Recombinant High Density Lipoprotein Research Articles

Adipose tissue targeted sequential delivery system regulating glycolipid metabolism for systemic obesity and its comorbidities

Obesity has emerged as a chronic, relapsing, progressive disease globally. Available methods including pharmacotherapy, surgery, and limotherapy, may lead to toxicities and gastrointestinal disturbances due to their lack of adipose tissue targetability. Increasing energy expenditure and reducing gluconeogenesis through browning of white adipose tissue (WAT) is a therapeutic target for obesity and its comorbidities. Here, we constructed a biomimetic discoidal recombinant high-density lipoprotein (rHDL) with high specificity for scavenger receptor class B type I (SR-BI). rHDL enables targeted delivery of combination drugs (RM) containing rosiglitazone (Rosi) and metformin (Met) to WAT, liver, and intestine, that express elevated levels of SR-BI, resulting in promoted browning of WAT, enhanced mitochondrial biogenesis, and adipocyte thermogenesis increase. For oral delivery, rHDL@RM was loaded in pH-senstive sodium alginate chitosan complex microspheres (MS), enabling stepwise release in the gastrointestinal tract, with mucosal penetration capability that facilitating longlasting lipid-lowering effect. Diet-induced obese (DIO) mice treated with rHDL@RM/MS showed 44.6 % reduction in body weight, with decreased serum glucose and lipid levels. Obesity comorbidities, including NAFLD, gut microbiome disorders, systemic lipid metabolism abnormalities, and chronic inflammation, were all effectively suppressed. Our designed rHDL@RM/MS oral-nanoplatform represents a valuable therapeutic strategy for painless treatment of systemic obesity and related comorbidities.

The effect of dual antioxidant modification on oxidative stress resistance and anti-dysfunction of non-split HDL and recombinant HDL

Recombinant high-density lipoprotein (rHDL) as anti-atherosclerosis (AS) vehicle has unique advantages including multiple anti-atherogenic functions and homing features to plaques. However, rHDL may be converted into dysfunctional forms due to complex treatment during preparation. Herein, oxidation-induced dysfunction of non-split HDL and rHDL was initially investigated. It was found that although both non-split HDL and rHDL showed oxidative dysfunction behavior, non-split HDL demonstrated superior oxidation defense compared to rHDL due to its intact composition and avoidance of overprocessing such as split and recombination. Unfortunately, in vivo oxidative stress could compromise the functionality of HDL. Therefore, surface engineering of non-split HDL and rHDL with cascade antioxidant enzyme analogues Ebselen and mitochondrial-targeted TPGS-Tempo was conducted to construct a dual-line defense HDL nano system (i.e., T@E-HDLs/rHDL), aiming to restore plaque redox balance and preserving the physiological function of HDL. Results indicated that both T@E-HDLs and rHDLs performed without distinction and exhibited greater resistance to oxidative stress damage as well as better functions than unmodified HDLs in macrophage foam cells. Overall, the modification of dual antioxidants strategy bridges the gap between non-split HDL and rHDL, and provides a promising resolution for the dilemmas of oxidative stress in plaques and HDL self dysfunction.

High-Density Lipoprotein Subclasses and Their Role in the Prevention and Treatment of Cardiovascular Disease: A Narrative Review.

The association between high-density lipoprotein cholesterol (HDL-C) and cardiovascular disease (CVD) is controversial. HDL-C is one content type of high-density lipoprotein (HDL). HDL consists of diverse proteins and lipids and can be classified into different subclasses based on size, shape, charge, and density, and can change dynamically in disease states. Therefore, HDL-C levels alone cannot represent HDLs' cardioprotective role. In this review, we summarized the methods for separating HDL subclasses, the studies on the association between HDL subclasses and cardiovascular risk (CVR), and the impact of lipid-modifying medications and nonpharmacological approaches (exercise training, dietary omega fatty acids, and low-density lipoprotein apheresis) on HDL subclasses. As HDL is a natural nanoplatform, recombinant HDLs (rHDLs) have been used as a delivery system in vivo by loading small interfering RNA, drugs, contrast agents, etc. Therefore, we further reviewed the HDL subclasses used in rHDLs and their advantages and disadvantages. This review would provide recommendations and guidance for future studies on HDL subclasses' cardioprotective roles.

Development of Recombinant High-Density Lipoprotein Platform with Innate Adipose Tissue-Targeting Abilities for Regional Fat Reduction.

As an escalating public health issue, obesity and overweight conditions are predispositions to various diseases and are exacerbated by concurrent chronic inflammation. Nonetheless, extant antiobesity pharmaceuticals (quercetin, capsaicin, catecholamine, etc.) manifest constrained efficacy alongside systemic toxic effects. Effective therapeutic approaches that selectively target adipose tissue, thereby enhancing local energy expenditure, surmounting the limitations of prevailing antiobesity modalities are highly expected. In this context, we developed a temperature-sensitive hydrogel loaded with recombinant high-density lipoprotein (rHDL) to achieve targeted delivery of resveratrol, an adipose browning activator, to adipose tissue. rHDL exhibits self-regulation on fat cell metabolism and demonstrates natural targeting toward scavenger receptor class B type I (SR-BI), which is highly expressed by fat cells, thereby achieving a synergistic effect for the treatment of obesity. Additionally, the dispersion of rHDL@Res in temperature-sensitive hydrogels, coupled with the regulation of their degradation and drug release rate, facilitated sustainable drug release at local adipose tissues over an extended period. Following 24 days' treatment regimen, obese mice exhibited improved metabolic status, resulting in a reduction of 68.2% of their inguinal white adipose tissue (ingWAT). Specifically, rHDL@Res/gel facilitated the conversion of fatty acids to phospholipids (PA, PC), expediting fat mobilization, mitigating triglyceride accumulation, and therefore facilitating adipose tissue reduction. Furthermore, rHDL@Res/gel demonstrated efficacy in attenuating obesity-induced inflammation and fostering angiogenesis in ingWAT. Collectively, this engineered local fat reduction platform demonstrated heightened effectiveness and safety through simultaneously targeting adipocytes, promoting WAT browning, regulating lipid metabolism, and controlling inflammation, showing promise for adipose-targeted therapy.

Effect of lipid-lowering therapy on platelet reactivity in patients treated with and without antiplatelet therapy.

Circulating lipoproteins may interact with platelets, increasing platelet sensitivity to aggregating agonists and their tendency towards activation and thrombus formation. In particular, patients with hypercholesterolemia exhibit a higher degree of platelet reactivity compared to normolipidemic. Moreover, accruing evidence report that lipid-lowering therapies can reduce thrombus formation, particularly in the absence of concomitant antiplatelet therapy. However, the underlying biological mechanism(s) explaining these clinical observations are not completely understood. Baseline platelet reactivity and high on-treatment platelet reactivity while on antiplatelet therapy (e.g., aspirin and clopidogrel) are associated with poor clinical outcomes. Therefore, strategies to reduce baseline platelet reactivity or improve the pharmacodynamic profile of antiplatelet therapies are an unmet clinical need. The potential use of lipid-lowering therapies for optimizing platelet reactivity provides several advantages as there is strong evidence that reducing circulating lipoproteins can improve clinical outcomes, and they may avoid the need for potent antiplatelet therapies that, although more effective, are associated with increased bleeding risk. This review will provide a systematic overview of the effects of lipid-lowering therapy on platelet reactivity in patients treated with and without antiplatelet therapy. We will focus on the potential biological mechanism(s) of action and the effect of statins, ezetimibe, proprotein convertase subtilisin/kexin 9 inhibitors, omega-3 fatty acids, and recombinant high-density lipoprotein on platelet reactivity. Ultimately, we will assess the current gaps in the literature and future perspective in the field.

Retardation of Aβ42 fibril formation by apolipoprotein A-I and recombinant HDL particles

The double nucleation mechanism of amyloid β (Aβ) peptide aggregation is retained from buffer to cerebrospinal fluid (CSF) but with reduced rate of all microscopic processes. Here, we used a bottom-up approach to identify retarding factors in CSF. We investigated the Aβ42 fibril formation as a function of time in the absence and presence of apolipoprotein A-I (ApoA-I), recombinant high-density lipoprotein (rHDL) particles, or lipid vesicles. A retardation was observed in the presence of ApoA-I or rHDL particles, most pronounced with ApoA-I, but not with lipid vesicles. Global kinetic analysis implies that rHDL interferes with secondary nucleation. The effect of ApoA-I could best be described as an interference with secondary and to a smaller extent primary nucleation. Using surface plasmon resonance and microfluidics diffusional sizing analyses, we find that both rHDL and ApoA-I interact with Aβ42 fibrils but not Aβ42 monomer, thus the effect on kinetics seems to involve interference with the catalytic surface for secondary nucleation. The Aβ42 fibrils were imaged using cryogenic-electron microscopy and found to be longer when formed in the presence of ApoA-I or rHDL, compared to formation in buffer. A retarding effect, as observed in CSF, could be replicated using a simpler system, from key components present in CSF but purified from a CSF-free host. However, the effect of CSF is stronger implying the presence of additional retarding factors.

#6078 MODULATION OF TRYPTOPHAN METABOLISM IN SEPSIS-INDUCED ACUTE KIDNEY INJURY (AKI): A NEW THERAPEUTIC APPROACH TO PREVENT NEUROCOGNITIVE IMPAIRMENT

Abstract Background and Aims Partial recovery following AKI could lead to long-term consequences that predispose to chronic dysfunction and may also accelerate progression of neurocognitive impairment. In this scenario, a tight crosstalk between kidney and brain named “kidney-brain axis” seems to play a pivotal role leading to detrimental outcome for AKI patients. Notably, many studies suggest a high relationship between kidney damage and brain dysfunction (cognitive impairment, taste and olfactory dysfunction, peripheral nerve dysfunction) even after the resolution of AKI. Furthermore, patients who suffered AKI may afterward show a disturbance of arousal, also called “brain fog”. Although major advances have been made in our understanding of the pathophysiology of AKI and brain dysfunction, there are no available preventive and therapeutic strategies in this field. Recent findings have revealed remarkable link existing between dyslipidemia/low High-density lipoprotein (HDL) levels and Kynurenine pathway (KP) alterations that lead to the production of neuroactive metabolites such as kynurenine (KYN) and quinolinic acid (QA) in AKI setting. Method Sepsis-induced AKI (SI-AKI) was induced in a porcine model by intravenous infusion of a saline solution containing 300 μg/kg of LPS. After LPS injection, 12 animals were treated with different doses of recombinant HDL (rHDL) (20–40 mg/kg) (rHDL group), while 6 animals did not receive treatment (LPS group). Animals were sacrificed after 24h from the start of experimental procedure. Results Endotoxemic pigs developed oliguric AKI with increased tubular and glomerular damage and interstitial inflammatory infiltrate. We found that rHDL treatment significantly decreased the inflammatory process and tubular damage, preventing AKI, especially in the rHDL 40 mg/kg group. Then, we evaluated the Indolamine-2,3-dioxygenase 1 (IDO1) enzyme activation, which is the first and rate-limiting step of KP, upregulated during inflammation in both sera and brain tissue, and has been linked to cognitive dysfunction. In our model, LPS induced an increased activation of the IDO-1 gene expression at brain level in endotoxemic animals, meanwhile it appears to be reduced in both treated arms (p<0.005). Furthermore, sera from the rHDL group showed a significant reduction in IDO1 activity (KYN/Trp ratio) (p<0.05) and QA levels (p<0.005) compared with the LPS group. Moreover, a significant decrease of both systemic and brain IL-6 levels was observed after rHDL treatments. Conclusion Our preliminary data indicated that HDL-enhancing therapies may decrease the inflammatory response, the retention of waste products and neuroactive compounds, improving renal and cognitive function in SI-AKI.

Correction to “Recombinant High-Density Lipoprotein Boosts the Therapeutic Efficacy of Mild Hypothermia in Traumatic Brain Injury”

Correction to “Recombinant High-Density Lipoprotein Boosts the Therapeutic Efficacy of Mild Hypothermia in Traumatic Brain Injury”

Steroid-loaded reconstituted high-density lipoprotein nanocarrier: A new treatment for systemic lupus erythematosus.

Glucocorticoids (GCs) are the most effective and commonly used drugs for the treatment of systemic lupus erythematosus (SLE). However, a large number of side effects occur after long-term or high-dose glucocorticoid treatment, which severely restricts the use of glucocorticoids. Reconstituted high-density lipoprotein (rHDL), an emerging nanocarrier, is promising for targeted delivery to sites of inflammation and macrophages. Here, we prepared a steroid-loaded recombinant high-density lipoprotein and evaluated its therapeutic efficacy in a murine macrophage cell line (RAW264.7) and a lupus (MRL/lpr mice) mouse model. The obtained corticosteroid-loaded nanomedicine, named PLP-CaP-rHDL, exhibited desirable characteristics. Pharmacodynamics studies revealed that the nanoparticles could significantly reduce the levels of inflammatory cytokines in the macrophages in vitro and also effectively alleviate lupus nephritis in MRL/lpr mice without causing obvious side effects at a dose of 0.25mg/kg. Thus, our newly developed steroid-loaded rHDL nanocarriers hold a great potential for anti-inflammatory therapy with reduced side effects and may provide a precise targeted therapy for SLE.

High-density lipoproteins: from quantitative measures to functional assessment and therapy (review of literature).

The antiatherogenic role of high-density lipoproteins (HDL) is associated primarily with their participation in the reverse transport of excess cholesterol from peripheral tissues to the liver. The efficiency of this mechanism depends on the ability of apolipoprotein A-I (apoA-I), the main protein component of HDL, to capture cholesterol from cells. It is known that the acceptor properties of this protein can change under the influence of various factors. This review discusses modern approaches aimed both at increasing the plasma level of HDL and preserving their native functional properties. As one of the key criteria of HDL functionality it is proposed to determine the ability of HDL to accept labeled cholesterol from macrophages. Studies have shown that injection of recombinant HDL or apoA-I mimetic peptides accelerates cholesterol efflux from peripheral tissues, improves vascular endothelial state, and leads to regression of atherosclerotic plaque. Thus, therapy with recombinant HDL/apoA-I may become an effective way to treat cardiovascular diseases caused by cholesterol accumulation in the vascular wall.

Salinomycin-Loaded High-Density Lipoprotein Exerts Promising Anti-Ovarian Cancer Effects by Inhibiting Epithelial-Mesenchymal Transition.

BackgroundEffective treatments for ovarian cancer remain elusive, and survival rates have long been considered grim. Ovarian cancer stem cells (OCSCs) and epithelial–mesenchymal transition (EMT) are associated with cancer progression and metastasis, as well as drug resistance and eventual treatment failure. Salinomycin (Sal) has an extensive effect on a variety of cancer stem cells (CSCs); however, its poor water solubility and toxicity to healthy tissues at high doses limit further research into its potential as an anti-cancer drug. We proposed a therapeutic strategy by constructing a tumor-targeting carrier that mimics high-density lipoprotein (HDL) to synthesize salinomycin-loaded high-density lipoprotein (S-HDL). This strategy helps reduce the side effects of salinomycin, thereby improving its clinical benefits.MethodsOCSCs were isolated from ovarian cancer cells (OCCs) and the uptake of HDL nanoparticles was observed using laser confocal microscopes. After the cell viability analysis revealed the inhibitory effect of S-HDL on OCCs and OCSCs, the main biological processes influenced by S-HDL were predicted with a transcriptome sequencing analysis and verified in vitro and in vivo.ResultsCellular uptake analysis showed that the HDL delivery system was able to significantly enhance the uptake of Sal by OCCs, tentatively validating the targeting role of recombinant HDL, so that S-HDL could reduce the toxicity of Sal and increase its anti-ovarian cancer effects. Conversely, S-HDL could exert anti-ovarian cancer effects by inhibiting the proliferation of OCCs and OCSCs, promoting apoptosis, blocking EMT, and suppressing stemness and angiogenesis-related protein expression in vitro and in vivo.ConclusionS-HDL had stronger anti-ovarian cancer effects than unencapsulated Sal. Thus, it may be a potential agent for ovarian cancer treatment in the future.

Macrophage membrane-camouflaged lipoprotein nanoparticles for effective obesity treatment based on a sustainable self-reinforcement strategy

Modern lifestyle has led to an increase in the incidence of obesity as a public health concern; however, current anti-obesity medications often show limited efficacy with severe side effects. Therapeutic drugs that are selectively delivered to adipose tissue and accelerate energy consumption are promising strategies to overcome the limitations of existing anti-obesity treatment approaches. Herein, a drug delivery platform based on a macrophage cell membrane (Ma)-camouflaged recombinant high-density lipoprotein (rHDL) that was further decorated with a P3 peptide was fabricated to realize targeted drug delivery to adipose tissue. By co-delivering rosiglitazone (Rosi), a peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist, and sildenafil (Sild), a phosphodiesterase type 5 (PDE5) inhibitor, a synergistic therapeutic outcome was achieved in the regulation of diet-induced obesity in a mice model. Body weight reduction and the metabolic status of obese mice were significantly improved after 28 days of treatment. More importantly, a sustainable self-reinforcement effect in multidose therapy was found after using this delivery system. The continuous treatment increased prohibitin (PHB) expression and capillary density in adipose tissue, which in turn improved the accumulation of the drugs in subsequent administration. Taken together, this constructed drug delivery system showed high effectiveness with good safety by combining two anti-obesity therapeutic agents, which exhibits promising research potential for adipose-targeted delivery. Statement of significanceTherapeutic strategies that directly target adipose tissue to increase energy consumption and regulate metabolism are promising but challenging. Herein, an adipose tissue-targeted delivery system was developed using a reconstituted high-density lipoprotein (rHDL) coated by a P3 peptide-decorated macrophage membrane. For the first time, we combined rosiglitazone (Rosi) and sildenafil (Sild) in the system and achieved synergy of adipose browning and angiogenesis for anti-obesity treatment. The therapy induced prohibitin expression and angiogenesis, which improved drug accumulation in adipose tissue in subsequent administrations. This resulted in a sustainable self-reinforcement effect with improved capacity for diet-induced obesity regulation. This study highlights the combination of adipose browning and angiogenesis in anti-obesity treatment and provides an innovative concept of enhancing adipose-targeted delivery.

Recombinant High-Density Lipoprotein Boosts the Therapeutic Efficacy of Mild Hypothermia in Traumatic Brain Injury.

Traumatic brain injury (TBI) leads to neuropsychiatric symptoms and increased risk of neurodegenerative disorders. Mild hypothermia is commonly used in patients suffering from severe TBI. However, its effect for long-term protection is limited, mostly because of its insufficient anti-inflammatory and neuroprotective efficacy and restricted treatment duration. Recombinant high-density lipoprotein (rHDL), which possesses anti-inflammatory and antioxidant activity and blood-brain barrier (BBB) permeability, was expected to potentially strengthen the therapeutic effect of mild hypothermia in TBI treatment. To test this hypothesis and optimize the regimen for combination therapy, the efficacy of mild hypothermia plus concurrent or sequential rHDL on oxidative stress, inflammatory reaction, and cell survival in the damaged brain cells was evaluated. It was found that the effect of combining mild hypothermia with concurrent rHDL was modest, as mild hypothermia inhibited the cellular uptake and lesion-site-targeting delivery of rHDL. In contrast, the combination of mild hypothermia with sequential rHDL more powerfully improved the anti-inflammatory and antioxidant activities, promoted nerve cell survival and BBB restoration, and ameliorated neurologic changes, which thus remarkably restored the spatial learning and memory ability of TBI mice. Collectively, these findings suggest that rHDL may serve as a novel nanomedicine for adjunctive therapy of TBI and highlight the importance of timing of combination therapy for optimal treatment outcome.

Anchoring β-CD on simvastatin-loaded rHDL for selective cholesterol crystals dissolution and enhanced anti-inflammatory effects in macrophage/foam cells

Macrophage/foam cells and cholesterol crystals (CCs) have been regarded as the central triggers of maladaptive inflammation in atherosclerotic plaque. Despite the tremendous progress of recombinant high-density lipoprotein (rHDL) serving for targeted drug delivery to alleviate inflammation in macrophage/foam cells, the active attempt to modulate/improve its CCs dissolution capacity remains poorly explored. The untreated CCs can seriously aggravate inflammation and threaten plaque stability. Based on the superb ability of β-cyclodextrin (β-CD) to bind CCs and promote cholesterol efflux, simvastatin-loaded discoidal-rHDL (ST-d-rHDL) anchored with β-CD (βCD-ST-d-rHDL) was constructed. We verified that βCD-ST-d-rHDL specifically bound and dissolved CCs extracellularly and intracellularly. Furthermore, anchoring β-CD onto the surface of ST-d-rHDL enhanced its cholesterol removal ability in RAW 264.7 cell-derived foam cells characterized by accelerated cholesterol efflux, reduced intracellular lipid deposition, and improved cell membrane fluidity/permeability. Finally, βCD-ST-d-rHDL exerted efficient drug delivery and effective anti-inflammatory effects in macrophage/foam cells. Collectively, anchoring β-CD onto the surface of ST-d-rHDL for selective CCs dissolution, accelerated cholesterol efflux, and improved drug delivery represents an effective strategy to enhance anti-inflammatory effects for the therapy of atherosclerosis.

Reactive oxygen species (ROS)-responsive size-reducible nanoassemblies for deeper atherosclerotic plaque penetration and enhanced macrophage-targeted drug delivery.

Nanoparticle-based therapeutics represent potential strategies for treating atherosclerosis; however, the complex plaque microenvironment poses a barrier for nanoparticles to target the dysfunctional cells. Here, we report reactive oxygen species (ROS)-responsive and size-reducible nanoassemblies, formed by multivalent host-guest interactions between β-cyclodextrins (β-CD)-anchored discoidal recombinant high-density lipoprotein (NP3ST) and hyaluronic acid-ferrocene (HA-Fc) conjugates. The HA-Fc/NP3ST nanoassemblies have extended blood circulation time, specifically accumulate in atherosclerotic plaque mediated by the HA receptors CD44 highly expressed in injured endothelium, rapidly disassemble in response to excess ROS in the intimal and release smaller NP3ST, allowing for further plaque penetration, macrophage-targeted cholesterol efflux and drug delivery. In vivo pharmacodynamicses in atherosclerotic mice shows that HA-Fc/NP3ST reduces plaque size by 53%, plaque lipid deposition by 63%, plaque macrophage content by 62% and local inflammatory factor level by 64% compared to the saline group. Meanwhile, HA-Fc/NP3ST alleviates systemic inflammation characterized by reduced serum inflammatory factor levels. Collectively, HA-Fc/NP3ST nanoassemblies with ROS-responsive and size-reducible properties exhibit a deeper penetration in atherosclerotic plaque and enhanced macrophage targeting ability, thus exerting effective cholesterol efflux and drug delivery for atherosclerosis therapy.

Dynamically enhancing plaque targeting via a positive feedback loop using multifunctional biomimetic nanoparticles for plaque regression

A paradigm shift from preventive therapy to aggressive plaque regression and eventual eradication is much needed to address increasing atherosclerotic burden and risks. Herein, we report a biologically inspired dual-targeting multifunctional recombinant high-density lipoprotein (rHDL)-mimicking core-shell nanoplatform. It is composed of an ATP-responsive ternary polyplexes core for SR-A siRNA and catalase complexation, and a phosphatidylserine-modified rHDL-based outer shell for SR-BI and CD36 targeting, in which pitavastatin is packaged. We demonstrated that dual-targeting biomimetic core-shell nanoparticles dynamically enhanced macrophage CD36 targeting in the plaques by establishing a positive feedback loop via the reciprocal regulation of SR-A and CD36. Positive feedback-enabled accumulation of the nanoparticles in the atherosclerotic plaques increased by 3.3-fold following 4-week repeated administration. A 3-month dosage regimen of the dual-targeting rHDL-mimicking nanoparticles reduced plaque areas by 65.8%, and decreased macrophages by 57.3%. Collectively, this work shows that dynamically enhancing plaque targeting via a positive feedback loop and dual action of cholesterol deposition inhibition and efflux enhancement accomplished with our novel multifunctional biomimetic nanoparticles provides a new way to regress plaques and alleviate the atherosclerotic burden.

Function of different proportions of apolipoprotein A-I cysteine mutants and apolipoprotein A-V on recombinant high-density lipoproteins in vitro.

To explore the anti-atherosclerotic effects of recombinant high-density lipoproteins (rHDL) of apolipoprotein AI wild-type (apoA-Iwt), apolipoprotein AI Milano (apoA-IM), apolipoprotein AI (N74C) (apoA-I (N74C) )and apolipoprotein AV (apoA-V). We constructed rHDL liposomes (rHDLs), which included apoA-Iwt, apoA-IM, and apoA-I (N74C), followed by the synthesis of rHDLs, with the indicated ratios of apoA-Iwt, apoA-IM, apoA-I (N74C) and apoA-V. We investigated the anti-atherosclerotic effects by experiments including the DMPC clearance assay and experiments that assessed the in vitro antioxidation against low-density lipoprotein, the cellular uptake of oxidized low-density lipoprotein (oxLDL) and the in vitro intracellular lipid accumulation. Electron microscopy results revealed that as more apoA-V was present in rHDLs, the particle size of rHDLs was larger. The DMPC clearance assay subsequently showed that rHDL protein mixtures could promote DMPC turbidity clearance when more apoA-V was included in the reaction mixtures, with apoAV-rHDL showing the strongest turbidity clearance ability (P<0.05 vs AI-rHDL). In vitro antioxidation against low-density lipoprotein assays indicated that rHDLs containing apoA-V had increasing oxidation resistance against low-density lipoprotein (LDL) with higher apoA-V contents. Finally, cellular uptake of oxLDL and intracellular lipids suggested an apparent oxidation resistance to LDL oxidation in vitro and a reduced intracellular lipid accumulation in THP-1-derived macrophages, with AIM-rHDL demonstrating the greatest ability to decrease intracellular lipid accumulation. Different proportions of apolipoprotein A-I cysteine mutants and apolipoprotein A-V of rHDL changed the lipid binding capacity, particle size, and antioxidant capacity. These changes may show a beneficial effect of rHDL on atherosclerosis.

The structure of apoA‐II on HDL reveals novel insights into its regulation of lipoprotein composition and function

High‐density lipoproteins (HDL) are compositionally and functionally diverse. HDL can host at least 95 different proteins and studies suggest many of these interactions are governed by specific conformations of HDL's primary scaffold protein, apolipoprotein (apo)A‐I. Previously, we showed that the presence of HDL's second most abundant scaffold protein, apoA‐II, impacts particle composition and function; presumably through interactions with apoA‐I. The goal of this study was to determine the structural conformation of apoA‐II on HDL to understand how its presence impacts HDL metabolism.We used chemical cross‐linking and mass spectrometry to derive spatial information on homogenous populations of recombinant HDL (rHDL) generated in vitro. The rHDL was created using a mixture of wild‐type and isotopically‐labeled apoA‐I to unambiguously identify the molecular span of the cross‐linked peptides; i.e. within a molecule of apoA‐I (intramolecular) or between two molecules of apoA‐I (intermolecular). Two rHDL populations were studied: (i) rHDL that contained only apoA‐I (LpA‐I) and (ii) rHDL containing both apoA‐I and apoA‐II (LpA‐I/A‐II).Cross‐linking and SDS‐PAGE showed both populations contained two molecules of apoA‐I while the LpA‐I/A‐II rHDL contained a single dimer of apoA‐II. Analysis by negative stain electron microscopy revealed both populations were discoidal in shape with no differences in size. The intermolecular cross‐linking pattern between apoA‐I molecules were consistent with the “double‐belt” model of apoA‐I on HDL and, surprisingly, the presence of apoA‐II didn't appear to perturb the pattern. However, we observed strong cross‐links between apoA‐II and the C‐terminus of apoA‐I.In a parallel approach, native LpA‐I and LpA‐I/A‐II HDL was isolated from human plasma using immunoaffinity and size exclusion chromatography. Shotgun proteomics and cross‐linking revealed accessory proteins were mostly associated with LpA‐I HDL and interactions were primarily localized to the N‐ and C‐termini of apoA‐I. As with the rHDL, we found apoA‐II specifically interacts with the C‐terminus of apoA‐I in the LpA‐I/A‐II particles. Taken together, this suggests that apoA‐II's compositional and functional effects on HDL are exerted via its interaction with the C‐terminus of apoA‐I possibly limiting its availability to interact with the accessory proteins. We have gone on to generate preliminary all‐atoms models of apoA‐II on HDL to illustrate this structural mechanism.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of the Combined Effect of Recombinant High-Density Lipoprotein Carrier and the Encapsulated Lovastatin in RAW264.7 Macrophage Cells Based on the Median-Effect Principle.

Recombinant high-density lipoprotein (rHDL) displays a similar anti-atherosclerotic effect with native HDL and could also be served as a carrier of cardiovascular drug for atherosclerotic plaque targeting. In our previous studies, rHDL has shown a more potent anti-atherosclerotic efficacy as compared to the other conventional nanoparticles with a payload of lovastatin (LS). Therefore, we hypothesized that a synergistic anti-atherosclerotic effect of the rHDL carrier and the encapsulated LS might exist. In this study, the dose-effect relationships and the combined effect of the rHDL and LS were quantitatively evaluated in RAW 264.7 macrophage cells using the median-effect analysis, in which the rHDL carrier was regarded as a drug combined. Median-effect analysis suggested that rHDL and LS exerted a desirable synergistic inhibition on the oxLDL internalization at a ratio of 6:1 ( Dm,LS: Dm,rHDL) in RAW 264.7 macrophage cells. About 50% of the reduction on the intracellular lipid contents was found when RAW264.7 cells were treated with LS-loaded rHDLs at their respective median-effect dose ( Dm) concentrations and a synergistic effect on the mediating cholesterol efflux was also observed, which verified the accuracy of the results obtained from the median-effect analysis. The mechanism underlying the synergistic effect of the rHDL carrier and the drug might be attributed to their potent inhibitory effects on SR-A expression. In conclusion, the median-effect analysis was proven to be a feasible method to quantitatively evaluate the synergistic effect of the biofunctional carrier and the drug encapsulated.

Targeting CD40-Induced TRAF6 Signaling in Macrophages Reduces Atherosclerosis

BackgroundDisrupting the costimulatory CD40-CD40L dyad reduces atherosclerosis, but can result in immune suppression. The authors recently identified small molecule inhibitors that block the interaction between CD40 and tumor necrosis factor receptor-associated factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity. ObjectivesThis study evaluates the potential of TRAF-STOP treatment in atherosclerosis. MethodsThe effects of TRAF-STOPs on atherosclerosis were investigated in apolipoprotein E deficient (Apoe−/−) mice. Recombinant high-density lipoprotein (rHDL) nanoparticles were used to target TRAF-STOPs to macrophages. ResultsTRAF-STOP treatment of young Apoe−/− mice reduced atherosclerosis by reducing CD40 and integrin expression in classical monocytes, thereby hampering monocyte recruitment. When Apoe−/− mice with established atherosclerosis were treated with TRAF-STOPs, plaque progression was halted, and plaques contained an increase in collagen, developed small necrotic cores, and contained only a few immune cells. TRAF-STOP treatment did not impair “classical” immune pathways of CD40, including T-cell proliferation and costimulation, Ig isotype switching, or germinal center formation, but reduced CD40 and β2-integrin expression in inflammatory monocytes. In vitro testing and transcriptional profiling showed that TRAF-STOPs are effective in reducing macrophage migration and activation, which could be attributed to reduced phosphorylation of signaling intermediates of the canonical NF-κB pathway. To target TRAF-STOPs specifically to macrophages, TRAF-STOP 6877002 was incorporated into rHDL nanoparticles. Six weeks of rHDL-6877002 treatment attenuated the initiation of atherosclerosis in Apoe−/− mice. ConclusionsTRAF-STOPs can overcome the current limitations of long-term CD40 inhibition in atherosclerosis and have the potential to become a future therapeutic for atherosclerosis.