Reduced binding of apoE4 to complement factor H promotes amyloid-ß oligomerization and neuroinflammation.

The APOE4 variant of apolipoprotein E (apoE) is the most prevalent genetic risk allele associated with late-onset Alzheimer's disease (AD). ApoE interacts with complement regulator factor H (FH), but the role of this interaction in AD pathogenesis is unknown. Here we elucidate the mechanism by which isoform-specific binding of apoE to FH alters Aß1-42-mediated neurotoxicity and clearance. Flow cytometry and transcriptomic analysis reveal that apoE and FH reduce binding of Aß1-42 to complement receptor 3 (CR3) and subsequent phagocytosis by microglia which alters expression of genes involved in AD. Moreover, FH forms complement-resistant oligomers with apoE/Aß1-42 complexes and the formation of these complexes is isoform specific with apoE2 and apoE3 showing higher affinity to FH than apoE4. These FH/apoE complexes reduce Aß1-42 oligomerization and toxicity, and colocalize with complement activator C1q deposited on Aß plaques in the brain. These findings provide an important mechanistic insight into AD pathogenesis and explain how the strongest genetic risk factor for AD predisposes for neuroinflammation in the early stages of the disease pathology.

Library screening identifies commercial drugs as potential structure correctors of abnormal apolipoprotein A-I.

AapoA-I, the main protein of high-density lipoprotein, plays a key role in the biogenesis and atheroprotective properties of high-density lipoprotein. We showed previously that a naturally occurring apoA-I mutation, L178P, induces major defects in protein's structural integrity and functions that may underlie the increased cardiovascular risk observed in carriers of the mutation. Here, a library of marketed drugs (956 compounds) was screened against apoA-I[L178P] to identify molecules that can stabilize the normal conformation of apoA-I. Screening was performed by the thermal shift assay in the presence of fluorescent dye SYPRO Orange. As an orthogonal assay, we monitored the change in fluorescence intensity of 8-anilinonaphthalene-1-sulfonic acid upon binding on hydrophobic sites on apoA-I. Screening identified four potential structure correctors. Subsequent analysis of the concentration-dependent effect of these compounds on secondary structure and thermodynamic stability of WT apoA-I and apoA-I[L178P] (assessed by thermal shift assay and circular dichroism spectroscopy), as well as on macrophage viability, narrowed the potential structure correctors to two, the drugs atorvastatin and bexarotene. Functional analysis showed that these two compounds can restore the defective capacity of apoA-I[L178P] to promote cholesterol removal from macrophages, an important step for atheroprotection. Computational docking suggested that both drugs target a positively charged cavity in apoA-I, formed between helix 1/2 and helix 5, and make extensive interactions that could underlie thermodynamic stabilization. Overall, our findings indicate that small molecules can correct defective apoA-I structure and function and may lead to novel therapeutic approaches for apoA-I-related dyslipidemias and increased cardiovascular risk.

Improvement of high-density lipoprotein atheroprotective properties in patients with systemic lupus erythematosus after belimumab treatment.

OBJECTIVE: Chronic inflammatory diseases, like Systemic Lupus Erythematosus (SLE), carry an increased risk for atherosclerosis and cardiovascular events, accompanied by impairment of atheroprotective properties of high-density lipoprotein (HDL). In SLE, serum BAFF (B cell-activating factor), a cytokine implicated in disease progression, has been correlated with subclinical atherosclerosis. We investigated the impact of treatment with belimumab -an anti-BAFF monoclonal antibody- on HDL atheroprotective properties and composition in SLE patients. METHODS: Serum samples were collected from 35 SLE patients with active disease despite conventional therapy, before and after 6-month add-on treatment with belimumab, and 26 matched healthy individuals. We measured cholesterol efflux and antioxidant capacities, paraoxonase-1 activity, serum amyloid A1, myeloperoxidase and lipid peroxidation product levels of HDL. LC-MS/MS was performed to analyze the HDL lipidome. RESULTS: Following treatment with belimumab, cholesterol efflux and antioxidant capacities of HDL were significantly increased in SLE patients and restored to levels of controls. HDL-associated paraoxonase-1 activity was also increased, whereas lipid peroxidation products were decreased following treatment. HDL cholesterol efflux and antioxidant capacities correlated negatively with the disease activity. Changes were noted in the HDL lipidome of SLE patients following belimumab treatment, as well as between SLE patients and healthy individuals, and specific changes in lipid species correlated with functional parameters of HDL. CONCLUSIONS: HDL of SLE patients with active disease displays impaired atheroprotective properties accompanied by distinct lipidomic signature compared with controls. Belimumab treatment may improve the HDL atheroprotective properties and modify the HDL lipidomic signature in SLE patients, thus potentially mitigating atherosclerosis development.

Curcumin-Loaded PnBA-b-POEGA Nanoformulations: A Study of Drug-Polymer Interactions and Release Behavior.

The current study focuses on the development of innovative and highly-stable curcumin (CUR)-based therapeutics by encapsulating CUR in biocompatible poly(n-butyl acrylate)-block-poly(oligo(ethylene glycol) methyl ether acrylate) (PnBA-b-POEGA) micelles. State-of-the-art methods were used to investigate the encapsulation of CUR in PnBA-b-POEGA micelles and the potential of ultrasound to enhance the release of encapsulated CUR. Dynamic light scattering (DLS), attenuated total reflection Fourier transform infrared (ATR-FTIR), and ultraviolet-visible (UV-Vis) spectroscopies confirmed the successful encapsulation of CUR within the hydrophobic domains of the copolymers, resulting in the formation of distinct and robust drug/polymer nanostructures. The exceptional stability of the CUR-loaded PnBA-b-POEGA nanocarriers over a period of 210 days was also demonstrated by proton nuclear magnetic resonance (1H-NMR) spectroscopy studies. A comprehensive 2D NMR characterization of the CUR-loaded nanocarriers authenticated the presence of CUR within the micelles, and unveiled the intricate nature of the drug-polymer intermolecular interactions. The UV-Vis results also indicated high encapsulation efficiency values for the CUR-loaded nanocarriers and revealed a significant influence of ultrasound on the release profile of CUR. The present research provides new understanding of the encapsulation and release mechanisms of CUR within biocompatible diblock copolymers and has significant implications for the advancement of safe and effective CUR-based therapeutics.

PCSK9 is minimally associated with HDL but impairs the anti-atherosclerotic HDL effects on endothelial cell activation.

Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) regulates the cell-surface localization of LDL receptors in hepatocytes and is associated with LDL and lipoprotein(a) [Lp(a)] uptake, reducing blood concentrations. However, the connection between PCSK9 and HDL is unclear. Here, we investigated the association of plasma PCSK9 with HDL subpopulations and examined the effects of PCSK9 on the atheroprotective function of HDL. We examined the association of PCSK9 with HDL in apoB-depleted plasma by ELISA, native PAGE, and immunoblotting. Our analyses showed that upon apoB-depletion, total circulating PCSK9 levels were 32% of those observed in normolipidemic plasma, and only 6% of PCSK9 in the apoB-depleted plasma, including both the mature and furin-cleaved forms, was associated with HDL. We also show human recombinant PCSK9 abolished the capacity of reconstituted HDL to reduce the formation of ROS in endothelial cells, while a PCSK9-blocking antibody enhanced the capacity of human HDL (in apoB-depleted plasma) to reduce ROS formation in endothelial cells and promote endothelial cell migration. Overall, our findings suggest that PCSK9 is only minimally associated with HDL particles, but PCSK9 in apoB-depleted plasma can affect the atheroprotective properties of HDL related to preservation of endothelial function. This study contributes to the elucidation of the pathophysiological role of plasma PCSK9 and highlights further the anti-atherosclerotic effect of PCSK9 inhibition.

Structure-function analysis of naturally occurring apolipoprotein A-I L144R, A164S and L178P mutants provides insight on their role on HDL levels and cardiovascular risk.

Naturally occurring point mutations in apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), may affect plasma HDL-cholesterol levels and cardiovascular risk. Here, we evaluated the effect of human apoA-I mutations L144R (associated with low HDL-cholesterol), L178P (associated with low HDL-cholesterol and increased cardiovascular risk) and A164S (associated with increased cardiovascular risk and mortality without low HDL-cholesterol) on the structural integrity and functions of lipid-free and lipoprotein-associated apoA-I in an effort to explain the phenotypes of subjects carrying these mutations. All three mutants, in lipid-free form, presented structural and thermodynamic aberrations, with apoA-I[L178P] presenting the greatest thermodynamic destabilization. Additionally, apoA-I[L178P] displayed reduced ABCA1-mediated cholesterol efflux capacity. When in reconstituted HDL (rHDL), apoA-I[L144R] and apoA-I[L178P] were more thermodynamically destabilized compared to wild-type apoA-I, both displayed reduced SR-BI-mediated cholesterol efflux capacity and apoA-I[L144R] showed severe LCAT activation defect. ApoA-I[A164S] was thermodynamically unaffected when in rHDL, but exhibited a series of functional defects. Specifically, it had reduced ABCG1-mediated cholesterol and 7-ketocholesterol efflux capacity, failed to reduce ROS formation in endothelial cells and had reduced capacity to induce endothelial cell migration. Mechanistically, the latter was due to decreased capacity of rHDL-apoA-I[A164S] to activate Akt kinase possibly by interacting with endothelial LOX-1 receptor. The impaired capacity of rHDL-apoA-I[A164S] to preserve endothelial function may be related to the increased cardiovascular risk for this mutation. Overall, our structure-function analysis of L144R, A164S and L178P apoA-I mutants provides insights on how HDL-cholesterol levels and/or atheroprotective properties of apoA-I/HDL are impaired in carriers of these mutations.

Identification and characterization of a rare variant in apolipoprotein A-IV, p.(V336M), and evaluation of HDL functionality in a Greek cohort with extreme HDL cholesterol levels.

High-Density Lipoprotein cholesterol (HDL-C) levels do not correlate well with Coronary Artery Disease (CAD) risk, while HDL functionality affects atherogenesis and is a better prognostic marker for CAD. Often, the extreme HDL-C levels have a multigenic origin. Here, we searched for single-nucleotide polymorphisms (SNPs) in ten genes of HDL metabolism in a Greek cohort with very low (<10th percentile, n = 13) or very high (>90th percentile, n = 21) HDL-C. We also evaluated the association between HDL-C levels, HDL functionality (anti-oxidant capacity) and CAD in the subjects of this cohort. Individuals with low HDL-C levels had higher triglyceride levels, lower apoA-I levels, decreased HDL anti-oxidant capacity and higher incidence of CAD compared with individuals with control or high HDL-C levels. With next generation sequencing we identified 18 exonic SNPs in 6 genes of HDL metabolism and for selected amino acid changes we performed computer-aided structural analysis and modeling. A previously uncharacterized rare apolipoprotein A-IV variant, apoA-IV [V336M], present in a subject with low HDL-C (14 mg/dL) and CAD, was expressed in recombinant form and structurally and functionally characterized. ApoA-IV [V336M] had similar α-helical content to WT apoA-IV but displayed a small thermodynamic stabilization by chemical unfolding analysis. ApoA-IV [V336M] was able to associate with phospholipids but presented reduced kinetics compared to WT apoA-IV. Overall, we identified a rare apoA-IV variant in a subject with low HDL levels and CAD with altered biophysical and phospholipid binding properties and showed that subjects with very low HDL-C presented with HDL dysfunction and higher incidence of CAD in a Greek cohort.

Novel 3-aryl-5-substituted-coumarin analogues: Synthesis and bioactivity profile.

Eighteen 3-aryl-5-substituted-coumarins-six 5-acetyloxy-derivatives, six 5-hydroxy-derivatives, and six 5-geranyloxy-derivatives-were synthesized, structurally characterized and their antioxidant activity, lipoxygenase inhibitory ability, as well as their cytotoxic activity against human neuroblastoma SK-N-SH and HeLa adenocarcinoma cell lines were evaluated. The 5-acetyloxy-compounds 3a-3f were found to be the best cytotoxic agents among all the compounds studied. The bromo-substituted coumarins 3a and 3b were remarkably active against HeLa cell line showing IC50 1.8 and 6.1 µM, respectively. Coumarin 5e possessing a geranyloxy-chain on position 5 of the coumarin scaffold presented dual bioactivity, while 5-geranyloxy-coumarin 5f was the most competent soybean lipoxygenase inhibitor of this series (IC50 10 µM). As shown by in silico docking studies, the studied molecules present allosteric interactions with soybean lipoxygenases.

The future of apolipoprotein E mimetic peptides in the prevention of cardiovascular disease.

PURPOSE OF REVIEW: This review aims to discuss the recent developments in the area of apolipoprotein E (apoE) mimetics and their therapeutic potential for treating cardiovascular disease, the leading cause of mortality worldwide. RECENT FINDINGS: Ongoing research efforts target the development of novel therapies that would not only reduce circulating levels of atherogenic lipoproteins, but could also increase high density lipoprotein cholesterol (HDL-C) levels and/or improve HDL function. Among them, synthetic peptides that mimic the structure of natural human apoE, a component of triglyceride-rich lipoproteins and HDL, have been designed and proven to be functionally similar to apoE. In specific, apoE mimetic peptides mediate hepatic clearance of circulating atherogenic lipoproteins, dramatically reduce plasma cholesterol, and lead to attenuation of atherosclerosis development in vivo. These peptides also exhibit pleiotropic antiatherogenic properties, such as macrophage cholesterol efflux capacity, as well as anti-inflammatory and antioxidative functions. SUMMARY: ApoE mimetics are undergoing preclinical and clinical evaluation with promising results to date that render them attractive candidates in cardiovascular disease prevention and treatment.

pH-Driven Morphological Diversity in Poly[n-Butyl Acrylate-block-(2-(Dimethylamino)Ethyl Acrylate)] Amphiphilic Copolymer Solutions.

Amphiphilic poly[n-butyl acrylate-block-(2-(dimethylamino)ethyl acrylate)] (PnBA-b-PDMAEA) block copolymers are synthesized by the reversible addition fragmentation chain transfer polymerization process. The pH-responsive self-assembly behavior in aqueous media is studied by dynamic, static, and electrophoretic light scattering and cryogenic transmission electron microscopy (Cryo-TEM) at different pHs. In particular, the PnBA40 -b-PDMAEA60 copolymer (where subscripts denote %wt composition of the components) shows remarkable morphological transitions in aqueous solutions of varying pH values forming, among others, an unusual and novel hierarchical vesicular morphology, as indicated by Cryo-TEM results. The observed transitions are attributed to synergistic effects involving alterations of the protonation degree of the PDMAEA block, in conjunction with the specific composition of the copolymer and the softness of PnBA blocks, with cumulative drive changes in the packing parameter of the copolymer system and result in the formation of various unexpected morphologies by simple pH changes.

ApoE isoforms and carboxyl-terminal-truncated apoE4 forms affect neuronal BACE1 levels and Aß production independently of their cholesterol efflux capacity.

The ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) initiates the production of amyloid-ß peptide (Aß), which is central to the pathogenesis of Alzheimer's disease (AD). Changes in brain cholesterol homeostasis have been suggested to affect Aß metabolism. Cholesterol homeostasis is maintained in the brain by apolipoprotein E (apoE). The apoE4 isoform constitutes the major risk factor for AD. Here, we investigated the effect of apoE forms on Aß generation and on BACE1 levels. We also examined the potential involvement in these processes of cholesterol transporters ABCG1 and ABCG4 or the lipoprotein receptor SR-BI, which are implicated in cholesterol efflux to apoE. It was found that reconstituted lipoprotein-associated apoE isoforms promoted the increase of Aß production and oligomerization and of BACE1 levels in human neuroblastoma SK-N-SH cells, with an apoE4 ≥ apoE3 > apoE2 potency rank order. Progressive carboxyl-terminal apoE4 deletions between residues 230-299 decreased the protein's ability to increase BACE1, while further truncations up to residue 166 prevented apoE4 from increasing BACE1 and Aß levels in SK-N-SH and primary mouse neuronal cells. ABCG1, but not ABCG4 or SR-BI, moderately increased Aß production and BACE1 levels in SK-N-SH cells. All apoE forms affected Aß production/oligomerization and BACE1 levels in a pattern that did not follow that of their capacity to promote ABCG1, ABCG4 or SR-BI-mediated cholesterol efflux. Overall, our data indicate that apoE-containing lipoprotein particles can have a direct effect on BACE1 levels and Aß secretion and possibly contribute to AD pathogenetic processes, independently of their capacity to promote cholesterol efflux.

Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy.

Lipoprotein glomerulopathy (LPG) is a rare renal disease, characterized by lipoprotein thrombi in glomerular capillaries. A series of apoE mutations have been associated with LPG development. We previously showed that three mutants based on apoE3 sequence, in which an arginine was substituted by proline, are thermodynamically destabilized and aggregation-prone. To examine whether other LPG-associated apoE3 mutations induce similar effects, we characterized three nonproline LPG-associated apoE3 mutations, namely, R25C (apoEKyoto), R114C (apoETsukuba), and A152D (apoELasVegas). All three apoE3 variants are found to have significantly reduced helical content and to be thermodynamically destabilized, both in lipid-free and lipoprotein-associated form, and to expose a larger portion of hydrophobic surface to the solvent compared with WT apoE3. Furthermore, all three apoE3 variants are aggregation-prone, as shown by dynamic light-scattering measurements and by their enhanced capacity to bind the amyloid probe thioflavin T. Overall, our data suggest that the LPG-associated apoE3 mutations R25C, R114C, and A152D induce protein misfolding, which may contribute to protein aggregation in glomerular capillaries. The similar effects of both LPG-associated proline and nonproline mutations on apoE3 structure suggest that the thermodynamic destabilization and enhanced aggregation of apoE3 may constitute a common underlying mechanism behind the pathogenesis of LPG.

Fatty acid-based lipidomics and membrane remodeling induced by apoE3 and apoE4 in human neuroblastoma cells.

Apolipoprotein E (apoE) is a major lipid carrier of the lipoprotein transport system that plays critical roles in various pathologies. Human apoE has three common isoforms, the apoE4 being associated with Alzheimer's disease. This is the first study in the literature investigating the effects of apoE (apoE3 and apoE4 isoforms) on membrane fatty acid profile in neuroblastoma SK-N-SH cells. Fatty acid analyses were carried out by gas chromatography of the corresponding methyl esters (FAME). We observed the occurrence of membrane fatty acid remodeling in the presence of each of the two apoE isoforms. ApoE3 increased the membrane level of stearic acid and dihomo-gamma-linolenic acid (DGLA), whereas apoE4 had opposite effects. Both apoE3 and apoE4 increased saturated and monounsaturated fatty acids (SFA and MUFA), omega-6/omega-3 ratio and decreased total polyunsaturated fatty acid (PUFA) amount, but with various intensities. Moreover, both apoE isoforms decreased membrane homeostasis indexes such as PUFA balance, unsaturation index and peroxidation index. Our results highlight membrane property changes connected to the apoE isoforms suggesting membrane lipidomics to be inserted in further model studies of apolipoproteins in health and disease.

Lipoprotein profiles in human heterozygote carriers of a functional mutation P297S in scavenger receptor class B1.

The scavenger receptor class B type 1 (SR-B1) is an important HDL receptor involved in cholesterol uptake and efflux, but its physiological role in human lipoprotein metabolism is not fully understood. Heterozygous carriers of the SR-B1(P297S) mutation are characterized by increased HDL cholesterol levels, impaired cholesterol efflux from macrophages and attenuated adrenal function. Here, the composition and function of lipoproteins were studied in SR-B1(P297S) heterozygotes.Lipoproteins from six SR-B1(P297S) carriers and six family controls were investigated. HDL and LDL/VLDL were isolated by ultracentrifugation and proteins were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. HDL antioxidant properties, paraoxonase 1 activities, apoA-I methionine oxidations and HDL cholesterol efflux capacity were assessed.Multivariate modeling separated carriers from controls based on lipoprotein composition. Protein analyses showed a significant enrichment of apoE in LDL/VLDL and of apoL-1 in HDL from heterozygotes compared to controls. The relative distribution of plasma apoE was increased in LDL and in lipid-free form. There were no significant differences in paraoxonase 1 activities, HDL antioxidant properties or HDL cholesterol efflux capacity but heterozygotes showed a significant increase of oxidized methionines in apoA-I.The SR-B1(P297S) mutation affects both HDL and LDL/VLDL protein compositions. The increase of apoE in carriers suggests a compensatory mechanism for attenuated SR-B1 mediated cholesterol uptake by HDL. Increased methionine oxidation may affect HDL function by reducing apoA-I binding to its targets. The results illustrate the complexity of lipoprotein metabolism that has to be taken into account in future therapeutic strategies aiming at targeting SR-B1.

Influence of Isoforms and Carboxyl-Terminal Truncations on the Capacity of Apolipoprotein E To Associate with and Activate Phospholipid Transfer Protein.

Phospholipid transfer protein (PLTP), a main protein in lipid and lipoprotein metabolism, exists in high-activity (HA-PLTP) and low-activity (LA-PLTP) forms in human plasma. Proper phospholipid transfer activity of PLTP is modulated by interactions with various apolipoproteins (apo) including apoE. The domains of apoE involved in interactions with PLTP are not known. Here we analyzed the capacity of recombinant apoE isoforms and apoE4 mutants with progressive carboxyl-terminal deletions to bind to and activate HA-PLTP and LA-PLTP. Our analyses demonstrated that lipid-free apoE isoforms bind to both HA-PLTP and LA-PLTP, resulting in phospholipid transfer activation, with apoE3 inducing the highest PLTP activation. The isoform-specific differences in apoE/PLTP binding and PLTP activation were abolished following apoE lipidation. Lipid-free apoE4[Δ(260-299)], apoE4[Δ(230-299)], apoE4[Δ(203-299)], and apoE4[Δ(186-299)] activated HA-PLTP by 120-160% compared to full-length apoE4. Lipid-free apoE4[Δ(186-299)] also activated LA-PLTP by 85% compared to full-length apoE4. All lipidated truncated apoE4 forms displayed a similar effect on HA-PLTP and LA-PLTP activity as full-length apoE4. Strikingly, lipid-free or lipidated full-length apoE4 and apoE4[Δ(186-299)] demonstrated similar binding capacity to LA-PLTP and HA-PLTP. Biophysical studies showed that the carboxyl-terminal truncations of apoE4 resulted in small changes of the structural or thermodynamic properties of lipidated apoE4, that were much less pronounced compared to changes observed previously for lipid-free apoE4. Overall, our findings show an isoform-dependent binding to and activation of PLTP by lipid-free apoE. Furthermore, the domain of apoE4 required for PLTP activation resides within its amino-terminal 1-185 region. The apoE/PLTP interactions can be modulated by the conformation and lipidation state of apoE.

Molecular basis for increased risk for late-onset Alzheimer disease due to the naturally occurring L28P mutation in apolipoprotein E4.

The apolipoprotein (apo) E4 isoform has consistently emerged as a susceptibility factor for late-onset Alzheimer disease (AD), although the exact mechanism is not clear. A rare apoE4 mutant, apoE4[L28P] Pittsburgh, burdens carriers with an added risk for late-onset AD and may be a useful tool for gaining insights into the role of apoE4 in disease pathogenesis. Toward this end, we evaluated the effect of the L28P mutation on the structural and functional properties of apoE4. ApoE4[L28P] was found to have significantly perturbed thermodynamic properties, to have reduced helical content, and to expose a larger portion of the hydrophobic surface to the solvent. Furthermore, this mutant is thermodynamically destabilized and more prone to proteolysis. When interacting with lipids, apoE4[L28P] formed populations of lipoprotein particles with structural defects. The structural perturbations brought about by the mutation were accompanied by aberrant functions associated with the pathogenesis of AD. Specifically, apoE4[L28P] promoted the cellular uptake of extracellular amyloid ß peptide 42 (Aß42) by human neuroblastoma SK-N-SH cells as well as by primary mouse neuronal cells and led to increased formation of intracellular reactive oxygen species that persisted for at least 24 h. Furthermore, lipoprotein particles containing apoE4[L28P] induced intracellular reactive oxygen species formation and reduced SK-N-SH cell viability. Overall, our findings suggest that the L28P mutation leads to significant structural and conformational perturbations in apoE4 and can induce functional defects associated with neuronal Aß42 accumulation and oxidative stress. We propose that these structural and functional changes underlie the observed added risk for AD development in carriers of apoE4[L28P].

Antisense oligonucleotides, microRNAs, and antibodies.

The specificity of Watson-Crick base pairing and the development of several chemical modifications to oligonucleotides have enabled the development of novel drug classes for the treatment of different human diseases. This review focuses on promising results of recent preclinical or clinical studies on targeting HDL metabolism and function by antisense oligonucleotides and miRNA-based therapies. Although many hurdles regarding basic mechanism of action, delivery, specificity, and toxicity need to be overcome, promising results from recent clinical trials and recent approval of these types of therapy to treat dyslipidemia suggest that the treatment of HDL dysfunction will benefit from these unique clinical opportunities. Moreover, an overview of monoclonal antibodies (mAbs) developed for the treatment of dyslipidemia and cardiovascular disease and currently being tested in clinical studies is provided. Initial studies have shown that these compounds are generally safe and well tolerated, but ongoing large clinical studies will assess their long-term safety and efficacy.

HDL biogenesis, remodeling, and catabolism.

In this chapter, we review how HDL is generated, remodeled, and catabolized in plasma. We describe key features of the proteins that participate in these processes, emphasizing how mutations in apolipoprotein A-I (apoA-I) and the other proteins affect HDL metabolism. The biogenesis of HDL initially requires functional interaction of apoA-I with the ATP-binding cassette transporter A1 (ABCA1) and subsequently interactions of the lipidated apoA-I forms with lecithin/cholesterol acyltransferase (LCAT). Mutations in these proteins either prevent or impair the formation and possibly the functionality of HDL. Remodeling and catabolism of HDL is the result of interactions of HDL with cell receptors and other membrane and plasma proteins including hepatic lipase (HL), endothelial lipase (EL), phospholipid transfer protein (PLTP), cholesteryl ester transfer protein (CETP), apolipoprotein M (apoM), scavenger receptor class B type I (SR-BI), ATP-binding cassette transporter G1 (ABCG1), the F1 subunit of ATPase (Ecto F1-ATPase), and the cubulin/megalin receptor. Similarly to apoA-I, apolipoprotein E and apolipoprotein A-IV were shown to form discrete HDL particles containing these apolipoproteins which may have important but still unexplored functions. Furthermore, several plasma proteins were found associated with HDL and may modulate its biological functions. The effect of these proteins on the functionality of HDL is the topic of ongoing research.

apoE3[K146N/R147W] acts as a dominant negative apoE form that prevents remnant clearance and inhibits the biogenesis of HDL.

The K146N/R147W substitutions in apoE3 were described in patients with a dominant form of type III hyperlipoproteinemia. The effects of these mutations on the in vivo functions of apoE were studied by adenovirus-mediated gene transfer in different mouse models. Expression of the apoE3[K146N/R147W] mutant in apoE-deficient (apoE(-/-)) or apoA-I-deficient (apoA-I(-/-))×apoE(-/-) mice exacerbated the hypercholesterolemia and increased plasma apoE and triglyceride levels. In apoE(-/-) mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and caused the accumulation of discoidal apoE-containing HDL. The WT apoE3 cleared the cholesterol of apoE(-/-) mice without induction of hypertriglyceridemia and promoted formation of spherical HDL. A unique property of the truncated apoE3[K146N/R147W]202 mutant, compared with similarly truncated apoE forms, is that it did not correct the hypercholesterolemia. The contribution of LPL and LCAT in the induction of the dyslipidemia was studied. Treatment of apoE(-/-) mice with apoE3[K146N/R147W] and LPL corrected the hypertriglyceridemia, but did not prevent the formation of discoidal HDL. Treatment with LCAT corrected hypertriglyceridemia and generated spherical HDL. The combined data indicate that the K146N/R147W substitutions convert the full-length and the truncated apoE3[K146N/R147W] mutant into a dominant negative ligand that prevents receptor-mediated remnant clearance, exacerbates the dyslipidemia, and inhibits the biogenesis of HDL.

A possible structural basis behind the pathogenic role of apolipoprotein E hereditary mutations associated with lipoprotein glomerulopathy.

Single amino acid mutations in apolipoprotein E (apoE) have been associated with the development of the rare kidney disease lipoprotein glomerulopathy (LPG). Although the genetic linkage to disease development is well established, the mechanism of pathogenesis is largely unknown, limiting therapeutic insight. Here, we summarize current knowledge in the field and focus on the possible effects of LPG-associated mutations on the structure of apoE. Recent findings have suggested that mutation-induced folding perturbations in apoE lead to structural destabilization and aggregation, effects that may underlie lipoprotein thrombi accumulation in the glomerulus, a hallmark of LPG. The recognition that structural destabilization may underlie the association between apoE mutations and LPG can be key for development of new innovative treatments for this rare disease.