Quantitative electroencephalography (qEEG), apolipoprotein A-I (APOA-I), and apolipoprotein epsilon 4 (APOE ɛ4) alleles for the diagnosis of mild cognitive impairment and Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is the most common type of dementia. Amnestic mild cognitive impairment (aMCI), a pre-dementia stage is an important stage for early diagnosis and intervention. This study aimed to investigate the diagnostic value of qEEG, APOA-I, and APOE ɛ4 allele in aMCI and AD patients and found the correlation between qEEG (Delta + Theta)/(Alpha + Beta) ratio (DTABR) and different cognitive domains. METHODS: All participants were divided into three groups: normal controls (NCs), aMCI, and AD, and all received quantitative electroencephalography (qEEG), neuropsychological scale assessment, apolipoprotein epsilon 4 (APOE ɛ4) alleles, and various blood lipid indicators. Different statistical methods were used for different data. RESULTS: The cognitive domains except executive ability were all negatively correlated with DTABR in different brain regions while executive ability was positively correlated with DTABR in several brain regions, although without statistical significance. The consequences confirmed that the DTABR of each brain area were related to MMSE, MoCA, instantaneous memory, and the language ability (p < 0.05), and the DTABR in the occipital area was relevant to all cognitive domains (p < 0.01) except executive function (p = 0.272). Also, occipital DTABR was most correlated with language domain when tested by VFT with a moderate level (r = 0.596, p < 0.001). There were significant differences in T3, T5, and P3 DTABR between both AD and NC and aMCI and NCs. As for aMCI diagnosis, the maximum AUC was achieved when using T3 combined with APOA-I and APOE ε4 (0.855) and the maximum AUC was achieved when using T5 combined with APOA-I and APOE ε4 (0.889) for AD diagnosis. CONCLUSION: These findings highlight that APOA-I, APOE ɛ4, and qEEG play an important role in aMCI and AD diagnosis. During AD continuum, qEEG DTABR should be taken into consideration for the early detection of AD risk.

Synergistic Anti-Inflammatory Activity of Lipid-Free Apolipoprotein (apo) A-I and CIGB-258 in Acute-Phase Zebrafish via Stabilization of the apoA-I Structure to Enhance Anti-Glycation and Antioxidant Activities.

CIGB-258, a 3 kDa peptide from heat shock protein 60, exhibits synergistic anti-inflammatory activity with apolipoprotein A-I (apoA-I) in reconstituted high-density lipoproteins (rHDLs) via stabilization of the rHDL structure. This study explored the interactions between CIGB-258 and apoA-I in the lipid-free state to assess their synergistic effects in the structural and functional enhancement of apoA-I and HDL. A co-treatment of lipid-free apoA-I and CIGB-258 inhibited the cupric ion-mediated oxidation of low-density lipoprotein (LDL) and a lowering of oxidized species in the dose-responsive manner of CIGB-258. The co-presence of CIGB-258 caused a blue shift in the wavelength of maximum fluorescence (WMF) of apoA-I with protection from proteolytic degradation. The addition of apoA-I:CIGB-258, with a molar ratio of 1:0.1, 1:0.5, and 1:1, to HDL2 and HDL3 remarkably enhanced the antioxidant ability against LDL oxidation up to two-fold higher than HDL alone. HDL-associated paraoxonase activities were elevated up to 28% by the co-addition of apoA-I and CIGB-258, which is linked to the suppression of Cu2+-mediated HDL oxidation with the slowest electromobility. Isothermal denaturation by a urea treatment showed that the co-presence of CIGB-258 attenuated the exposure of intrinsic tryptophan (Trp) and increased the mid-points of denaturation from 2.33 M for apoA-I alone to 2.57 M for an apoA-I:CIGB-258 mixture with a molar ratio of 1:0.5. The addition of CIGB-258 to apoA-I protected the carboxymethyllysine (CML)-facilitated glycation of apoA-I with the prevention of Trp exposure. A co-treatment of apoA-I and CIGB-258 synergistically safeguarded zebrafish embryos from acute death by CML-toxicity, suppressing oxidative stress and apoptosis. In adult zebrafish, the co-treatment of apoA-I+CIGB-258 exerted the highest anti-inflammatory activity with a higher recovery of swimming ability and survivability than apoA-I alone or CIGB-258 alone. A co-injection of apoA-I and CIGB-258 led to the lowest infiltration of neutrophils and interleukin (IL)-6 generation in hepatic tissue, with the lowest serum triglyceride, aspartate transaminase, and alanine transaminase levels in plasma. In conclusion, the co-presence of CIGB-258 ameliorated the beneficial functionalities of apoA-I, such as antioxidant and anti-glycation activities, by enhancing the structural stabilization and protection of apoA-I. The combination of apoA-I and CIGB-258 synergistically enforced the anti-inflammatory effect against CML toxicity in embryos and adult zebrafish.

Application of Weighted Gene Co-Expression Network Analysis to Metabolomic Data from an ApoA-I Knockout Mouse Model.

As the ability to collect profiling data in metabolomics increases substantially with the advances in Liquid Chromatography-Mass Spectrometry (LC-MS) instruments, it is urgent to develop new and powerful data analysis approaches to match the big data collected and to extract as much meaningful information as possible from tens of thousands of molecular features. Here, we applied weighted gene co-expression network analysis (WGCNA), an algorithm popularly used in microarray or RNA sequencing, to plasma metabolomic data and demonstrated several advantages of WGCNA over conventional statistical approaches such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). By using WGCNA, a large number of molecular features were clustered into a few modules to reduce the dimension of a dataset, the impact of phenotypic traits such as diet type and genotype on the plasma metabolome was evaluated quantitatively, and hub metabolites were found based on the network graph. Our work revealed that WGCNA is a very powerful tool to decipher, interpret, and visualize metabolomic datasets.

Is reverse cholesterol transport regulated by active cholesterol?

This review considers the hypothesis that a small portion of plasma membrane cholesterol regulates reverse cholesterol transport in coordination with overall cellular homeostasis. It appears that almost all of the plasma membrane cholesterol is held in stoichiometric complexes with bilayer phospholipids. The minor fraction of cholesterol that exceeds the complexation capacity of the phospholipids is called active cholesterol. It has an elevated chemical activity and circulates among the organelles. It also moves down its chemical activity gradient to plasma HDL, facilitated by the activity of ABCA1, ABCG1, and SR-BI. ABCA1 initiates this process by perturbing the organization of the plasma membrane bilayer, thereby priming its phospholipids for translocation to apoA-I to form nascent HDL. The active excess sterol and that activated by ABCA1 itself follow the phospholipids to the nascent HDL. ABCG1 similarly rearranges the bilayer and sends additional active cholesterol to nascent HDL, while SR-BI simply facilitates the equilibration of the active sterol between plasma membranes and plasma proteins. Active cholesterol also flows downhill to cytoplasmic membranes where it serves both as a feedback signal to homeostatic ER proteins and as the substrate for the synthesis of mitochondrial 27-hydroxycholesterol (27HC). 27HC binds the LXR and promotes the expression of the aforementioned transport proteins. 27HC-LXR also activates ABCA1 by competitively displacing its inhibitor, unliganded LXR. § Considerable indirect evidence suggests that active cholesterol serves as both a substrate and a feedback signal for reverse cholesterol transport. Direct tests of this novel hypothesis are proposed.

Role of enterocyte Enpp2 and autotaxin in regulating lipopolysaccharide levels, systemic inflammation, and atherosclerosis.

Conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA) by autotaxin, a secreted phospholipase D, is a major pathway for producing LPA. We previously reported that feeding Ldlr-/- mice standard mouse chow supplemented with unsaturated LPA or lysophosphatidylcholine qualitatively mimicked the dyslipidemia and atherosclerosis induced by feeding a Western diet (WD). Here, we report that adding unsaturated LPA to standard mouse chow also increased the content of reactive oxygen species and oxidized phospholipids (OxPLs) in jejunum mucus. To determine the role of intestinal autotaxin, enterocyte-specific Ldlr-/-/Enpp2 KO (intestinal KO) mice were generated. In control mice, the WD increased enterocyte Enpp2 expression and raised autotaxin levels. Ex vivo, addition of OxPL to jejunum from Ldlr-/- mice on a chow diet induced expression of Enpp2. In control mice, the WD raised OxPL levels in jejunum mucus and decreased gene expression in enterocytes for a number of peptides and proteins that affect antimicrobial activity. On the WD, the control mice developed elevated levels of lipopolysaccharide in jejunum mucus and plasma, with increased dyslipidemia and increased atherosclerosis. All these changes were reduced in the intestinal KO mice. We conclude that the WD increases the formation of intestinal OxPL, which i) induce enterocyte Enpp2 and autotaxin resulting in higher enterocyte LPA levels; that ii) contribute to the formation of reactive oxygen species that help to maintain the high OxPL levels; iii) decrease intestinal antimicrobial activity; and iv) raise plasma lipopolysaccharide levels that promote systemic inflammation and enhance atherosclerosis.

Beneficial effects of recombinant CER-001 high-density lipoprotein infusion in sepsis: results from a bench to bedside translational research project.

BACKGROUND: Sepsis is characterized by a dysregulated immune response and metabolic alterations, including decreased high-density lipoprotein cholesterol (HDL-C) levels. HDL exhibits beneficial properties, such as lipopolysaccharides (LPS) scavenging, exerting anti-inflammatory effects and providing endothelial protection. We investigated the effects of CER-001, an engineered HDL-mimetic, in a swine model of LPS-induced acute kidney injury (AKI) and a Phase 2a clinical trial, aiming to better understand its molecular basis in systemic inflammation and renal function. METHODS: We carried out a translational approach to study the effects of HDL administration on sepsis. Sterile systemic inflammation was induced in pigs by LPS infusion. Animals were randomized into LPS (n = 6), CER20 (single dose of CER-001 20 mg/kg; n = 6), and CER20 × 2 (two doses of CER-001 20 mg/kg; n = 6) groups. Survival rate, endothelial dysfunction biomarkers, pro-inflammatory mediators, LPS, and apolipoprotein A-I (ApoA-I) levels were assessed. Renal and liver histology and biochemistry were analyzed. Subsequently, we performed an open-label, randomized, dose-ranging (Phase 2a) study included 20 patients with sepsis due to intra-abdominal infection or urosepsis, randomized into Group A (conventional treatment, n = 5), Group B (CER-001 5 mg/kg BID, n = 5), Group C (CER-001 10 mg/kg BID, n = 5), and Group D (CER-001 20 mg/kg BID, n = 5). Primary outcomes were safety and efficacy in preventing AKI onset and severity; secondary outcomes include changes in inflammatory and endothelial dysfunction markers. RESULTS: CER-001 increased median survival, reduced inflammatory mediators, complement activation, and endothelial dysfunction in endotoxemic pigs. It enhanced LPS elimination through the bile and preserved liver and renal parenchyma. In the clinical study, CER-001 was well-tolerated with no serious adverse events related to study treatment. Rapid ApoA-I normalization was associated with enhanced LPS removal and immunomodulation with improvement of clinical outcomes, independently of the type and gravity of the sepsis. CER-001-treated patients had reduced risk for the onset and progression to severe AKI (stage 2 or 3) and, in a subset of critically ill patients, a reduced need for organ support and shorter ICU length of stay. CONCLUSIONS: CER-001 shows promise as a therapeutic strategy for sepsis management, improving outcomes and mitigating inflammation and organ damage. TRIAL REGISTRATION: The study was approved by the Agenzia Italiana del Farmaco (AIFA) and by the Local Ethic Committee (N° EUDRACT 2020-004202-60, Protocol CER-001- SEP_AKI_01) and was added to the EU Clinical Trials Register on January 13, 2021.

Apolipoprotein A-I (ApoA-I) protects Nile tilapia (Oreochromis niloticus) against bacterial infection.

Apolipoprotein A-I (ApoA-I) is a lipoprotein involved in a variety of physiological and pathological processes. However, the immunomodulatory functions of ApoA-I in fish are not well understood. In this study, ApoA-I from Nile tilapia (Oreochromis niloticus) (On-ApoA-I) was identified, and its function in bacterial infection was investigated. The open reading frame of On-ApoA-I is 792 bp, which codes for a protein containing 263 amino acids. On-ApoA-I shared over 60% sequence similarity with other teleost fish and more than 20% with mammalian ApoA-I. On-ApoA-I was found to be highly expressed in the liver and significantly induced during Streptococcus agalactiae infection by qRT‒PCR analysis. Furthermore, invivo studies revealed that recombinant On-ApoA-I protein could suppress inflammation and apoptosis and improve the likelihood of surviving bacterial infection. Additionally, On-ApoA-I showed invitro antimicrobial properties against Gram-positive and Gram-negative bacteria. These findings offer a theoretical basis for further investigations into the role of ApoA-I in fish immunology.

Cholesterol Metabolic Profiling of HDL in Women with Late-Onset Preeclampsia.

A specific feature of dyslipidemia in pregnancy is increased high-density lipoprotein (HDL) cholesterol concentration, which is probably associated with maternal endothelium protection. However, preeclampsia is most often associated with low HDL cholesterol, and the mechanisms behind this change are scarcely explored. We aimed to investigate changes in HDL metabolism in risky pregnancies and those complicated by late-onset preeclampsia. We analyze cholesterol synthesis (cholesterol precursors: desmosterol, 7-dehydrocholesterol, and lathosterol) and absorption markers (phytosterols: campesterol and ß-sitosterol) within HDL particles (NCSHDL), the activities of principal modulators of HDL cholesterol's content, and major HDL functional proteins levels in mid and late pregnancy. On the basis of the pregnancy outcome, participants were classified into the risk group (RG) (70 women) and the preeclampsia group (PG) (20 women). HDL cholesterol was lower in PG in the second trimester compared to RG (p < 0.05) and followed by lower levels of cholesterol absorption markers (p < 0.001 for campesterolHDL and p < 0.05 for ß-sitosterolHDL). Lowering of HDL cholesterol between trimesters in RG (p < 0.05) was accompanied by a decrease in HDL phytosterol content (p < 0.001), apolipoprotein A-I (apoA-I) concentration (p < 0.05), and paraoxonase 1 (PON1) (p < 0.001), lecithin-cholesterol acyltransferase (LCAT) (p < 0.05), and cholesterol ester transfer protein (CETP) activities (p < 0.05). These longitudinal changes were absent in PG. Development of late-onset preeclampsia is preceded by the appearance of lower HDL cholesterol and NCSHDL in the second trimester. We propose that reduced capacity for intestinal HDL synthesis, decreased LCAT activity, and impaired capacity for HDL-mediated cholesterol efflux could be the contributing mechanisms resulting in lower HDL cholesterol.

Cuban Sugar Cane Wax Alcohol Exhibited Enhanced Antioxidant, Anti-Glycation and Anti-Inflammatory Activity in Reconstituted High-Density Lipoprotein (rHDL) with Improved Structural and Functional Correlations: Comparison of Various Policosanols.

Policosanols from various sources, such as sugar cane, rice bran, and insects, have been marketed to prevent dyslipidemia, diabetes, and hypertension by increasing the blood high-density lipoproteins cholesterol (HDL-C) levels. On the other hand, there has been no study on how each policosanol influences the quality of HDL particles and their functionality. Reconstituted high-density lipoproteins (rHDLs) with apolipoprotein (apo) A-I and each policosanol were synthesized using the sodium cholate dialysis method to compare the policosanols in lipoprotein metabolism. Each rHDL was compared regarding the particle size and shape, antioxidant activity, and anti-inflammatory activity in vitro and in zebrafish embryos. This study compared four policosanols including one policosanol from Cuba (Raydel® policosanol) and three policosanols from China (Xi'an Natural sugar cane, Xi'an Realin sugar cane, and Shaanxi rice bran). The synthesis of rHDLs with various policosanols (PCO) from Cuba or China using a molar ratio of 95:5:1:1 with palmitoyloleoyl phosphatidylcholine (POPC): free cholesterol (FC): apoA-I:PCO (wt:wt) showed that rHDL containing Cuban policosanol (rHDL-1) showed the largest particle size and the most distinct particle shape. The rHDL-1 showed a 23% larger particle diameter and increased apoA-I molecular weight with a 1.9 nm blue shift of the maximum wavelength fluorescence than rHDL alone (rHDL-0). Other rHDLs containing Chinese policosanols (rHDL-2, rHDL-3, and rHDL-4) showed similar particle sizes with an rHDL-0 and 1.1-1.3 nm blue shift of wavelength maximum fluorescence (WMF). Among all rHDLs, the rHDL-1 showed the strongest antioxidant ability to inhibit cupric ion-mediated LDL oxidation. The rHDL-1-treated LDL showed the most distinct band intensity and particle morphology compared with the other rHDLs. The rHDL-1 also exerted the highest anti-glycation activity to inhibit the fructose-mediated glycation of human HDL2 with the protection of apoA-I from proteolytic degradation. At the same time, other rHDLs showed a loss of anti-glycation activity with severe degradation. A microinjection of each rHDL alone showed that rHDL-1 had the highest survivability of approximately 85 ± 3%, with the fastest developmental speed and morphology. In contrast, rHDL-3 showed the lowest survivability, around 71 ± 5%, with the slowest developmental speed. A microinjection of carboxymethyllysine (CML), a pro-inflammatory advanced glycated end product, into zebrafish embryos resulted in severe embryo death of approximately 30 ± 3% and developmental defects with the slowest developmental speed. On the other hand, the phosphate buffered saline (PBS)-injected embryo showed 83 ± 3% survivability. A co-injection of CML and each rHDL into adult zebrafish showed that rHDL-1 (Cuban policosanol) induced the highest survivability, around 85 ± 3%, while rHDL-0 showed 67 ± 7% survivability. In addition, rHDL-2, rHDL-3, and rHDL-4 showed 67 ± 5%, 62 ± 37, and 71 ± 6% survivability, respectively, with a slower developmental speed and morphology. In conclusion, Cuban policosanol showed the strongest ability to form rHDLs with the most distinct morphology and the largest size. The rHDL-containing Cuban policosanol (rHDL-1) showed the strongest antioxidant ability against LDL oxidation, anti-glycation activity to protect apoA-I from degradation, and the highest anti-inflammatory activity to protect embryo death under the presence of CML.

CIGB-258 Exerts Potent Anti-Inflammatory Activity against Carboxymethyllysine-Induced Acute Inflammation in Hyperlipidemic Zebrafish via the Protection of Apolipoprotein A-I.

Inflammation and atherosclerosis are intimately associated via the production of dysfunctional high-density lipoproteins (HDL) and modification of apolipoprotein (apo) A-I. A putative interaction between CIGB-258 and apoA-I was investigated to provide mechanistic insight into the protection of HDL. The protective activity of CIGB-258 was tested in the CML-mediated glycation of apoA-I. The in vivo anti-inflammatory efficacy was compared in paralyzed hyperlipidemic zebrafish and its embryo in the presence of CML. Treatment of CML induced greater glycation extent of HDL/apoA-I and proteolytic degradation of apoA-I. In the presence of CML, however, co-treatment of CIGB-258 inhibited the glycation of apoA-I and protected the degradation of apoA-I, exerting enhanced ferric ion reduction ability. Microinjection of CML (500 ng) into zebrafish embryos resulted in acute death with the lowest survivability with severe developmental defects with interleukin (IL)-6 production. Conversely, a co-injection of CIGB-258 or Tocilizumab produced the highest survivability with a normal development speed and morphology. In hyperlipidemic zebrafish, intraperitoneal injection of CML (500 µg) caused the complete loss of swimming ability and severe acute death with only 13% survivability 3 h post-injection. A co-injection of the CIGB-258 resulted in a 2.2-fold faster recovery of swimming ability than CML alone, with higher survivability of approximately 57%. These results suggest that CIGB-258 protected hyperlipidemic zebrafish from the acute neurotoxicity of CML. Histological analysis showed that the CIGB-258 group had 37% lower infiltration of neutrophils in hepatic tissue and 70% lower fatty liver changes than those of the CML-alone group. The CIGB-258 group exhibited the smallest IL-6 expression in the liver and the lowest blood triglyceride level. CIGB-258 displayed potent anti-inflammatory activity in hyperlipidemic zebrafish by inhibiting apoA-I glycation, promoting rapid recovery from the paralysis of CML toxicity and suppression of IL-6, and lowering fatty liver changes.

Cuban Policosanol (Raydel®) Potently Protects the Liver, Ovary, and Testis with an Improvement in Dyslipidemia in Hyperlipidemic Zebrafish: A Comparative Study with Three Chinese Policosanols.

Many policosanols from different sources, such as sugar cane and rice bran, have been marketed worldwide to improve blood lipid profiles. But so far, no comparative study has commenced elucidating the effect of different policosanols to improve the blood lipid profile and other beneficial effects. This study compared the efficacy of four different policosanols, including one sugar cane wax alcohol from Cuba (Raydel®) and three policosanols from China (Xi'an Natural sugar cane, Xi'an Realin sugar cane, and Shaanxi rice bran), to treat dyslipidemia in hyperlipidemic zebrafish. After 12 weeks of consumption of each policosanol (final 0.1% in diet, wt/wt) and a high-cholesterol diet (HCD, final 4%, wt/wt), the Raydel policosanol group and the Xi'an Natural policosanol group showed the highest survivability, of approximately 81%. In contrast, the Xi'an Realin policosanol and the Shaanxi policosanol groups showed 57% and 67% survivability, respectively. Among the five HCD groups, the Raydel policosanol group showed the lowest serum total cholesterol (TC, p < 0.001 versus HCD control) and triglyceride (p < 0.001 versus HCD control), with the highest percentage of high-density lipoproteins-cholesterol in TC. The Raydel policosanol group also showed the lowest serum aspartate aminotransferase and alanine aminotransferase levels, with the least infiltration of inflammatory cells and interleukin-6 production in hepatocytes with a marked reduction in reactive oxygen species (ROS) production and fatty liver changes. In the ovary, the Raydel policosanol group also showed the highest content of mature vitellogenic oocytes with the lowest production of reactive oxygen species and cellular apoptosis in ovarian cells. In the testes, the Raydel policosanol group also showed the healthiest morphology for spermatogenesis, with the lowest interstitial area and reactive oxygen species production in testicular cells. Conclusively, among the tested policosanols, Cuba (Raydel®) policosanol exhibited a comparatively better effect in maintaining zebrafish body weight, survivability, blood lipid profile, hepatic function biomarkers, fatty liver changes, ROS generation, inflammation, and restoration of the cell morphology in ovaries and testes affected by the HCD consumption.

Engineering a New IFN-ApoA-I Fusion Protein with Low Toxicity and Prolonged Action.

Recombinant human interferon alpha-2b (rIFN) is widely used in antiviral and anticancer immunotherapy. However, the high efficiency of interferon therapy is accompanied by a number of side effects; this problem requires the design of a new class of interferon molecules with reduced cytotoxicity. In this work, IFN was modified via genetic engineering methods by merging it with the blood plasma protein apolipoprotein A-I in order to reduce acute toxicity and improve the pharmacokinetics of IFN. The chimeric protein was obtained via biosynthesis in the yeast P. pastoris. The yield of ryIFN-ApoA-I protein when cultivated on a shaker in flasks was 30 mg/L; protein purification was carried out using reverse-phase chromatography to a purity of 95-97%. The chimeric protein demonstrated complete preservation of the biological activity of IFN in the model of vesicular stomatitis virus and SARS-CoV-2. In addition, the chimeric form had reduced cytotoxicity towards Vero cells and increased cell viability under viral load conditions compared with commercial IFN-a2b preparations. Analysis of the pharmacokinetic profile of ryIFN-ApoA-I after a single subcutaneous injection in mice showed a 1.8-fold increased half-life of the chimeric protein compared with ryIFN.

Apolipoprotein mimetics in cancer.

Peptides have many advantages over traditional therapeutics, including small molecules and other biologics, because of their low toxicity and immunogenicity, while still exhibiting efficacy. This review discusses the benefits and mechanism of action of apolipoprotein mimetic peptides in tumor biology and their potential utility in treating various cancers. Among lipoproteins in the circulation, high-density lipoprotein (HDL) and its constituents including apolipoprotein A-I (apoA-I; the predominant protein in HDL), apoJ, and apoE, harbor anti-tumorigenic activities. Peptides that mimic apoA-I function have been developed through molecular mimicry of the amphipathic α-helices of apoA-I. Oral apoA-I mimetic peptides remodel HDL, promote cholesterol efflux, sequester oxidized lipids, and activate anti-inflammatory processes. ApoA-I and apoJ mimetic peptides ameliorate various metrics of cancer progression and have demonstrated efficacy in preclinical models in the inhibition of ovarian, colon, breast, and metastatic lung cancers. Apolipoprotein mimetic peptides are poorly absorbed when administered orally and rapidly degraded when injected into the circulation. The small intestine is the major site of action for apoA-I mimetic peptides and recent studies suggest that modulation of immune cells in the lamina propria of the small intestine is, in part, a potential mechanism of action. Finally, several recent studies underscore the use of reconstituted HDL as target-specific nanoparticles carrying poorly soluble or unstable therapeutics to tumors even across the blood-brain barrier. Preclinical studies suggest that these versatile recombinant lipoprotein based nanoparticles and apolipoprotein mimetics can serve as safe, novel drug delivery, and therapeutic agents for the treatment of a number of cancers.

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.

Oxidized phospholipids cause changes in jejunum mucus that induce dysbiosis and systemic inflammation.

We previously reported that adding a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to a Western diet (WD) ameliorated systemic inflammation. To determine the mechanism(s) responsible for these observations, Ldlr-/- mice were fed chow, a WD, or WD plus Tg6F. We found that a WD altered the taxonomic composition of bacteria in jejunum mucus. For example, Akkermansia muciniphila virtually disappeared, while overall bacteria numbers and lipopolysaccharide (LPS) levels increased. In addition, gut permeability increased, as did the content of reactive oxygen species and oxidized phospholipids in jejunum mucus in WD-fed mice. Moreover, gene expression in the jejunum decreased for multiple peptides and proteins that are secreted into the mucous layer of the jejunum that act to limit bacteria numbers and their interaction with enterocytes including regenerating islet-derived proteins, defensins, mucin 2, surfactant A, and apoA-I. Following WD, gene expression also decreased for Il36γ, Il23, and Il22, cytokines critical for antimicrobial activity. WD decreased expression of both Atoh1 and Gfi1, genes required for the formation of goblet and Paneth cells, and immunohistochemistry revealed decreased numbers of goblet and Paneth cells. Adding Tg6F ameliorated these WD-mediated changes. Adding oxidized phospholipids ex vivo to the jejunum from mice fed a chow diet reproduced the changes in gene expression in vivo that occurred when the mice were fed WD and were prevented with addition of 6F peptide. We conclude that Tg6F ameliorates the WD-mediated increase in oxidized phospholipids that cause changes in jejunum mucus, which induce dysbiosis and systemic inflammation.

Evolutionary and structural constraints influencing apolipoprotein A-I amyloid behavior.

Apolipoprotein A-I (apoA-I) has a key function in the reverse cholesterol transport. However, aggregation of apoA-I single point mutants can lead to hereditary amyloid pathology. Although several studies have tackled the biophysical and structural consequences introduced by these mutations, there is little information addressing the relationship between the evolutionary and structural features that contribute to the amyloid behavior of apoA-I. We combined evolutionary studies, in silico mutagenesis and molecular dynamics (MD) simulations to provide a comprehensive analysis of the conservation and pathogenic role of the aggregation-prone regions (APRs) present in apoA-I. Sequence analysis demonstrated that among the four amyloidogenic regions described for human apoA-I, only two (APR1 and APR4) are evolutionary conserved across different species of Sarcopterygii. Moreover, stability analysis carried out with the FoldX engine showed that APR1 contributes to the marginal stability of apoA-I. Structural properties of full-length apoA-I models suggest that aggregation is avoided by placing APRs into highly packed and rigid portions of its native fold. Compared to silent variants extracted from the gnomAD database, the thermodynamic and pathogenic impact of amyloid mutations showed evidence of a higher destabilizing effect. MD simulations of the amyloid variant G26R evidenced the partial unfolding of the alpha-helix bundle with the concomitant exposure of APR1 to the solvent, suggesting an insight into the early steps involved in its aggregation. Our findings highlight APR1 as a relevant component for apoA-I structural integrity and emphasize a destabilizing effect of amyloid variants that leads to the exposure of this region.

Using adenovirus-mediated gene transfer to study the effect of myeloperoxidase on plasma lipid levels, HDL structure and functionality in mice expressing human apoA-I forms.

Apolipoprotein A-I (apoA-I), the main protein component of High-Density Lipoprotein (HDL), is modified in plasma and the arterial wall by various enzymes. Myeloperoxidase (MPO), a leukocyte-derived peroxidase, is highly expressed during inflammation and associates with HDL reducing its functionality and contributing to atherosclerosis. In the present study we sought to explore further the effect of MPO on HDL structure and functionality in vivo using adenovirus-mediated gene transfer of human MPO combined with human apoA-I forms containing substitutions at MPO-sensitive sites or wild type apoA-I. We found that overexpression of MPO in mice significantly increased plasma apoA-I and HDL levels without affecting the expression of genes involved in HDL biogenesis or catabolism in the liver. Overexpression of MPO in the liver reduced the expression of pro-inflammatory genes and increased or did not affect the expression of anti-inflammatory genes suggesting that MPO had no toxic effects in this organ. In the plasma of mice overexpressing MPO, no significant alterations in HDL size or electrophoretic mobility was observed with the exception of mice expressing apoA-I (M148A) which showed enriched pre-ß relative to α HDL particles, suggesting that the apoA-I (M148A) mutation may interfere with HDL remodelling. Overexpression of MPO was associated with reduced anti-oxidant capacity of HDL particles in all mice. Interestingly, HDL particles bearing apoA-I (Y192A) showed enhanced ABCA1-dependent cholesterol efflux from macrophages which was not affected by MPO and these mice had reduced levels of LDL-c. These findings provide new insights on the role of specific amino acid residues of apoA-I in HDL structure and function following modification by MPO. This knowledge may facilitate the development of novel therapies based on improved HDL forms for patients with chronic diseases that are characterized by dysfunctional HDL.

Boosting Cholesterol Efflux from Foam Cells by Sequential Administration of rHDL to Deliver MicroRNA and to Remove Cholesterol in a Triple-Cell 2D Atherosclerosis Model.

Cardiovascular disease, the leading cause of mortality worldwide, is primarily caused by atherosclerosis, which is characterized by lipid and inflammatory cell accumulation in blood vessels and carotid intima thickening. Although disease management has improved significantly, new therapeutic strategies focused on accelerating atherosclerosis regression must be developed. Atherosclerosis models mimicking in vivo-like conditions provide essential information for research and new advances toward clinical application. New nanotechnology-based therapeutic opportunities have emerged with apoA-I nanoparticles (recombinant/reconstituted high-density lipoproteins, rHDL) as ideal carriers to deliver molecules and the discovery that microRNAs participate in atherosclerosis establishment and progression. Here, a therapeutic strategy to improve cholesterol efflux is developed based on a two-step administration of rHDL consisting of a first dose of antagomiR-33a-loaded rHDLs to induce adenosine triphosphate-binding cassette transporters A1 overexpression, followed by a second dose of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine rHDLs, which efficiently remove cholesterol from foam cells. A triple-cell 2D-atheroma plaque model reflecting the cellular complexity of atherosclerosis is used to improve efficiency of the nanoparticles in promoting cholesterol efflux. The results show that sequential administration of rHDL potentiates cholesterol efflux indicating that this approach may be used in vivo to more efficiently target atherosclerotic lesions and improve prognosis of the disease.

HDL is the primary transporter for carotenoids from liver to retinal pigment epithelium in transgenic ApoA-I-/-/Bco2-/- mice.

Supplementation with antioxidant carotenoids is a therapeutic strategy to protect against age-related macular degeneration (AMD); however, the transport mechanism of carotenoids from the liver to the retina is still not fully understood. Here, we investigate if HDL serves as the primary transporter for the macular carotenoids. ApoA-I, the key apolipoprotein of HDL, was genetically deleted from BCO2 knockout (Bco2-/-) mice, a macular pigment mouse model capable of accumulating carotenoids in the retina. We then conducted a feeding experiment with a mixed carotenoid chow (lutein:zeaxanthin:ß-carotene = 1:1:1) for one month. HPLC data demonstrated that the total carotenoids were increased in the livers but decreased in the serum, retinal pigment epithelium (RPE)/choroids, and retinas of ApoA-I-/-/Bco2-/- mice compared to Bco2-/- mice. In detail, ApoA-I deficiency caused a significant increase of ß-carotene but not lutein and zeaxanthin in the liver, decreased all three carotenoids in the serum, blocked the majority of zeaxanthin and ß-carotene transport to the RPE/choroid, and dramatically reduced ß-carotene and zeaxanthin but not lutein in the retina. Furthermore, surface plasmon resonance spectroscopy (SPR) data showed that the binding affinity between ApoA-I and ß-carotene â‰« zeaxanthin > lutein. Our results show that carotenoids are transported from the liver to the eye mainly by HDL, and ApoA-I may be involved in the selective delivery of macular carotenoids to the RPE.

ApoA-I Infusion Therapies Following Acute Coronary Syndrome: Past, Present, and Future.

PURPOSE OF REVIEW: The elevated adverse cardiovascular event rate among patients with low high-density lipoprotein cholesterol (HDL-C) formed the basis for the hypothesis that elevating HDL-C would reduce those events. Attempts to raise endogenous HDL-C levels, however, have consistently failed to show improvements in cardiovascular outcomes. However, steady-state HDL-C concentration does not reflect the function of this complex family of particles. Indeed, HDL functions correlate only weakly with serum HDL-C concentration. Thus, the field has pivoted from simply raising the quantity of HDL-C to a focus on improving the putative anti-atherosclerotic functions of HDL particles. Such functions include the ability of HDL to promote the efflux of cholesterol from cholesterol-laden macrophages. Apolipoprotein A-I (apoA-I), the signature apoprotein of HDL, may facilitate the removal of cholesterol from atherosclerotic plaque, reduce the lesional lipid content and might thus stabilize vulnerable plaques, thereby reducing the risk of cardiac events. Infusion of preparations of apoA-I may improve cholesterol efflux capacity (CEC). This review summarizes the development of apoA-I therapies, compares their structural and functional properties and discusses the findings of previous studies including their limitations, and how CSL112, currently being tested in a phase III trial, may overcome these challenges. RECENT FINDINGS: Three major ApoA-I-based approaches (MDCO-216, CER-001, and CSL111/CSL112) have aimed to enhance reverse cholesterol transport. These three therapies differ considerably in both lipid and protein composition. MDCO-216 contains recombinant ApoA-I Milano, CER-001 contains recombinant wild-type human ApoA-I, and CSL111/CSL112 contains native ApoA-I isolated from human plasma. Two of the three agents studied to date (apoA-1 Milano and CER-001) have undergone evaluation by intravascular ultrasound imaging, a technique that gauges lesion volume well but does not assess other important variables that may relate to clinical outcomes. ApoA-1 Milano and CER-001 reduce lecithin-cholesterol acyltransferase (LCAT) activity, potentially impairing the function of HDL in reverse cholesterol transport. Furthermore, apoA-I Milano can compete with and alter the function of the recipient's endogenous apoA-I. In contrast to these agents, CSL112, a particle formulated using human plasma apoA-I and phosphatidylcholine, increases LCAT activity and does not lead to the malfunction of endogenous apoA-I. CSL112 robustly increases cholesterol efflux, promotes reverse cholesterol transport, and now is being tested in a phase III clinical trial. Phase II-b studies of MDCO-216 and CER-001 failed to produce a significant reduction in coronary plaque volume as assessed by IVUS. However, the investigation to determine whether the direct infusion of a reconstituted apoA-I reduces post-myocardial infarction coronary events is being tested using CSL112, which is dosed at a higher level than MDCO-216 and CER-001 and has more favorable pharmacodynamics.