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-1 protected hepatic ischaemia-reperfusion injury through suppressing macrophage pyroptosis via TLR4-NF-κB pathway.

BACKGROUND AND AIMS: Apolipoprotein A-1 (ApoA-1), the major apolipoprotein of high-density lipoprotein, plays anti-atherogenic role in cardiovascular diseases and exerts anti-inflammation effect in various inflammatory and infectious diseases. However, the role and mechanism of ApoA-1 in hepatic ischaemia-reperfusion (I/R) injury is unknown. METHODS: In this study, we measured ApoA-1 expression in human liver grafts after transplantation. Mice partial hepatic I/R injury model was made in ApoA-1 knockout mice, ApoA-1 mimetic peptide D-4F treatment mice and corresponding control mice to examine the effect of ApoA-1 on liver damage, inflammation response and cell death. Primary hepatocytes and macrophages were isolated for in vitro study. RESULTS: The results showed that ApoA-1 expression was down-regulated in human liver grafts after transplantation and mice livers subjected to hepatic I/R injury. ApoA-1 deficiency aggravated liver damage and inflammation response induced by hepatic I/R injury. Interestingly, we found that ApoA-1 deficiency increased pyroptosis instead of apoptosis during acute phase of hepatic I/R injury, which mainly occurred in macrophages rather than hepatocytes. The inhibition of pyroptosis compensated for the adverse impact of ApoA-1 deficiency. Furthermore, the up-regulated pyroptosis process was testified to be mediated by ApoA-1 through TLR4-NF-κB pathway and TLR4 inhibition significantly improved hepatic I/R injury. In addition, we confirmed that D-4F ameliorated hepatic I/R injury. CONCLUSIONS: Our study has identified the protective role of ApoA-1 in hepatic I/R injury through inhibiting pyroptosis in macrophages via TLR4-NF-κB pathway. The effect of ApoA-1 may provide a novel therapeutic approach for hepatic I/R injury.

Systematic evaluation of the effect of different apolipoprotein A-I mimetic peptides on the performance of synthetic high-density lipoproteins in vitro and in vivo.

Synthetic high-density lipoproteins nanomedicine (sHDL) composed of apolipoprotein A-I (ApoA-I) mimetic peptides and lipids have shown very promising results for the treatment of various cardiovascular diseases. Numerous efforts have also been made to design different ApoA-I mimetic peptides to improve the potency of sHDL, especially the efficiency of reverse cholesterol transport. However, the way in which ApoA-I mimetic peptides affect the properties of sHDL, including stability, cholesterol efflux, cholesterol esterification, elimination in vivo, and the relationship of these properties, is still poorly understood. Revealing the effect of these factors on the potency of sHDL is important for the design of better ApoA-I mimetic peptides. In this study, three widely used ApoA-I mimetic peptides with different sequences, lengths, LCAT activation and lipid binding affinities were used for the preparation of sHDL and were evaluated in terms of physical/chemical properties, cholesterol efflux, cholesterol esterification, remodeling, and pharmacokinetics/pharmacodynamics. Our results showed that ApoA-I mimetic peptides with the highest cholesterol efflux and cholesterol esterification in vitro did not exhibit the highest cholesterol mobilization in vivo. Further analysis indicated that other factors, such as pharmacokinetics and remodeling of sHDL, need to be considered in order to predict the efficiency of cholesterol mobilization in vivo. Thus, our study highlights the importance of using the overall performance, rather than in vitro results alone, as the blueprint for the design and optimization of ApoA-I mimetic peptides.

Apolipoprotein A-I Inhibits Transendothelial Transport of Apolipoprotein B-Carrying Lipoproteins and Enhances Its Associated High-Density Lipoprotein Formation.

Caveola-located scavenger receptor type B class I (SR-BI) and activin receptor-like kinase-1 (ALK1) are involved in transendothelial transport of apolipoprotein B-carrying lipoproteins (apoB-LPs). Transport of apoB-LPs though mouse aortic endothelial cells (MAECs) is associated with apoE-carrying high-density lipoprotein (HDL)-like particle formation and apoAI induces raft-located proteins to shift to non-raft membranes by upregulation of ATP-binding cassette transporter A1 (ABCA1). To investigate apoAI's effect on transendothelial transport of apoB-LPs, MAECs and human coronary artery endothelial cells (HCAECs) were treated with apoB-LPs ± apoAI. Our data demonstrated that apoAI neither altered SR-BI and ALK1 expression nor affected apoB-LP binding to MAECs. ApoAI inhibited MAEC uptake, transcellular transport, and intracellular accumulation of apoB-LPs and accelerated their resecretion in MAECs. ApoAI enhanced transendothelial apoB-LP transport-associated HDL-like particle formation, upregulated ABCA1 expression, shifted SR-BI and ALK1 to the non-raft membrane in MAECs, inhibited transcellular transport of apoB-LPs, and enhanced associated HDL-like particle formation in HCAECs. ABCA1 knockdown attenuated apoAI-induced membrane SR-BI and ALK1 relocation and diminished apoAI's effect on transendothelial apoB-LP transport and HDL-like particle formation in MAECs. This suggests that upregulation of ABCA1 expression is a mechanism, whereby apoAI provokes caveola-located receptor relocation, inhibits transendothelial apoB-LP transport, and promotes associated HDL-like particle formation.

Analysis of the orientation of cholesterol in high-density lipoprotein nanodiscs using solid-state NMR.

Cholesterol is an essential component of eukaryotic cellular membranes that regulates the order and phase behaviour of dynamic lipid bilayers. Although cholesterol performs many vital physiological roles, hypercholesterolaemia and the accumulation of cholesterol in atherosclerotic plaques can increase the risk of coronary heart disease morbidity. The risk is mitigated by the transportation of cholesterol from peripheral tissue to the liver by high-density lipoprotein (HDL), 6-20 nm-diameter particles of lipid bilayers constrained by an annular belt of the protein apolipoprotein A-I (apoA-I). Information on the dynamics and orientation of cholesterol in HDL is pertinent to the essential role of HDL in cholesterol cycling. This work investigates whether the molecular orientation of cholesterol in HDL differs from that in the unconstrained lipid bilayers of multilamellar vesicles (MLVs). Solid-state NMR (ssNMR) measurements of dynamically-averaged 13C-13C and 13C-1H dipolar couplings were used to determine the average orientation of triple 13C-labelled cholesterol in palmitoyloleoylphosphatidylcholine (POPC) lipid bilayers in reconstituted HDL (rHDL) nanodiscs and in MLVs. Individual 13C-13C dipolar couplings were measured from [2,3,4-13C3]cholesterol in a one-dimensional NMR experiment, by using a novel application of a method to excite double quantum coherence at rotational resonance. The measured dipolar couplings were compared with average values calculated from orientational distributions of cholesterol generated using a Gaussian probability density function. The data were consistent with small differences in the average orientation of cholesterol in rHDL and MLVs, which may reflect the effects of the constrained and unconstrained lipid bilayers in the two environments. The calculated distributions of cholesterol in rHDL and MLVs that were consistent with the NMR data also agreed well with orientational distributions extracted from previous molecular dynamics simulations of HDL nanodiscs and planar POPC bilayers.

Apolipoprotein A-I, elevated in trauma patients, inhibits platelet activation and decreases clot strength.

Apolipoprotein A-I (ApoA-I) is elevated in the plasma of a subgroup of trauma patients with systemic hyperfibrinolysis. We hypothesize that apoA-I inhibits platelet activation and clot formation. The effects of apoA-I on human platelet activation and clot formation were assessed by whole blood thrombelastography (TEG), platelet aggregometry, P-selectin surface expression, microfluidic adhesion, and Akt phosphorylation. Mouse models of carotid artery thrombosis and pulmonary embolism were used to assess the effects of apoA-I in vivo. The ApoA-1 receptor was investigated with transgenic mice knockouts (KO) for the scavenger receptor class B member 1 (SR-BI). Compared to controls, exogenous human apoA-I inhibited arachidonic acid and collagen-mediated human and mouse platelet aggregation, decreased P-selectin surface expression and Akt activation, resulting in diminished clot strength and increased clot lysis by TEG. ApoA-I also decreased platelet aggregate size formed on a collagen surface under flow. In vivo, apoA-I delayed vessel occlusion in an arterial thrombosis model and conferred a survival advantage in a pulmonary embolism model. SR-BI KO mice significantly reduced apoA-I inhibition of platelet aggregation versus wild-type platelets. Exogenous human apoA-I inhibits platelet activation, decreases clot strength and stability, and protects mice from arterial and venous thrombosis via the SR-BI receptor.

HDL (High-Density Lipoprotein) and ApoA-1 (Apolipoprotein A-1) Potentially Modulate Pancreatic α-Cell Glucagon Secretion.

OBJECTIVE: Subjects with low levels of HDL (high-density lipoprotein) and ApoA-1 (apolipoprotein A-1) have increased risk to develop type 2 diabetes. HDL levels are an independent predictor of ß-cell function and positively modulate it. Type 2 diabetes is characterized by defects in both ß and α-cell function, but the effect of HDL and ApoA1 on α-cell function is unknown. Approach and Results: We observed a significant negative correlation (r=-0.422, P<0.0001) between HDL levels and fasting glucagon in a cohort of 132 Italian subjects. In a multivariable regression analysis including potential confounders such as age, sex, BMI, triglycerides, total cholesterol, fasting and 2-hour postload glucose, and fasting insulin, the association between HDL and fasting glucagon remained statistically significant (ß=-0.318, P=0.006). CD1 mice treated with HDL or ApoA-1 for 3 consecutive days showed a 32% (P<0.001) and 23% (P<0.05) reduction, respectively, in glucagon levels following insulin-induced hypoglycemia, compared with controls. Treatment of pancreatic αTC1 clone 6 cells with HDL or ApoA-1 for 24 hours resulted in a significant reduction of glucagon expression (P<0.04) and secretion (P<0.01) after an hypoglycemic stimulus and increased Akt (RAC-alpha serine/threonine-protein kinase) and FoxO1 (forkhead/winged helix box gene, group O-1) phosphorylation. Pretreatment with Akt inhibitor VIII, PI3K (phosphatidylinositol 3-kinase) inhibitor LY294002, and HDL receptor SCARB-1 (scavenger receptor class B type 1) inhibitor BLT-1 (block lipid transport-1) restored αTC1 cell response to low glucose levels. CONCLUSIONS: These results support the notion that HDL and ApoA-1 modulate glucagon expression and secretion by binding their cognate receptor SCARB-1, and activating the PI3K/Akt/FoxO1 signaling cascade in an in vitro α-cell model. Overall, these results raise the hypothesis that HDL and ApoA-1 may have a role in modulating glucagon secretion.

Structural and Functional Impairments of Reconstituted High-Density Lipoprotein by Incorporation of Recombinant ß-Amyloid42.

Beta (ß)-amyloid (Aß) is a causative protein of Alzheimer's disease (AD). In the pathogenesis of AD, the apolipoprotein (apo) A-I and high-density lipoprotein (HDL) metabolism is essential for the clearance of Aß. In this study, recombinant Aß42 was expressed and purified via the pET-30a expression vector and E.coli production system to elucidate the physiological effects of Aß on HDL metabolism. The recombinant human Aß protein (51 aa) was purified to at least 95% purity and characterized in either the lipid-free and lipid-bound states with apoA-I. Aß was incorporated into the reconstituted HDL (rHDL) (molar ratio 95:5:1, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):cholesterol:apoA-I) with various apoA-I:Aß ratios from 1:0 to 1:0.5, 1:1 and 1:2. With an increasing molar ratio of Aß, the α-helicity of apoA-I was decreased from 62% to 36% with a red shift of the Trp wavelength maximum fluorescence from 337 to 340 nm in apoA-I. The glycation reaction of apoA-I was accelerated further by the addition of Aß. The treatment of fructose and Aß caused more multimerization of apoA-I in the lipid-free state and in HDL. The phospholipid-binding ability of apoA-I was impaired severely by the addition of Aß in a dose-dependent manner. The phagocytosis of LDL into macrophages was accelerated more by the presence of Aß with the production of more oxidized species. Aß severely impaired tissue regeneration, and a microinjection of Aß enhanced embryotoxicity. In conclusion, the beneficial functions of apoA-I and HDL were severely impaired by the addition of Aß via its detrimental effect on secondary structure. The impairment of HDL functionality occurred more synergistically by means of the co-addition of fructose and Aß.

Protective Effect of ApoA1 (Apolipoprotein A1)-Milano in a Rat Model of Large Vessel Occlusion Stroke.

Background and Purpose- Previous experimental studies found that the infusion of human purified nascent HDL (high-density lipoprotein) significantly reduced infarct volume and hemorrhagic transformation rate by decreasing neutrophil recruitment. ApoA1-M (apolipoprotein A1-Milano) is a natural variant of human ApoA1 that confers protection against atherosclerosis. Recombinant ApoA1-M has been formulated as a complex with phospholipids to mimic the properties of nascent HDL. The aim of this study was to assess the impact of intravenous ApoA1-M in a transient middle cerebral artery occlusion stroke model in rats. Methods- In a first experiment, rats were subjected to 120-minute transient middle cerebral artery occlusion and intravenous ApoA1-M was infused immediately or 4 hours after occlusion. In a second experiment, rats were subjected to 240-minute transient middle cerebral artery occlusion and intravenous ApoA1-M was infused with or without recombinant tPA (tissue-type plasminogen activator) immediately after recanalization. Primary outcome criteria were the infarct volume and hemorrhagic transformation rate measured at 24 hours. Platelets, coagulation, and neutrophil activation biomarkers were measured in brain homogenates and plasma. Additional in vitro experiments studied the effects of ApoA1-M on platelet aggregation and platelet-neutrophil interactions. Results- The infusion of ApoA1-M immediately or 4 hours after 120-minute transient middle cerebral artery occlusion significantly reduced the infarct volume compared with saline (P=0.034 and P=0.036, respectively). Compared with tPA alone, co-administration of ApoA1-M and tPA showed similar rates of hemorrhagic transformation. ApoA1-M had no significant inhibition effect on neutrophil activation biomarkers. Platelet activation was slightly decreased in rats treated with ApoA1-M compared with saline. In vitro, the incubation of human and rat platelet-rich plasma with ApoA1-M significantly reduced ADP-induced platelet aggregation (P=0.001 and P=0.02, respectively). Conclusions- ApoA1-Milano significantly decreased the infarct volume through an inhibition of platelet aggregation but did not reduce hemorrhagic transformation and neutrophils activation as expected after previous experimental studies with nascent HDL. Visual Overview- An online visual overview is available for this article.

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

Apolipoprotein A-I is an anti-inflammatory, antioxidative, cardioprotective, anti-tumorigenic, and anti-diabetic in mammals. Apolipoprotein A-I also regulates innate immune defense mechanisms in vertebrates and invertebrates. Apolipoproteins A-I from mammals and several teleosts display antibacterial activities against Gram negative and Gram positive bacteria. The present study describes strategies to obtain high amounts of soluble purified recombinant Apolipoprotein A-I of Labeo rohita, an Indian major carp (rLrApoA-I). The study also reports its detailed structural and functional characterization i.e. antimicrobial activity against a number of important marine and fresh water bacterial pathogens. The rLrApoA-I was expressed in Escherichia coli BL21(DE3) pLysS expression host as a soluble protein under optimized conditions. The yield of purified rLrApoA-I was ~ 75 mg/L from soluble fraction using metal ion affinity chromatography. The authenticity of the rLrApoA-I was confirmed by MALDI-TOF-MS analysis. The secondary structure analysis showed rLrApoA-I to be predominantly alpha helical, an evolutionary conserved characteristic across mammals and teleosts. The purified rLrApoA-I exhibited antimicrobial activity as evident from inhibition of growth of a number of bacteria namely Aeromonas hydrophila, A. liquefaciens, A. culicicola, A. sobria, Vibrio harveyi, V. parahaemolyticus and Edwardsiella tarda in a dose-dependent manner. Minimum bactericidal concentration for A. liquefaciens, A. culicicola, and A. sobria, was determined to be 25 µg/ml or 0.81 µM whereas for A. hydrophila, E. tarda, V. parahaemolyticus and V. harveyi, it was determined to be 100 µg/ml or 3.23 µM. These data strongly suggest that recombinant ApoA-I from Labeo rohita could play a role in primary defense against fish pathogen. Further, at temperature ≥ 55 °C, though a loss in secondary structure was observed, no effect on its antibacterial activity was observed. This is of significance as the antibacterial activity is not likely to be lost even if the protein is subjected to high temperatures during transport.

Apolipoprotein A-I Supports MSCs Survival under Stress Conditions.

Clinical trials have shown the safety of mesenchymal stem/stromal cells (MSCs) transplantation, but the effectiveness of these treatments is limited. Since, transplanted MSCs will undergo metabolic disturbances in the bloodstream, we investigated the influence of blood plasmas of type 2 diabetes (T2D) patients on MSCs viability and examined whether apolipoprotein A-I (apoA-I) could protect cells from stressful conditions of serum deprivation (SD), hypoxia, and elevated concentrations of reactive oxygen species (ROS). ApoA-I exhibits anti-inflammatory, immune activities, improves glycemic control, and is suitable for T2D patients but its influence on MSCs remains unknown. For the first time we have shown that apoA-I decreases intracellular ROS and supports proliferative rate of MSCs, thereby increasing cell count in oxidation conditions. ApoA-I did not influence cell cycle when MSCs were predominantly in the G0/G1 phases under conditions of SD/hypoxia, activated proliferation rapidly, and reduced apoptosis during MSCs transition to the oxygenation or oxidation conditions. Finally, it was found that the blood plasma of T2D individuals had a cytotoxic effect on MSСs in 39% of cases and had a wide variability of antioxidant properties. ApoA-I protects cells under all adverse conditions and can increase the efficiency of MSCs transplantation in T2D patients.

Lipoprotein modulation of proteinuric renal injury.

High-density lipoprotein (HDL) and its main protein, apolipoprotein AI (apoAI), have established benefits in various cells, but whether these cytoprotective effects of HDL pertain to renal cells is unclear. We investigated the in vitro consequences of exposing damaged podocytes to normal apoAI, HDL, and apoAI mimetic (L-4F), and the in vivo effects of L-4F on kidney and atherosclerotic injury in a podocyte-specific injury model of proteinuria. In vitro, primary mouse podocytes were injured by puromycin aminonucleoside (PAN). Cellular viability, migration, production of reactive oxygen species (ROS), apoptosis, and the underlying signaling pathway were assessed. In vivo, we used a proteinuric model, Nphs1-hCD25 transgenic (NEP25+) mice, which express human CD25 on podocytes. Podocyte injury was induced by using immunotoxin (LMB2) and generated a proteinuric atherosclerosis model, NEP25+:apoE-/- mice, was generated by mating apoE-deficient (apoE-/-) mice with NEP25+ mice. Animals received L-4F or control vehicle. Renal function, podocyte injury, and atherosclerosis were assessed. PAN reduced podocyte viability, migration, and increased ROS production, all significantly lessened by apoAI, HDL, and L-4F. L-4F attenuated podocyte apoptosis and diminished PAN-induced inactivation of Janus family protein kinase-2/signal transducers and activators of transcription 3. In NEP25+ mice, L-4F significantly lessened overall proteinuria, and preserved podocyte expression of synaptopodin and cell density. Proteinuric NEP25+:apoE-/- mice had more atherosclerosis than non-proteinuric apoE-/- mice, and these lesions were significantly decreased by L-4F. Normal human apoAI, HDL, and apoAI mimetic protect against podocyte damage. ApoAI mimetic provides in vivo beneficial effects on podocytes that culminate in reduced albuminuria and atherosclerosis. The results suggest supplemental apoAI/apoAI mimetic may be a novel candidate to lessen podocyte damage and its complications.

Treatment with apolipoprotein A1 protects mice against doxorubicin-induced cardiotoxicity in a scavenger receptor class B, type I-dependent manner.

Doxorubicin, an agent used to treat a variety of cancers, is cardiotoxic by triggering cardiomyocyte apoptosis. We previously showed that treating cultured cardiomyocytes with human high-density lipoprotein in vitro or transgenic overexpression of human apolipoprotein A1, its main structural protein, protects against doxorubicin-induced cardiomyocyte apoptosis in a manner dependent on the scavenger receptor class B type I [Durham KK, Chathely KM, Mak KC, Momen A, Thomas CT, Zhao YY, MacDonald ME, Curtis JM, Husain M, Trigatti BL. HDL protects against doxorubicin-induced cardiotoxicity in a scavenger receptor class B type 1-, phosphatidylinositol 3-kinase-, and Akt-dependent manner. Am J Physiol Heart Circ Physiol 314: H31-H44, 2018]. This was due to high-density lipoprotein-induced activation of Akt signaling in cardiomyocytes. We now demonstrate that mice lacking the scavenger receptor class B, type I exhibit increased sensitivity to doxorubicin-induced cardiomyocyte apoptosis in vivo. Cardiomyocytes expressing scavenger receptor class B, type I are protected from doxorubicin-induced apoptosis by preincubation with high-density lipoprotein isolated from wild-type mice, whereas high-density lipoprotein from scavenger receptor class B, type 1 knockout mice is less effective. Cardiomyocytes from scavenger receptor class B, type I knockout mice, however, are not protected by high-density lipoprotein in vitro, and hearts from knockout mice are more sensitive to doxorubicin in vivo. Pharmacological administration of purified apolipoprotein A1 dramatically protected wild-type mice from doxorubicin-induced cardiotoxicity and left ventricular dysfunction, whereas this protection was lost in scavenger receptor class B, type I-deficient mice. This demonstrates, at least in mice, that high-density lipoprotein therapy can confer protection against doxorubicin-induced cardiomyocyte apoptosis in a manner mediated by the scavenger receptor class B, type I. NEW & NOTEWORTHY We show that scavenger receptor class B, type I (SR-B1) mediates HDL-dependent protection against doxorubicin-induced cardiomyocyte apoptosis and that this is a property of SR-B1 in cardiomyocytes in vitro and in hearts in vivo. We also demonstrate that pharmacological treatment with apolipoprotein A1, the major HDL structural protein, protects mice against doxorubicin-induced cardiomyocyte apoptosis and left ventricular dysfunction in an SR-B1-dependent manner. This suggests that HDL-targeted pharmacological therapy may hold promise for protecting against the deleterious, cardiotoxic side effects of this commonly used chemotherapeutic drug.

Effect of high-density lipoprotein on oocyte maturation and bovine embryo development in vitro.

High-density lipoprotein (HDL) is the main lipoprotein in the follicular fluid, and it has anti-inflammatory, antioxidant and cryoprotectant properties. The anti-inflammatory potential and antioxidant potential are derived from its lipid composition, especially the apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1). The aim of this study was to evaluate the effect of HDL during in vitro maturation (IVM) on oocyte maturation and early bovine embryo development. For this, cumulus-oocyte complexes (COCs) were obtained from bovine ovaries collected at a local slaughterhouse. COCs (n = 2,250) were allocated into three groups (n = 50 COCs/group) according to the addition of HDL protein (HDL-P) during IVM for 22 hr: 0 (control), 50 and 150 mg/dl. After IVM, COCs were inseminated (in vitro fertilization) and cultivated for 7 days. Total cholesterol concentration, total protein, triglycerides and ApoAI concentrations on IVM medium increased proportionally to HDL-P addition. However, PON1 activity was not detected in any treatment. The addition of HDL-P did not affect nuclear maturation rate, endogenous reactive oxygen species and glutathione levels in COCs (p > 0.05). The highest HDL-P concentration (150 mg/dl) decreased cleavage and blastocyst rate (p < 0.05). Moreover, the HDL-P 150 mg/dl group had lower cellular count/blastocyst than the 50 mg/dl group (p < 0.05). However, the addition of HDL-P did not affect relative gene expression of evaluated genes. In conclusion, the complex HDL/ApoAI obtained from human plasma, in the absence of PON1 activity during in vitro oocyte maturation, decreased initial embryo development.

Effective Treatment of Diabetic Cardiomyopathy and Heart Failure with Reconstituted HDL (Milano) in Mice.

The risk of heart failure (HF) is prominently increased in patients with type 2 diabetes mellitus. The objectives of this study were to establish a murine model of diabetic cardiomyopathy induced by feeding a high-sugar/high-fat (HSHF) diet and to evaluate the effect of reconstituted HDLMilano administration on established HF in this model. The HSHF diet was initiated at the age of 12 weeks and continued for 16 weeks. To investigate the effect of reconstituted HDLMilano on HF, eight intraperitoneal administrations of MDCO-216 (100 mg/kg protein concentration) or of an identical volume of control buffer were executed with a 48-h interval starting at the age of 28 weeks. The HSHF diet-induced obesity, hyperinsulinemia, and type 2 diabetes mellitus. Diabetic cardiomyopathy was present in HSHF diet mice as evidenced by cardiac hypertrophy, increased interstitial and perivascular fibrosis, and decreased myocardial capillary density. Pressure-volume loop analysis indicated the presence of both systolic and diastolic dysfunction and of decreased cardiac output in HSHF diet mice. Treatment with MDCO-216 reversed pathological remodelling and cardiac dysfunction and normalized wet lung weight, indicating effective treatment of HF. No effect of control buffer injection was observed. In conclusion, reconstituted HDLMilano reverses HF in type 2 diabetic mice.

D-4F, an apolipoprotein A-I mimetic, suppresses IL-4 induced macrophage alternative activation and pro-fibrotic TGF-ß1 expression.

Context: We reported that D-4F, an apolipoprotein A-I (Apo A-I) mimetic polypeptide with 18 d-amino acids, suppressed IL-4 induced macrophage alternative activation and TGF-ß1 expression in phorbol 12-myristate 13-acetate (PMA) treated human acute monocytic leukemia cells (THP-1). Objective: Macrophage alternative activation, TGF-ß1 and epithelial-mesenchymal transition (EMT) are intensively involved in pulmonary fibrosis. Recent studies demonstrated that Apo A-I resolved established pulmonary fibrotic nodules, and D-4F inhibited TGF-ß1 induced EMT in alveolar cells. Therefore, this study evaluated the effects of D-4F on IL-4 induced macrophage alternative activation and TGF-ß1 expression. Materials and methods: THP-1 cells were simulated with PMA (100 ng/mL) for 48 h and treated with medium control, IL-4 (20 ng/mL) alone, or IL-4 (20 ng/mL) in the presence of D-4F (1, 5, and 10 µg/mL) for 24 and 48 h. Flow cytometry, RT-PCR and ELISA evaluations were performed to investigate the subsequent effects of D-4F. Results: Compared to stimulation with IL-4 alone, 1, 5, and 10 µg/mL of D-4F reduced alternative activation by 45.38%, 59.98%, and 60.10%, increased TNF-α mRNA levels by 8%, 11%, and 16% and decreased TGF-ß1 mRNA levels by 21%, 37%, and 39%, respectively (all p ≤ 0.05). In addition, TNF-α protein levels increased from 388 pg/mL (IL-4 alone) to 429, 475, and 487 pg/mL (1, 5, and 10 µg/mL D-4F), while TGF-ß1 protein levels dropped from 27.01 pg/mL (IL-4 alone) to 19.15, 12.27, and 10.47 pg/mL (1, 5, and 10 µg/mL D-4F). Conclusion: D-4F suppressed IL-4 induced macrophage alternative activation and pro-fibrotic TGF-ß1 expression.

Apolipoprotein A-I Mimetic Peptides: Discordance Between In Vitro and In Vivo Properties-Brief Report.

OBJECTIVE: Apolipoprotein A-I (apoA-I) mimetic peptides have antiatherogenic properties of high-density lipoprotein in vitro and have been shown to inhibit atherosclerosis in vivo. It is unclear, however, if each in vitro antiatherogenic property of these peptides translates to a corresponding activity in vivo, and if so, which of these contributes most to reduce atherosclerosis. APPROACH AND RESULTS: The effect of 7 apoA-I mimetic peptides, which were developed to selectively reproduce a specific component of the antiatherogenic properties of apoA-I, on the development of atherosclerosis was investigated in apolipoprotein E-deficient mice fed a high-fat diet for 4 or 12 weeks. The peptides include those that selectively upregulate cholesterol efflux, or are anti-inflammatory, or have antioxidation properties. All the peptides studied effectively inhibited the in vivo development of atherosclerosis in this model to the same extent. However, none of the peptides had the same selective effect in vivo as they had exhibited in vitro. None of the tested peptides affected plasma lipoprotein profile; capacity of plasma to support cholesterol efflux was increased modestly and similarly for all peptides. CONCLUSIONS: There is a discordance between the selective in vitro and in vivo functional properties of apoA-I mimetic peptides, and the in vivo antiatherosclerotic effect of apoA-I-mimetic peptides is independent of their in vitro functional profile. Comparing the properties of apoA-I mimetic peptides in plasma rather than in the lipid-free state is better for predicting their in vivo effects on atherosclerosis.

ApoA-1 Mimetic Peptide ELK-2A2K2E Decreases Inflammatory Factor Levels Through the ABCA1-JAK2-STAT3-TTP Axis in THP-1-Derived Macrophages.

OBJECTIVE: The aim of this study was to determine whether the apolipoprotein A-1 (apoA-1) mimetic peptide ELK-2A2K2E regulates inflammatory cytokine expression through activating the adenosine triphosphate-binding cassette transporter A1 (ABCA1)-janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3)-tristetraprolin (TTP) signaling pathway in THP-1 macrophage-derived foam cells. METHODS AND RESULTS: The cells were treated with the apoA-1 mimetic peptide ELK-2A2K2E at different concentrations (0, 20, 40, and 80 µg/mL) or incubated with ELK-2A2K2E (40 µg/mL) for different times (0, 6, 12, and 24 hours). Our results showed that the levels of the cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), were decreased at both concentration- and time-dependent manners. When the cells were exposed to lipopolysaccharides and actinomycin D, ELK-2A2K2E significantly decreased the mRNA stability of inflammatory cytokines at different time points (0, 30, 60, and 120 minutes) by increasing TTP expression as analyzed by real-time quantitative polymerase chain reaction. The effect of ELK-2A2K2E on TTP was obviously blocked by the inhibition of the JAK-STAT3 pathway. Furthermore, we found that ELK-2A2K2E activated the JAK-STAT3-TTP pathway through the upregulation of ABCA1 and then decreased inflammatory cytokine expression. CONCLUSIONS: ApoA-I mimetic peptide ELK-2A2K2E increases the degradation of TNF-α, IL-6, and MCP-1 mRNA and reduces the levels of inflammatory cytokines through activating the JAK2-STAT3-TTP signaling pathway that is dependent on the upregulation of ABCA1.

High-Density Lipoprotein Functionality as a New Pharmacological Target on Cardiovascular Disease: Unifying Mechanism That Explains High-Density Lipoprotein Protection Toward the Progression of Atherosclerosis.

The formation of the atherosclerotic plaque that is characterized by the accumulation of abnormal amounts of cholesterol-loaded macrophages in the artery wall is mediated by both inflammatory events and alterations of lipid/lipoprotein metabolism. Reverse transport of cholesterol opposes the formation and development of atherosclerotic plaque by promoting high density lipoprotein (HDL)-mediated removal of cholesterol from peripheral macrophages and its delivery back to the liver for excretion into the bile. Although an inverse association between HDL plasma levels and the risk of cardiovascular disease (CVD) has been demonstrated over the years, several studies have recently shown that the antiatherogenic functions of HDL seem to be mediated by their functionality, not always associated with their plasma concentrations. Therefore, assessment of HDL function, evaluated as the capacity to promote cell cholesterol efflux, may offer a better prediction of CVD than HDL levels alone. In agreement with this idea, it has recently been shown that the assessment of serum cholesterol efflux capacity (CEC), as a metric of HDL functionality, may represent a predictor of atherosclerosis extent in humans. The purpose of this narrative review is to summarize the current evidence concerning the role of cholesterol efflux capacity that is important for evaluating CVD risk, focusing on pharmacological evidences and its relationship with inflammation. We conclude that HDL therapeutics are a promising area of investigation but strategies for identifying efficacy must move beyond the idea of simply raising static HDL-cholesterol levels and toward methods of measuring the dynamics of HDL particle remodeling and the generation of lipid-free apolipoprotein A-I (apoA-I). In this way, apoA-I, unlike mature HDL, can promote the greatest extent of cholesterol efflux relieving cellular cholesterol toxicity and the inflammation it causes.

HDL Mimetics Infusion and Regression of Atherosclerosis: Is It Still Considered a Valid Therapeutic Option?

PURPOSE OF REVIEW: This review aims to summarize and discuss the recent findings in the field of using HDL mimetics for the treatment of patients with coronary artery disease. RECENT FINDINGS: Following the largely disappointing results with the cholesteryl ester transfer protein inhibitors, focus moved to HDL functionality rather than absolute HDL cholesterol values. A number of HDL/apoA-I mimicking molecules were developed, aiming to enhance reverse cholesterol transport that has been associated with an atheroprotective effect. Three HDL mimetics have made the step from bench-testing to clinical trials in humans and are discussed here: apoA-I Milano, CSL-112, and CER-001. Unfortunately, with the exception of CSL-112 where the results of the clinical trial are not yet known, none of the agents was able to demonstrate a clinical benefit. HDL mimetics have failed to date to prove a beneficial effect in clinical practice. Reverse cholesterol transport remains a challenging therapeutic pathway to be explored.