline as studied by intravenous ultrasound [4]. However, perhaps due to the prohibitive cost of producing human or recombinant apoA-I, this technology has not

HDL and Cardiovascular Risk

High-density lipoprotein cholesterol (HDL-C) has been dubbed the good cholesterol because it is thought to reflect the ability of HDL particles to remove excess cholesterol molecules from peripheral cells (including those in atherosclerotic plaques) for return to the liver. Not surprisingly, then, HDL-C has frequently been assumed to be a biomarker of HDL function, consistent with the inverse relationship in observational studies between plasma levels of HDL-C and risk of coronary artery disease. Recently, Voight et al have challenged this assumption by showing that genetically elevated HDL-C did not protect against myocardial infarction. This finding has fueled a lively discussion in the lay, scientific, and medical press about the relationship between HDL-C and HDL function, and the potential effectiveness of various HDL-C raising strategies.

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.

Phospholipid nanodisc engineering for drug delivery systems

Biocompatible mesoscale nanoparticles (5-100 nm in diameter) are attractive tools for drug delivery. Among them are several types of liposomes and polymer micelles already in clinical trial or use. Generally, biocompatibility of such particles is achieved by coating them with polyethylene glycol (PEG). Without PEG coating, particles are quickly trapped in the reticuloendothelial system when intravenously administered. However, recent studies have revealed several potential problems with PEG coating, including antigenicity and restriction of cellular uptake. This has motivated the development of alternative drug and gene delivery vehicles, including chemically and genetically engineered high-density lipoprotein (HDL)-like nanodiscs or "bicelles". HDL is a naturally occurring mesoscale nanoparticle that normally ferries cholesterol around in the body. Its initial "nascent" form is thought to be a simple 10 nm disc of phospholipids in a bilayer, and can be easily synthesized in vitro by mixing recombinant apoA-I proteins with various phospholipids. In this review, the use of synthetic HDL-like phospholipid nanodiscs as biocompatible drug carriers is summarized, focussing on manufacturing, size-control, drug loading and cell targeting.

Abstract 411: An Anion-Exchange Chromatography Isolated Subfraction of Mouse Apolipoprotein A-I Is Unable to Activate Cellular Cholesterol Release from Mouse Peritoneal Macrophage Foam Cells

Aim: Human and mouse plasma apoA-I separate on anion exchange chromatography (6M urea) in distinct subfractions. To investigate the nature of this diversity, we analyzed the protein subfractions by IEF and Urea-PAGE, and compared their ability to generate HDL in vitro. Methods: Human and mouse plasma HDLs were delipidated then applied to an DE52 ion-exchange column and eluted by 30-60mM Tris-HCl, pH7.80 containing 6M urea. Mouse, human, and recombinant apoA-Is were analyzed by IEF (Ettan IPGphor3) followed by CBB protein staining. Urea-PAGE analysis was performed with 14% acrylamide gel in 6M urea buffer. ApoA-Is abilities to efflux cholesterol from mouse peritoneal foam cells and to shift the size of the ACAT-accessible cholesterol pool were determined. Results: Human plasma and recombinant human apoA-I showed two apoA-I subtypes by IEF and Urea-PAGE analysis; whereas HDL from C57BL/6 and 129SV mice contained three apoA-Is at pI=5.37, 5.31, 5.27 (mP3-1, mP2, mP3-3), and pI=5.44, 5.37, 5.32, respectively. Subfractions mP2 and mP3-3 were isolated on a DE52 column and, despite their difference in pI, were able to mediate cellular cholesterol efflux at levels similar to human plasma apoA-Is. Subfraction mP3-1, with the highest pI, eluted between mP2 and mP3-3 on DE-52 column. Interestingly, for both mouse strains, mP3-1 showed very poor cholesterol efflux and the incorporation of 14C-oleic acid to cellular CE was significantly higher when compared to mP2 and mP3-3. Conclusion: We found that differences in the pI of anion-exchange chromatography apoA-I subfractions did not directly affect HDL generation except for mouse subtype mP3-1. mP3-1 binds strongly to the anionic DE52 resin, probably by hydrophobic interaction, and is unable to mediate cellular cholesterol efflux. Further investigation of the nature of the differences in these apoA-I subtypes will provide insights on factors that may be implicated in the biogenesis of HDL in vivo.

Abstract 263: Isotopic Labeling and Chemical Cross-Linking Reveals Intermolecular Contacts and Dynamics of Apolipoprotein A-I Structure in Reconstituted HDL Particles

Apolipoprotein (apo)A-I has been proposed to adopt a number of different, but related, structures when in contact with lipid. The technique of chemical cross-linking has recently been used to determine the spatial relationships between two molecules of apoA-I in reconstituted high density lipoprotein (rHDL) particles. However, this technique is limited in that it cannot unequivocally distinguish between intramolecular and intermolecular contacts. To address this issue, we have produced two forms of recombinant apoA-I that contain the naturally occurring isotope of nitrogen (N14) or a stable isotope (N15). These forms were mixed 1:1 and then used to produce reconstituted HDL particles with synthetic lipids. The resulting mass shifts (readily detectable in the mass spectrometer) were exploited to unambiguously distinguish between intramolecular (N14 to N14 or N15 to N15) and intermolecular (N14 to N15) cross-linked peptides. An additional benefit of this approach was the ability to identify cross-links with high certainty without the need for peptide fragmentation, allowing for dramatic increases in method sensitivity. We studied highly homogeneous rHDL particles made with the fully saturated phospholipid palmitoyl steroyl phosphatidylcholine (PSPC) to minimize apoA-I conformational dynamics. These particles were 98Å in diameter, contained two molecules of apoA-I, approximately 155 molecules of PC, and were discoidal in shape by cryo EM. We identified 30 cross-links (17 intramolecular, 13 intermolecular) that were overall consistent with the double belt model in which both apoA-I molecules wrap around a bilayer of lipids in an antiparallel orientation. Unambiguous intramolecular linkages between the N- and C-terminal regions of apoA-I were inconsistent with the ‘double super helix’ variant of the belt model. We identified several low abundance cross-links that suggest that the N-terminus may be conformationally dynamic and may spend some time folded back across the molecule as proposed for the ‘belt and buckle’ belt model.

Myeloperoxidase Targets Apolipoprotein A-I, the Major High Density Lipoprotein Protein, for Site-Specific Oxidation in Human Atherosclerotic Lesions

Oxidative damage by myeloperoxidase (MPO) has been proposed to deprive HDL of its cardioprotective effects. In vitro studies reveal that MPO chlorinates and nitrates specific tyrosine residues of apoA-I, the major HDL protein. After Tyr-192 is chlorinated, apoA-I is less able to promote cholesterol efflux by the ABCA1 pathway. To investigate the potential role of this pathway in vivo, we used tandem mass spectrometry with selected reaction monitoring to quantify the regiospecific oxidation of apoA-I. This approach demonstrated that Tyr-192 is the major chlorination site in apoA-I in both plasma and lesion HDL of humans. We also found that Tyr-192 is the major nitration site in apoA-I of circulating HDL but that Tyr-18 is the major site in lesion HDL. Levels of 3-nitrotyrosine strongly correlated with levels of 3-chlorotyrosine in lesion HDL, and Tyr-18 of apoA-I was the major nitration site in HDL exposed to MPO in vitro, suggesting that MPO is the major pathway for chlorination and nitration of HDL in human atherosclerotic tissue. These observations may have implications for treating cardiovascular disease, because recombinant apoA-I is under investigation as a therapeutic agent and mutant forms of apoA-I that resist oxidation might be more cardioprotective than the native form.

Plasmonics for Treatment of Atherosclerosis: Results of NANOM-FIM Trial

Background: Atheroregression becomes an attractive target for cardiovascular treatment. Some clinical trials have demonstrated that intensive therapy with rosuvastatin or recombinant ApoA-I Milano can partially reduce the total atheroma volume (TAV) up to 6.38 mm 3 or 14.1 mm 3 respectively. Our previous bench studies of selected nanotechnologies documented TAV reduction up to unprecedented 79.4 mm 3 . Methods: The completed observational three arms (n=180) first-in-man trial (the NANOM FIM trial) assessed (NCT01270139) the safety and feasibility of two delivery techniques for nanoparticles (NP), and plasmonic photothermal therapy (PPTT). Patients were assigned to receive either (1) nano-intervention with delivery of silica-gold NP in bioengineered on-artery patch (n=60), or (2) nano-intervention with delivery of silica-gold iron-bearing NP with targeted micro-bubbles or stem cells using magnetic navigation system (n=60) versus (3) stent implantation (n=60). The primary outcome was TAV at 12 months. Results: The mean TAV reduction at 12 months in nano group was 60.3 mm 3 (SD 39.5; min 41.9 mm 3 , max 94.2 mm 3 ; p<0.05) up to mean 37.8% (95% CI: 31.1%, 51.7%; p<0.05) plaque burden. The analysis of the event free survival of the ongoing clinical follow-up shows the significantly lower risk of cardiovascular death in nano group when compared with others (91.7% vs 81.7% and 80% respectively; p<0.05) with no cases of the target lesion-related complications. Conslusions: PPTT using silica-gold NP associated with significant regression of coronary atherosclerosis.

Exogenous expression of human apoA-I enhances cardiac differentiation of pluripotent stem cells.

The cardioprotective effects of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A1 (apoA-I) are well documented, but their effects in the direction of the cardiac differentiation of embryonic stem cells are unknown. We evaluated the effects of exogenous apoA-I expression on cardiac differentiation of ESCs and maturation of ESC-derived cardiomyocytes. We stably over-expressed full-length human apoA-I cDNA with lentivirus (LV)-mediated gene transfer in undifferentiated mouse ESCs and human induced pluripotent stem cells. Upon cardiac differentiation, we observed a significantly higher percentage of beating embryoid bodies, an increased number of cardiomyocytes as determined by flow cytometry, and expression of cardiac markers including α-myosin heavy chain, β-myosin heavy chain and myosin light chain 2 ventricular transcripts in LV-apoA-I transduced ESCs compared with control (LV-GFP). In the presence of noggin, a BMP4 antagonist, activation of BMP4-SMAD signaling cascade in apoA-I transduced ESCs completely abolished the apoA-I stimulated cardiac differentiation. Furthermore, co-application of recombinant apoA-I and BMP4 synergistically increased the percentage of beating EBs derived from untransduced D3 ESCs. These together suggests that that pro-cardiogenic apoA-I is mediated via the BMP4-SMAD signaling pathway. Functionally, cardiomyocytes derived from the apoA-I-transduced cells exhibited improved calcium handling properties in both non-caffeine and caffeine-induced calcium transient, suggesting that apoA-I plays a role in enhancing cardiac maturation. This increased cardiac differentiation and maturation has also been observed in human iPSCs, providing further evidence of the beneficial effects of apoA-I in promoting cardiac differentiation. In Conclusion, we present novel experimental evidence that apoA-I enhances cardiac differentiation of ESCs and iPSCs and promotes maturation of the calcium handling property of ESC-derived cardiomyocytes via the BMP4/SMAD signaling pathway.