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High‐density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A‐I (ApoA‐I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA‐I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA‐I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA‐I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small‐angle x‐ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA‐I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid–protein interactions and their behavior. [ABSTRACT FROM AUTHOR]

Hypoxia and hypo-high-density lipoproteinemia (hypo-HDLemia) are proposed risk factors for rotator cuff tear. HDL is recognized for its potential benefits in ischemia-driven angiogenesis and wound healing. Nevertheless, research on the potential benefits of reconstituted HDL (rHDL) on human rotator cuff fibroblasts (RCFs) under hypoxia is limited. This study investigates the cytoprotective and regenerative effects of rHDL, as well as N-acetylcysteine (NAC), vitamin C (Vit C), and HDL on human RCFs under hypoxic conditions. Sixth-passage human RCFs were divided into normoxia, hypoxia, and hypoxia groups pretreated with antioxidants (NAC, Vit C, rHDL, HDL). Hypoxia was induced by 1000 µM CoCl2. In the hypoxia group compared to the normoxia group, there were significant increases in hypoxia-inducible factor-1α (HIF-1α), heme oxygenase-1 (HO-1), and Bcl-2/E1B-19kDa interacting protein 3 (BNIP3) expressions, along with reduced cell viability, elevated reactive oxygen species (ROS) production, apoptosis rate, expressions of cleaved caspase-3, cleaved poly ADP-ribose polymerase-1 (PARP-1), vascular endothelial growth factors (VEGF), and matrix metalloproteinase-2 (MMP-2), as well as decreased collagen I and III production, and markedly lower cell proliferative activity (p ≤ 0.039). These responses were significantly mitigated by pretreatment with rHDL (p ≤ 0.046). This study suggests that rHDL can enhance cell proliferation and collagen I and III production while reducing apoptosis in human RCFs under hypoxic conditions. [ABSTRACT FROM AUTHOR]

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body.

Hypoxia and hypo-high-density lipoproteinemia (hypo-HDLemia) are proposed risk factors for rotator cuff tear. HDL is recognized for its potential benefits in ischemia-driven angiogenesis and wound healing. Nevertheless, research on the potential benefits of reconstituted HDL (rHDL) on human rotator cuff fibroblasts (RCFs) under hypoxia is limited. This study investigates the cytoprotective and regenerative effects of rHDL, as well as N-acetylcysteine (NAC), vitamin C (Vit C), and HDL on human RCFs under hypoxic conditions. Sixth-passage human RCFs were divided into normoxia, hypoxia, and hypoxia groups pretreated with antioxidants (NAC, Vit C, rHDL, HDL). Hypoxia was induced by 1000 µM CoCl2. In the hypoxia group compared to the normoxia group, there were significant increases in hypoxia-inducible factor-1α (HIF-1α), heme oxygenase-1 (HO-1), and Bcl-2/E1B-19kDa interacting protein 3 (BNIP3) expressions, along with reduced cell viability, elevated reactive oxygen species (ROS) production, apoptosis rate, expressions of cleaved caspase-3, cleaved poly ADP-ribose polymerase-1 (PARP-1), vascular endothelial growth factors (VEGF), and matrix metalloproteinase-2 (MMP-2), as well as decreased collagen I and III production, and markedly lower cell proliferative activity (p ≤ 0.039). These responses were significantly mitigated by pretreatment with rHDL (p ≤ 0.046). This study suggests that rHDL can enhance cell proliferation and collagen I and III production while reducing apoptosis in human RCFs under hypoxic conditions.

Low levels of high-density lipoprotein (HDL) and impaired HDL functionality have been consistently associated with increased susceptibility to infection and its serious consequences. This has been attributed to the critical role of HDL in maintaining cellular lipid homeostasis, which is essential for the proper functioning of immune and structural cells. HDL, a multifunctional particle, exerts pleiotropic effects in host defense against pathogens. It functions as a natural nanoparticle, capable of sequestering and neutralizing potentially harmful substances like bacterial lipopolysaccharides. HDL possesses antiviral activity, preventing viruses from entering or fusing with host cells, thereby halting their replication cycle. Understanding the complex relationship between HDL and the immune system may reveal innovative targets for developing new treatments to combat infectious diseases and improve patient outcomes. This review aims to emphasize the role of HDL in influencing the course of bacterial and viral infections and its and its therapeutic potential. [ABSTRACT FROM AUTHOR]

High-density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A-I (ApoA-I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA-I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA-I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA-I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small-angle x-ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA-I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid-protein interactions and their behavior., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Abstract Background ApoJ/clusterin is a multifunctional protein highly expressed in the brain. The implication of ApoJ in β-amyloid (Aβ) fibrillization and clearance in the context of Alzheimer’s disease has been widely studied, although the source and concentration of ApoJ that promotes or inhibits Aβ cerebral accumulation is not clear yet. ApoJ is abundant in plasma and approximately 20% can appear bound to HDL-particles. In this regard, the impact of plasmatic ApoJ and its lipidation status on cerebral β-amyloidosis is still not known. Hence, our main objective was to study the effect of a peripheral increase of free ApoJ or reconstituted HDL particles containing ApoJ in an experimental model of cerebral β-amyloidosis. Methods Fourteen-month-old APP23 transgenic mice were subjected to subchronic intravenous treatment with rHDL-rApoJ nanodiscs or free rApoJ for 1 month. Aβ concentration and distribution in the brain, as well as Aβ levels in plasma and CSF, were determined after treatments. Other features associated to AD pathology, such as neuronal loss and neuroinflammation, were also evaluated. Results Both ApoJ-based treatments prevented the Aβ accumulation in cerebral arteries and induced a decrease in total brain insoluble Aβ42 levels. The peripheral treatment with rApoJ also induced an increase in the Aβ40 levels in CSF, whereas the concentration remained unaltered in plasma. At all the endpoints studied, the lipidation of rApoJ did not enhance the protective properties of free rApoJ. The effects obtained after subchronic treatment with free rApoJ were accompanied by a reduction in hippocampal neuronal loss and an enhancement of the expression of a phagocytic marker in microglial cells surrounding Aβ deposits. Finally, despite the activation of this phagocytic phenotype, treatments did not induce a global neuroinflammatory status. In fact, free rApoJ treatment was able to reduce the levels of interleukin-17 (IL17) and keratinocyte chemoattractant (KC) chemokine in the brain. Conclusions Our results demonstrate that an increase in circulating human rApoJ induces a reduction of insoluble Aβ and CAA load in the brain of APP23 mice. Thus, our study suggests that peripheral interventions, based on treatments with multifunctional physiological chaperones, offer therapeutic opportunities to regulate the cerebral Aβ load.

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body.

Coenzyme Q10 (CoQ10) is a strongly hydrophobic lipid that functions in the electron transport chain and as an antioxidant. CoQ10 was conferred with aqueous solubility by incorporation into nanoparticles containing phosphatidylcholine (PtdCho) and apolipoprotein (apo) A‐I. These particles, termed CoQ10 nanodisks (ND), contain 1.0 mg CoQ10/5 mg PtdCho/2 mg apoA‐I (97% CoQ10 solubilization efficiency). UV/Vis absorbance spectroscopy of CoQ10 ND revealed a characteristic absorbance peak centered at 275 nm. Incorporation of CoQ10 into ND resulted in quenching of apoA‐I tryptophan fluorescence emission. Gel filtration chromatography of CoQ10 ND gave rise to a single major absorbance peak and HPLC of material extracted from this peak confirmed the presence of CoQ10. Incubation of cultured cells with CoQ10 ND, but not empty ND, resulted in a significant increase in the CoQ10 content of mitochondria as well as enhanced oxidative phosphorylation, as observed by a ~24% increase in maximal oxygen consumption rate. Collectively, a facile method to solubilize significant quantities of CoQ10 in lipid nanoparticles has been developed. The availability of CoQ10 ND provides a novel means to investigate biochemical aspects of CoQ10 uptake by cells and/or administer it to subjects deficient in this key lipid as a result of inborn errors of metabolism, statin therapy, or otherwise. [ABSTRACT FROM AUTHOR]

Apolipoproteins are critical structural and functional components of lipoproteins, which are large supramolecular assemblies composed predominantly of lipids and proteins, and other biomolecules such as nucleic acids. A signature feature of apolipoproteins is the preponderance of amphipathic α-helical motifs that dictate their ability to make extensive non-covalent inter- or intra-molecular helix–helix interactions in lipid-free states or helix–lipid interactions with hydrophobic biomolecules in lipid-associated states. This review focuses on the latter ability of apolipoproteins, which has been capitalized on to reconstitute synthetic nanoscale binary/ternary lipoprotein complexes composed of apolipoproteins/peptides and lipids that mimic native high-density lipoproteins (HDLs) with the goal to transport drugs. It traces the historical development of our understanding of these nanostructures and how the cholesterol accepting property of HDL has been reconfigured to develop them as drug-loading platforms. The review provides the structural perspective of these platforms with different types of apolipoproteins and an overview of their synthesis. It also examines the cargo that have been loaded into the core for therapeutic and imaging purposes. Finally, it lays out the merits and challenges associated with apolipoprotein-based nanostructures with a future perspective calling for a need to develop “zip-code”-based delivery for therapeutic and diagnostic applications.

ATP-binding cassette transporter A1 (ABCA1) mediates formation of disc-shaped high-density lipoprotein (HDL) from cell lipid and lipid-free apolipoprotein A-I (apo A-I). Discoidal HDL particles are heterogeneous in physicochemical characteristics for reasons that are understood incompletely. Discoidal lipoprotein particles similar in characteristics and heterogeneity to cell-formed discoidal HDL can be reconstituted from purified lipids and apo A-I by cell-free, physicochemical methods. The heterogeneity of reconstituted HDL (rHDL) is sensitive to the lipid composition of the starting lipid/apo A-I mixture. To determine whether the heterogeneity of cell-formed HDL is similarly sensitive to changes in cell lipids, we investigated four compounds that have well-established effects on cell lipid metabolism and ABCA1-mediated cell cholesterol efflux. 2-Bromopalmitate, D609, monensin and U18666A decreased formation of the larger-sized, but dramatically increased formation of the smaller-sized HDL. 2-Bromopalmitate did not appear to affect ABCA1 activity, subcellular localization or oligomerization, but induced dissolution of the cholesterol-phospholipid complexes in the plasma membrane. Arachidonic and linoleic acids shifted HDL formation to the smaller-sized species. Tangier disease mutations and inhibitors of ABCA1 activity wheat germ agglutinin and AG 490 reduced formation of both larger-sized and smaller-sized HDL. The effect of probucol was similar to the effect of 2-bromopalmitate. Taking rHDL formation as a paradigm, we propose that ABCA1 mutations and activity inhibitors reduce the amount of cell lipid available for HDL formation, and the compounds in the 2-bromopalmitate group and the polyunsaturated fatty acids change cell lipid composition from one that favors formation of the larger-sized HDL particles to one that favors formation of the smaller-sized species. [ABSTRACT FROM AUTHOR]

Heart failure with preserved ejection fraction (HFpEF) represents a major unmet therapeutic need. This study investigated whether feeding coconut oil (CC diet) for 26 weeks in female C57BL/6N mice induces HFpEF and evaluated the effect of reconstituted high-density lipoprotein (HDL)Milano (MDCO-216) administration on established HFpEF. Eight intraperitoneal injections of MDCO-216 (100 mg/kg protein concentration) or of an equivalent volume of control buffer were executed with a 48-h interval starting at 26 weeks after the initiation of the diet. Feeding the CC diet for 26 weeks induced pathological left ventricular hypertrophy characterized by a 17.1% (p < 0.0001) lower myocardial capillary density and markedly (p < 0.0001) increased interstitial fibrosis compared to standard chow (SC) diet mice. Parameters of systolic and diastolic function were significantly impaired in CC diet mice resulting in a reduced stroke volume, decreased cardiac output, and impaired ventriculo-arterial coupling. However, ejection fraction was preserved. Administration of MDCO-216 in CC diet mice reduced cardiac hypertrophy, increased capillary density (p < 0.01), and reduced interstitial fibrosis (p < 0.01). MDCO-216 treatment completely normalized cardiac function, lowered myocardial acetyl-coenzyme A carboxylase levels, and decreased myocardial transforming growth factor-β1 in CC diet mice. In conclusion, the CC diet induced HFpEF. Reconstituted HDLMilano reversed pathological remodeling and functional cardiac abnormalities.

The molecular mechanism by which nascent HDL forms via the interaction of apolipoprotein A-I (apoA-I) and transmembrane ABCA1 is poorly understood. Here, because ABCA1 has been reported to localize to acidic intracellular compartments, including the Golgi and endosome, we studied the interaction of apoA-I with model membranes under acidic conditions. Pure phosphatidylcholine liposomes were persistent against apoA-I at pH levels above 5.0, but were progressively transformed into reconstituted HDLs (rHDLs) by apoA-I at lower pH. Circular dichroism spectral measurements and 8-anilino-1-naphthalenesulfonic acid fluorescence measurements of lipid-free apoA-I ascribed this accelerated rHDL formation to the conformational change of the protein into a rather hydrophobic α-helical structure under acidic conditions. The addition of phosphatidylserine (PS) increased acidity at the bilayer surface and enabled the formation of discoidal rHDLs even at the pH of the endosome and slightly lower pH of the Golgi. These results suggest the following new scenario of nascent HDL formation: ABCA1, which colocalizes with apoA-I in acidic intracellular compartments, including the Golgi and endosome, increases acidity at the membrane surface on the luminal side by PS translocase activity and causes apoA-I to form nascent HDL.

Although many acute and chronic diseases are managed via pharmacological means, challenges remain regarding appropriate drug targeting and maintenance of therapeutic levels within target tissues. Advances in nanotechnology will overcome these challenges through the development of lipidic particles, including liposomes, lipoproteins, and reconstituted high-density lipoproteins (rHDL) that are potential carriers of water-soluble, hydrophobic, and amphiphilic molecules. Herein we summarize the properties of human plasma lipoproteins and rHDL, identify the physicochemical determinants of lipid transfer between phospholipid surfaces, and discuss strategies for increasing the plasma half-life of lipoprotein- and liposome-associated molecules. [ABSTRACT FROM AUTHOR]

Many lines of evidence suggest a protective role for high-density lipoprotein (HDL) and its major apolipoprotein (apo)A-I in Alzheimer's Disease (AD). HDL/apoA-I particles are produced by the liver and intestine and, in addition to removing excess cholesterol from the body, are increasingly recognized to have vasoprotective functions. Here we tested the ability of reconstituted HDL (rHDL) consisting of human apoA-I reconstituted with soy phosphatidylcholine for its ability to lower amyloid beta (Aβ) levels in symptomatic APP/PS1 mice, a well-characterized preclinical model of amyloidosis. Animals were treated intravenously either with four weekly doses (chronic study) or a single dose of 60 mg/kg of rHDL (acute study). The major finding of our acute study is that soluble brain Aβ40 and Aβ42 levels were significantly reduced within 24 h of a single dose of rHDL. By contrast, no changes were observed in our chronic study with respect to soluble or deposited Aβ levels in animals assessed 7 days after the final weekly dose of rHDL, suggesting that beneficial effects diminish as rHDL is cleared from the body. Further, rHDL-treated animals showed no change in amyloid burden, cerebrospinal fluid (CSF) Aβ levels, neuroinflammation, or endothelial activation in the chronic study, suggesting that the pathology-modifying effects of rHDL may indeed be acute and may be specific to the soluble Aβ pool. That systemic administration of rHDL can acutely modify brain Aβ levels provides support for further investigation of the therapeutic potential of apoA-I-based agents for AD. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock. [ABSTRACT FROM AUTHOR]

Among unsaturated fatty acids, epidemiologic studies have demonstrated that ω-6 (linoleic acid) and ω-3 (linolenic acid) fatty acids show different associations with risk of cardiovascular disease (CVD), although its molecular mechanisms remain unclear. To determine why consumption of ω-3 fatty acid is associated with lower risk of CVD, we investigated the biological functions of ω-6 (linoleic acid) and ω-3 (linolenic acid) in reconstituted HDL (rHDL) after encapsulation using human cells and zebrafish embryo. Apolipoprotein A-I (apoA-I) is the principal protein behind the beneficial functions of HDL, which include potent anti-oxidant, anti-inflammatory, and anti-atherosclerotic activities in blood. Several rHDLs were synthesized with apoA-I and different molar ratio of ω-6 or ω-3 fatty acid. Both fatty acids showed similar solubility in rHDL up to a molar ratio of 95:5:1:10 (palmitoyloleoyl phosphatidylcholine:cholesterol:apoA-I:fatty acid). Although both rHDL showed similar structural properties and α-helical contents, ω-6-rHDL showed loss of anti-oxidant ability against LDL oxidation. Uptake of acetylated LDL into macrophages was inhibited by ω-3-rHDL but not ω-6-rHDL, suggesting that ω-6-rHDL has higher pro-atherosclerotic activity. ω-3-rHDL showed more enhanced cholesterol efflux activity with less accumulation of triglyceride in the macrophage. ω-6-rHDL caused more senescence in human dermal fibroblast cells with cytotoxicity, while ω-3-rHDL treatment inhibited the senescence. In zebrafish embryo survivability, ω-3-rHDL-injected embryos showed 86±3% survival, whereas ω-6-rHDL-injected ones showed 72±2% survival as well as an elevated inflammatory response in zebrafish embryos. In conclusion, ω-6-rHDL and ω-3-rHDL show different physiological activities in atherosclerosis, inflammation, and cellular senescence. [ABSTRACT FROM AUTHOR]

Val156 of apolipoprotein A-I (apoA-I) was found to be a key amino acid in the structure and function of high density lipoprotein (HDL) (J. Biol. Chem., 275: 26821–26827, 2000). To determine more precisely the functions of the individual amino acids proximal to Val156, serial point mutants of proapoA-I, including V156K, D157K, and A158E, were overexpressed and purified to at least 95% purity. In the lipid-free state, A158E exhibited the most profound self-associative patterns and the least pronounced dimyristoyl phosphatidylcholine (DMPC) clearance activities. In the lipid-bound state, A158E formed a larger reconstituted HDL (rHDL) with palmitoyloleoyl phosphatidylcholine (POPC), ∼120 Å, whereas other mutants and the wild type (WT) formed 97 Å of POPC-rHDL. Cross-linking analysis revealed that A158E-rHDL harbored at least four protein molecules in the particle, while other rHDL conformations contained only two protein molecules. All of the POPC-rHDL produced smaller HDL, around 78 Å, after 24 h of incubation in the presence of low density lipoprotein at 37°C. V156K and A158E exhibited decreased lecithin:cholesterol acyltransferase activation activity in the POPC-rHDL state, showing

Mast cell chymase, a chymotrypsin-like neutral protease, can proteolyze HDL3. Here we studied the ability of rat and human chymase to proteolyze discoidal preβ-migrating reconstituted HDL particles (rHDLs) containing either apolipoprotein A-I (apoA-I) or apoA-II. Both chymases cleaved apoA-I in rHDL at identical sites, either at the N-terminus (Tyr18 or Phe33) or at the C-terminus (Phe225), so generating three major truncated polypeptides that remained bound to the rHDL. The cleavage sites were independent of the size of the rHDL particles, but small particles were more susceptible to degradation than bigger ones. Chymase-induced truncation of apoA-I yielded functionally compromised rHDL with reduced ability to promote cellular cholesterol efflux. In sharp contrast to apoA-I, apoA-II was resistant to degradation. However, when apoA-II was present in rHDL that also contained apoA-I, it was degraded by chymase.We conclude that chymase reduces the ability of apoA-I in discoidal rHDL particles to induce cholesterol efflux by cleaving off either its amino- or carboxy-terminal portion. This observation supports the concept that limited extracellular proteolysis of apoA-I is one pathophysiologic mechanism leading to the generation and maintenance of foam cells in atherosclerotic lesions.

Apolipoprotein A-I (apoA-I) is an important ligand for the high density lipoprotein (HDL) scavenger receptor class B type I (SR-BI). SR-BI binds both free and lipoprotein-associated apoA-I, but the effects of particle size, composition, and apolipoprotein conformation on HDL binding to SR-BI are not understood. We have studied the effect of apoA-I conformation on particle binding using native HDL and reconstituted HDL particles of defined composition and size. SR-BI expressed in transfected Chinese hamster ovary cells was shown to bind human HDL2 with greater affinity than HDL3, suggesting that HDL size, composition, and possibly apolipoprotein conformation influence HDL binding to SR-BI. To discriminate between these factors, SR-BI binding was studied further using reconstituted l-α-palmitoyloleoyl-phosphatidylcholine-containing HDL particles having identical components and equal amounts of apoA-I, but differing in size (7.8 vs. 9.6 nm in diameter) and apoA-I conformation. The affinity of binding to SR-BI was significantly greater (50-fold) for the larger (9.6-nm) particle than for the 7.8-nm particle. We conclude that differences in apoA-I conformation in different-sized particles markedly influence apoA-I recognition by SR-BI. Preferential binding of larger HDL particles to SR-BI would promote productive selective cholesteryl ester uptake from larger cholesteryl ester-rich HDL over lipid-poor HDL. —de Beer, M. C., D. M. Durbin, L. Cai, A. Jonas, F. C. de Beer, and D. R. van der Westhuyzen. Apolipoprotein A-I conformation markedly influences HDL interaction with scavenger receptor BI.

Non-HDL-cholesterol is well recognised as a primary causal risk factor in cardiovascular disease. However, despite consistent epidemiological evidence for an inverse association between HDL-C and coronary heart disease, clinical trials aimed at raising HDL-C (AIM-HIGH, HPS2-THRIVE, dal-OUTCOMES) failed to meet their primary goals. This systematic review and meta-analysis investigated the effects of established and novel treatment strategies, specifically targeting HDL, on inhibition of atherosclerosis in cholesteryl ester transfer protein-expressing animals, and the prevention of clinical events in randomised controlled trials. Linear regression analyses using data from preclinical studies revealed associations for TC and non-HDL-C and lesion area ( R 2 =0.258, P =0.045; R 2 =0.760, P <0.001), but not for HDL-C ( R 2 =0.030, P =0.556). In clinical trials, non-fatal myocardial infarction risk was significantly less in the treatment group with pooled odd ratios of 0.87 [0.81; 0.94] for all trials and 0.85 [0.78; 0.93] after excluding some trials due to off-target adverse events, whereas all-cause mortality was not affected (OR 1.05 [0.99–1.10]). Meta-regression analyses revealed a trend towards an association between between-group differences in absolute change from baseline in LDL-C and non-fatal myocardial infarction ( P =0.066), whereas no correlation was found for HDL-C ( P =0.955). We conclude that the protective role of lowering LDL-C and non-HDL-C is well-established. The contribution of raising HDL-C on inhibition of atherosclerosis and the prevention of cardiovascular disease remains undefined and may be dependent on the mode of action of HDL-C-modification. Nonetheless, treatment strategies aimed at improving HDL function and raising apolipoprotein A-I may be worth exploring. [ABSTRACT FROM AUTHOR]

Previous studies have shown that both high density lipoproteins (HDL) isolated from human plasma and reconstituted HDL (rHDL) are effective inhibitors of adhesion molecule expression in human endothelial cells. In this study rHDL have been used to investigate whether HDL particle shape, size, apolipoprotein composition or lipid composition are important determinants of the ability of HDL to inhibit the TNF-α induced expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). On the basis of these studies it is possible to draw several firm conclusions. i) Neither phospholipid-containing vesicles nor lipid-free apolipoprotein (apo) A-I inhibit VCAM-1 expression in HUVECs. ii) Simple discoidal complexes containing only phospholipid and apoA-I (discoidal (A-I)rHDL) are sufficient to inhibit the TNF-α-induced expression of VCAM-1 in HUVECs. iii) Spherical apoA-I-containing rHDL (spherical (A-I)rHDL) are superior to discoidal (A-I)rHDL as inhibitors. iv) The particle size of spherical (A-I)rHDL has no influence on the inhibition. v) Spherical rHDL that contain apoA-I are superior as inhibitors of VCAM-1 to those containing apoA-II when the rHDL preparations are equated for apolipoprotein molarity. However, when compared at equivalent particle molarities, this difference is no longer apparent. vi) Replacement of cholesteryl esters with triglyceride in the core of spherical (A-I)rHDL has no effect on the ability of these particles to inhibit VCAM-1 expression. From these results it is tempting to speculate that variations in inhibitory activity may contribute to the variations observed in the anti-atherogenicity of different HDL subpopulations.—Baker, P. W., K-A. Rye, J. R. Gamble, M. A. Vadas, and P. J. Barter. Ability of reconstituted high density lipoproteins to inhibit cytokine-induced expression of vascular cell adhesion molecule-1 in human umbilical vein endothelial cells. J. Lipid Res. 1999. 40: 345–353.

CSL112 is apoA-I purified from human plasma and reconstituted with phosphatidylcholine (PC) to form high-density lipoprotein (HDL)-particles suitable for infusion. CSL112 is in development for the potential treatment of acute coronary syndromes (ACS) by optimizing cholesterol efflux. This study assesses the pharmacokinetics (PK), safety and tolerability of CSL112. Repeat doses of CSL112 or placebo were administered intravenously once- (3.4 g or 6.8 g) or twice-weekly (3.4 g) to healthy subjects in a placebo-controlled, randomized (3 CSL112: 1 placebo), ascending-dose study (NCT01281774). Twenty-seven subjects received CSL112 and nine received placebo. Study endpoints included plasma apoA-I and PC concentrations and specific PK parameters. CSL112 infusions immediately produced robust increases in apoA-I concentration in a dose-proportional manner, reaching levels higher than observed with currently available or investigational HDL products. After infusion of CSL112, apoA-I levels remained above baseline for approximately 3 days. Multiple infusions of CSL112 were safe and well tolerated with no evidence of major organ toxicity or immunogenicity. CSL112 may provide a novel option to rapidly transport cholesterol from atherosclerotic plaque to the liver and reduce early recurrent events following ACS. The data presented here support continued clinical development of CSL112 in patient populations. [ABSTRACT FROM AUTHOR]

Nascent high-density lipoprotein (HDL) particles arise in different sizes. We have sought to uncover factors that control this size heterogeneity. Gel filtration, native PAGE, and protein cross-linking were used to analyze the size heterogeneity of nascent HDL produced by BHK-ABCA1, RAW 264.7, J774, and HepG2 cells under different levels of two factors considered as a ratio, the availability of apolipoprotein AI (apoAI) -accessible cell lipid, and concentration of extracellular lipid-free apoAI. Increases in the available cell lipid:apoAI ratio due to either elevated ATPbinding cassette transporter A1 (ABCA1) expression and activity or raised cell density (i.e., increasing numerator) shifted the production of nascent HDL from smaller particles with fewer apoAI molecules per particle and fewer molecules of choline-phospholipid and cholesterol per apoAI molecule to larger particles that contained more apoAI and more lipid per molecule of apoAI. A further shift to larger particles was observed in BHK-ABCA1 cells when the available cell lipid:apoAI ratio was raised still higher by decreasing the apoAI concentration (i.e., the denominator). These changes in nascent HDL biogenesis were reminiscent of the transition that occurs in the size composition of reconstituted HDL in response to an increasing initial lipid: apoAI molar ratio. Thus, the ratio of available cell lipid:apoAI is a fundamental cause of nascent HDL size heterogeneity, and rHDL formation is a good model of nascent HDL biogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Reduction in low-density lipoprotein cholesterol (LDL-C) levels has been associated with a 25–30% reduction in cardiovascular disease risk. However, there still remains a significant and quantifiable risk. Since epidemiologic data have demonstrated that low levels of high-density lipoprotein cholesterol (HDL-C) are associated with an increased risk for cardiovascular disease, novel therapeutic agents are currently being developed to either raise HDL-C levels or enhance HDL functionality. This review will highlight some of these therapeutic agents including cholesteryl ester transfer protein inhibitors, apolipoprotein A-I mimetics, RVX-208, and apolipoprotein A-I based infusion therapies. For each therapeutic class, an overview of the mechanism of action, pharmacokinetic data, and efficacy/safety evidence will be provided. [Copyright &y& Elsevier]

Dilauryl fluorescein (DLF) is a lipid soluble molecule that becomes fluorescent when lauric acid is removed by hydrolysis The purpose of these studies was to evaluate DLF as a potential probe for the function of reconstituted high-density lipoproteins (rHDL) as hydrophobic drug transport vehicles. The DLF containing rHDL nanoparticles were characterized regarding their physical/chemical properties, including molecular diameter, molecular weight, chemical composition, and buoyant density. We investigated the uptake of DLF from rHDL in cells that overexpress the scavenger receptor (SR-B1), known to facilitate the selective cellular uptake of cholesteryl esters from HDL. These studies show that DLF can be incorporated into rHDL and redistributed in the plasma compartment. In addition, these studies demonstrated an enhanced uptake and hydrolysis of DLF from rHDL by cells that overexpress the SR-B1 receptor, suggesting the involvement of a receptor mediated mechanism. The incorporation of DLF into the rHDL nanoparticles appear to protect against hydrolysis in the systemic circulation based on the lower rate of rHDL/DLF hydrolysis compared with the free DLF during incubation with human plasma. DLF may thus be used as a probe to track the movement and metabolism of HDL core constituents, including cancer chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Studies of "pre β" high density lipoprotein (HDL) and reconstituted HDL (rHDL) have contributed to our understanding of the Reverse Cholesterol Transport pathway. The relative ease with which discoidal rHDL can be generated in vitro has led to novel applications including a) infusion of rHDL into patients to promote regression of atherosclerosis; b) use of rHDL as a miniature membrane for integration of transmembrane proteins in a native-like conformation and c) incorporation of hydrophobic bioactive molecules into rHDL, creating a delivery device. The present review is focused on bioactive agent containing rHDL. The broad array of hydrophobic bioactive molecules successfully incorporated into these particles is discussed, as well as the use of natural lipids and synthetic lipid analogs to confer distinctive binding activity. This technology remains in its infancy with the full potential of these simple, yet elegant, nanoparticles still to be discovered. [Display omitted] • Reconstituted HDL exists as a discoidal bilayer surrounded by an apolipoprotein. • Classically, rHDL has been used in studies of Reverse Cholesterol Transport. • rHDL formulation with bioactive agents creates "nanodisks" (ND) delivery vehicles. • Altering the lipid, scaffold and/or bioactive agent of ND confers novel functions. • ND harboring nickel chelating lipids bind His-tagged proteins. [ABSTRACT FROM AUTHOR]

Abstract: Tangier disease (TD) is a rare familial disorder with mutations in the ATP-binding cassette transporter A1 (ABCA1) gene. It results in extremely low levels of HDL cholesterol. Since TD is a genetic disorder, a therapeutic approach to TD has not been established. We report a typical case of TD with a homozygous novel point mutation in the ABCA1 gene by using genomic DNA sequencing. Primary monocyte-derived macrophages of blood from a patient with TD and normolipidemic subjects were compared for cholesterol efflux. The macrophages from the TD patient showed no apoA-I-mediated cholesterol efflux. In contrast, POPC/apoA-I discs were able to take up cholesterol from macrophages from both the TD and normolipidemic subject. Capillary isotachophoresis (cITP), which separates lipoprotein into subfractions according to electrophoretic mobility, was used to characterize plasma lipoprotein subfractions. Both slow-migrating HDL (sHDL) and slow-migrating LDL (sLDL; unmodified LDL) subfractions were extremely low in the patient with TD. After incubation of plasma from the TD patient with POPC/apoA-I discs, sHDL and sLDL subfractions rapidly appeared. In conclusion, POPC/apoA-I discs not only have beneficial effects on cholesterol efflux, but also have potential as a lipoprotein modulator in patients with TD. [Copyright &y& Elsevier]

Background Two mutants of apolipoprotein (apo) A-I, V156K and A158E, showed markedly different structural and functional properties in lipid-free and lipid-bound states in the authors’ earlier report. The physiological activities of these mutants were compared with the wild-type (WT) and R173C mutant using in vitro and in vivo experiments. Materials and methods A reconstituted high-density lipoprotein (rHDL) with palmitoyloleoyl phosphatidylcholine (POPC), combined with each of the apoA-I variants, was injected into the tail-veins of hypercholesterolaemic mice (C57BL6/J), which had been fed a high cholesterol and high fat (HCHF; 0·5% cholesterol, 15% lard, 0·1% sodium cholate) diet for 23 weeks, once at 0 h and then every 24 h, at a dosage of 30 mg apoA-I kg−1 of body-weight. Results The V156K-rHDL and R173C-rHDL exhibited significantly stronger anti-oxidant activity against copper-mediated low-density lipoprotein (LDL) oxidation than did A158E in an apolipoprotein state. The mice injected with WT-rHDL or A158E-rHDL showed abrupt increases in total cholesterol concentrations (47% and 38%, respectively) as compared with the levels before injection, whereas the mice injected with V156K-rHDL and R173C-rHDL did not. Injection with V156K-rHDL improved serum lipids and anti-oxidative activities compared with the injection of WT-rHDL. Injection of WT-rHDL or A158E-rHDL increased serum interleukin-6 (IL-6) to 90–110 pg mL−1, whereas the injection of V156K-rHDL or R173C-rHDL increased serum IL-6 to 17–25 pg mL−1 only. Conclusion The V156K-rHDL and R173C-rHDL displayed potent beneficial effects, including anti-oxidant and anti-inflammatory activity from both in vitro and in vivo evaluations, whereas the WT-rHDL and A158E-rHDL did not. [ABSTRACT FROM AUTHOR]

Apolipoproteins are critical structural and functional components of lipoproteins, which are large supramolecular assemblies composed predominantly of lipids and proteins, and other biomolecules such as nucleic acids. A signature feature of apolipoproteins is the preponderance of amphipathic α-helical motifs that dictate their ability to make extensive non-covalent inter- or intra-molecular helix–helix interactions in lipid-free states or helix–lipid interactions with hydrophobic biomolecules in lipid-associated states. This review focuses on the latter ability of apolipoproteins, which has been capitalized on to reconstitute synthetic nanoscale binary/ternary lipoprotein complexes composed of apolipoproteins/peptides and lipids that mimic native high-density lipoproteins (HDLs) with the goal to transport drugs. It traces the historical development of our understanding of these nanostructures and how the cholesterol accepting property of HDL has been reconfigured to develop them as drug-loading platforms. The review provides the structural perspective of these platforms with different types of apolipoproteins and an overview of their synthesis. It also examines the cargo that have been loaded into the core for therapeutic and imaging purposes. Finally, it lays out the merits and challenges associated with apolipoprotein-based nanostructures with a future perspective calling for a need to develop "zip-code"-based delivery for therapeutic and diagnostic applications. [ABSTRACT FROM AUTHOR]

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer DH declared a past co-authorship with one of the authors VF., (Copyright © 2021 Waldie, Sebastiani, Moulin, Del Giudice, Paracini, Roosen-Runge, Gerelli, Prevost, Voss, Darwish, Yepuri, Pichler, Maric, Forsyth, Haertlein and Cárdenas.)

Coenzyme Q 10 (CoQ 10 ) is a strongly hydrophobic lipid that functions in the electron transport chain and as an antioxidant. CoQ 10 was conferred with aqueous solubility by incorporation into nanoparticles containing phosphatidylcholine (PtdCho) and apolipoprotein (apo) A-I. These particles, termed CoQ 10 nanodisks (ND), contain 1.0 mg CoQ 10 /5 mg PtdCho/2 mg apoA-I (97% CoQ 10 solubilization efficiency). UV/Vis absorbance spectroscopy of CoQ 10 ND revealed a characteristic absorbance peak centered at 275 nm. Incorporation of CoQ 10 into ND resulted in quenching of apoA-I tryptophan fluorescence emission. Gel filtration chromatography of CoQ 10 ND gave rise to a single major absorbance peak and HPLC of material extracted from this peak confirmed the presence of CoQ 10 . Incubation of cultured cells with CoQ 10 ND, but not empty ND, resulted in a significant increase in the CoQ 10 content of mitochondria as well as enhanced oxidative phosphorylation, as observed by a ~24% increase in maximal oxygen consumption rate. Collectively, a facile method to solubilize significant quantities of CoQ 10 in lipid nanoparticles has been developed. The availability of CoQ 10 ND provides a novel means to investigate biochemical aspects of CoQ 10 uptake by cells and/or administer it to subjects deficient in this key lipid as a result of inborn errors of metabolism, statin therapy, or otherwise., (© 2020 AOCS.)

Atherosclerosis affects millions of people worldwide. However, the wide variety of limitations in the current therapeutic options leaves much to be desired in future lipid-lowering therapies. For example, although statins, which are the first-line treatment for coronary heart disease (CHD), reduce the risk of cardiovascular events in a large percentage of patients, they lead to optimal levels of low density lipoprotein-cholesterol (LDL-C) in only about one-third of patients. A new promising research direction against atherosclerosis aims to improve lipoprotein metabolism. Novel therapeutic approaches are being developed to increase the levels of functional high density lipoprotein (HDL) particles. This review aims to highlight the atheroprotective potential of the in vitro synthesized reconstituted HDL particles containing apolipoprotein E (apoE) as their sole apolipoprotein component (rHDL-apoE). For this purpose, we provide: (1) a summary of the atheroprotective properties of native plasma HDL and its apolipoprotein components, apolipoprotein A-I (apoA-I) and apoE; (2) an overview of the anti-atherogenic functions of rHDL-apoA-I and apoA-I-containing HDL, i.e., natural HDL isolated from transgenic Apoa1−/− × Apoe−/− mice overexpressing human apoA-I (HDL-apoA-I); and (3) the latest developments and therapeutic potential of HDL-apoE and rHDL-apoE. Novel rHDL formulations containing apoE could possibly present enhanced biological functions, leading to improved therapeutic efficacy against atherosclerosis. [ABSTRACT FROM AUTHOR]

Heart failure with preserved ejection fraction (HFpEF) represents a major unmet therapeutic need. This study investigated whether feeding coconut oil (CC diet) for 26 weeks in female C57BL/6N mice induces HFpEF and evaluated the effect of reconstituted high-density lipoprotein (HDL)Milano (MDCO-216) administration on established HFpEF. Eight intraperitoneal injections of MDCO-216 (100 mg/kg protein concentration) or of an equivalent volume of control buffer were executed with a 48-h interval starting at 26 weeks after the initiation of the diet. Feeding the CC diet for 26 weeks induced pathological left ventricular hypertrophy characterized by a 17.1% (p < 0.0001) lower myocardial capillary density and markedly (p < 0.0001) increased interstitial fibrosis compared to standard chow (SC) diet mice. Parameters of systolic and diastolic function were significantly impaired in CC diet mice resulting in a reduced stroke volume, decreased cardiac output, and impaired ventriculo-arterial coupling. However, ejection fraction was preserved. Administration of MDCO-216 in CC diet mice reduced cardiac hypertrophy, increased capillary density (p < 0.01), and reduced interstitial fibrosis (p < 0.01). MDCO-216 treatment completely normalized cardiac function, lowered myocardial acetyl-coenzyme A carboxylase levels, and decreased myocardial transforming growth factor-β1 in CC diet mice. In conclusion, the CC diet induced HFpEF. Reconstituted HDLMilano reversed pathological remodeling and functional cardiac abnormalities. [ABSTRACT FROM AUTHOR]