Native HDL Research Articles

Abstract 20918: Cytokines Involved in Arterial Wall Inflammation Are Transported by High Density Lipoprotein Particles

Introduction: Inflammation plays an important role in the induction of CAD and the progression to deadly coronary syndromes. Cytokines are key mediators of inflammation, both in pro- and anti-inflammatory capacities. HDL has a vital role in the maintenance of cardiovascular health, and recent discoveries highlight the diversity of HDL functions to include the transport of lipid signaling molecules, microRNAs, and other effectors of metabolic regulation. Given the importance of HDL in transporting molecules involved in cardiovascular health, we questioned if HDL has a direct role in transporting cytokines and effecting inflammatory responses in CAD. Hypothesis: HDL plays an important role in the transport of cytokines involved in CAD. Methods: To explore the role of HDL in arterial inflammation, we profiled the abundance of 35 cytokines in plasma and HDL of men and women with and without CAD. We analyzed whole plasma and native HDL isolated using a selected-affinity immunosorption technique from patients who suffered myocardial infarction at an early age (<60 years, male n=19; female n=20) and control patients (male n=10, female n=10). Samples were assayed by multiplex array technology for quantification of cytokines. Results: Of 35 cytokines analyzed, 25 appear to be significantly transported in HDL. Three cytokines (IL-12, MCP-1, and IL-RA) are excluded from HDL and are only detectable in plasma. IL-3 and IL-7 are predominately associated with HDL and are elevated in both males and females with MI. There are differences in several HDL-associated cytokines that appear to be sex-dependent. Conclusions: We have discovered that a number of cytokines are transported in HDL complexes. Significant elevations or decreases in a number of these cytokines are observed in patients with CAD, and vary in abundance between males and females.

Dysfunctional high-density lipoprotein from HIV+ individuals promotes monocyte-derived foam cell formation in vitro.

The role of high-density lipoprotein (HDL) function in HIV-related atherosclerotic cardiovascular disease (CVD) is unclear. HDLs isolated from HIV [HIV(+)HDL] and HIV-uninfected individuals [HIV(-)HDL] were assessed for HDL function and ability to promote monocyte-derived foam cell formation (MDFCF; a key event in HIV-related CVD) ex vivo. Using an established in-vitro model of atherogenesis and plasma samples from an established cross-sectional study of virologically suppressed HIV men on stable effective antiretroviral therapy and with low CVD risk (median age: 42 years; n = 10), we explored the impact of native HDL [HIV(+)HDL] on MDFCF. In this exploratory study, we selected HIV(+)HDL known to be dysfunctional based on two independent measures of impaired HDL function: antioxidant (high HDLox) ability of HDL to release apolipoprotein A-I (ApoA-I) (low HDL-ApoA-I exchange). Five healthy men matched by age and race to the HIV group were included. Given that oxidation of HDL leads to abnormal HDL function, we also compared proatherogenic effects of HIV(+)HDL vs. chemically derived HDLox. The ex-vivo atherogenesis assay was performed using lipoproteins (purchased or isolated from plasma using ultracentrifugation) and monocytes purified via negative selection from healthy donors. HIV(+)HDL known to have reduced antioxidant function and rate of HDL/ApoAI exchange promoted MDFCF to a greater extent than HDL (33.0 vs. 26.2% foam cells; P = 0.015). HDL oxidized in vitro also enhanced foam cell formation as compared with nonoxidized HDL (P < 0.01). Dysfunctional HDL in virologically suppressed HIV individuals may potentiate atherosclerosis in HIV infection by promoting MDFCF.The role of HDL function in HIV-related atherosclerotic CVD is unclear. HDL isolated from HIV [HIV(+)HDL] and HIV-uninfected individuals [HIV(-)HDL] were assessed for HDL function and ability to promote foam cell formation ex vivo. HIV(+)HDL known to have reduced antioxidant function and rate of HDL/ApoA1 exchange promoted MDFCF to a greater extent than HDL(-)HDL (33.0 vs. 26.2% foam cells.Subject codes: Inflammation, Lipids and Cholesterol, Vascular Biology, Oxidant Stress, Atherosclerosis.

High-density lipoproteins attenuate high glucose-impaired endothelial cell signaling and functions: potential implications for improved vascular repair in diabetes

BackgroundAbnormalities of endothelial cell function are proposed to be a critical factor underlying adverse cardiovascular outcomes in the setting of hyperglycaemia. While high-density lipoproteins (HDL) have been demonstrated to be cardioprotective, the impact on the endothelium in hyperglycaemia has not been fully elucidated.MethodsHuman umbilical vein endothelial cells (HUVECs) were exposed to high-glucose conditions using dextrose, the main isoform of glucose, and native HDL. HUVEC proliferation and migration were determined. The key signalling pathways that regulate endothelial cell function were also characterized.ResultsIncreasing concentrations of dextrose resulted in significant reductions in HUVEC proliferation, this was attenuated by coincubation with HDL. In support of this, HDL was also found to rescue dextrose impaired expression of PCNA and the activation (phosphorylation) of the key transcription factor for proliferation ERK. Dextrose also dose-dependently inhibited HUVEC migration, which was mitigated by co-incubation with HDL. Consistent with this, HDL prevented dextrose-induced inhibition of p38 phosphorylation, responsible for cell migration. Finally, phosphorylation of the pro-survival transcription factor Akt was dose-dependently inhibited by dextrose, however, this was completely rescued by co-administration with HDL.ConclusionDextrose-induced hyperglycaemia causes the impairment of endothelial cell proliferation and migration and inhibits the activation of ERK, p38 and Akt pathways. The protective effects of HDL in this milieu highlights the potential for HDL to improve vascular repair in patients with impaired glucose homeostasis.

Rerouting Native HDL to Predetermined Receptors for Improved Tumor-Targeted Gene Silencing Therapy.

High-density lipoprotein (HDL) is an outstanding biocompatible nanovector for tumor-targeted delivery of multimodel drugs in cancer therapy. However, this seemingly promising delivery platform demonstrates an adverse accumulation in liver and adrenal due to the primary expression of natural target scavenger receptor class B type I (SR-BI), which overexpressed in malignant cells as well. Therefore, we endowed native HDLs with rerouting capacity, that is, enabling HDLs to get away from natural receptors (SR-BI) to selectively alternate tumor-rich receptors. The αvβ3-integrin specific cyclic-RGDyk peptide was conjugated with HDL-protein component apolipoprotein A-I (apoA-I), demonstrating high substitution degree of 26.2%. Afterward, RGD-modified apoA-I was introduced to fabricate cholesterol siRNA-loaded HDL nanoparticles (RGD-HDL/Ch-siRNA) for specific affinity with tumor angiogenesis and αvβ3 integrin on tumor surface. After preparation, RGD-HDL/Ch-siRNA shared desirable particle size, efficient siRNA protection during blood circulation, and favorable proton sponge effect. αvβ3 integrin-associated superior rerouting capacity, endocytosis pathway, and rapid endolysosome escape were confirmed both in vitro and in vivo. For targeted gene silencing therapy, Pokemon-specific siRNA (siPokemon) was introduced as RNA interference candidate; the enhanced antitumor efficacy and decreased Pokemon expression level were commendably confirmed by tumor growth inhibition, survival period extension, and western blot analysis. Collectively, cyclic-RGDyk modification endows native HDLs with rerouting capacity to specific αvβ3 integrin receptor, which provides a promising strategy to extend malignancy targeting potential of native HDL to a broader purview.

Secretory phospholipase A2 modified HDL rapidly and potently suppresses platelet activation

Levels of secretory phospholipases A2 (sPLA2) highly increase under acute and chronic inflammatory conditions. sPLA2 is mainly associated with high-density lipoproteins (HDL) and generates bioactive lysophospholipids implicated in acute and chronic inflammatory processes. Unexpectedly, pharmacological inhibition of sPLA2 in patients with acute coronary syndrome was associated with an increased risk of myocardial infarction and stroke. Given that platelets are key players in thrombosis and inflammation, we hypothesized that sPLA2-induced hydrolysis of HDL-associated phospholipids (sPLA2-HDL) generates modified HDL particles that affect platelet function. We observed that sPLA2-HDL potently and rapidly inhibited platelet aggregation induced by several agonists, P-selectin expression, GPIIb/IIIa activation and superoxide production, whereas native HDL showed little effects. sPLA2-HDL suppressed the agonist-induced rise of intracellular Ca2+ levels and phosphorylation of Akt and ERK1/2, which trigger key steps in promoting platelet activation. Importantly, sPLA2 in the absence of HDL showed no effects, whereas enrichment of HDL with lysophosphatidylcholines containing saturated fatty acids (the main sPLA2 products) mimicked sPLA2-HDL activities. Our findings suggest that sPLA2 generates lysophosphatidylcholine-enriched HDL particles that modulate platelet function under inflammatory conditions.

Glycation of high-density lipoprotein triggers oxidative stress and promotes the proliferation and migration of vascular smooth muscle cells.

BackgroundIn type 2 diabetes mellitus (T2DM), high-density lipoprotein (HDL) impairs its anti-atherogenic properties and even develops to a pro-inflammatory and pro-atherogenic phenotype because of abnormal compositions and modifications. In this study, we examined the effects and the related mechanisms of glycation of HDL on the proliferation and migration of vascular smooth muscle cells (VSMCs).Methods & ResultsGlycated HDL (G-HDL) was modified with D-glucose (25 mmol/L) in vitro. Diabetic HDL (D-HDL) was isolated from T2DM patients. Rat VSMCs were isolated from the thoracic aortas. Human VSMCs were obtained from ScienCell Research Laboratories. Alpha-actin was detected through immunofluorescence. VSMC proliferation was assayed by Cell Count. VSMC migration was determined by transwell chamber and scratch-wound assay. Intracellular reactive oxygen species (ROS) was detected based on ROS-mediated 2′,7′-dichlorofluorescein (DCFH-DA) fluorescence. Compared to native HDL (N-HDL), G-HDL remarkably promoted VSMC proliferation and migration in the dose and time-dependent manners. In addition, G-HDL enhanced ROS generation in VSMCs. However, the ROS scavenger, N-acetylcysteine, efficiently decreased ROS production and subsequently inhibited the proliferation of VSMCs induced by G-HDL. Similarly, D-HDL from T2DM patients also promoted ROS release and VSMC proliferation and migration.ConclusionsHDL either glycated in vitro or isolated from T2DM patients triggered VSMC proliferation, migration, and oxidative stress. These results might partly interpret the higher morbidity of cardiovascular disease in T2DM patients.

Abstract 397: Aldehyde-Modified Hdl Has CD36-dependent Pro-Atherogenic Effects in Macrophages

The role of high density lipoproteins (HDL) in protecting against cardiovascular disease is compromised when HDL undergoes modification during conditions of oxidative stress; however, the mechanisms underlying these changes in HDL function are not well defined. Reactive aldehydes such as acrolein (a major component in cigarette smoke) or major products of lipid peroxidation such as 4-hydroxynonenal (HNE) or malondialdehyde (MDA) are known to oxidize HDL in cardiovascular disease. To test the hypothesis that modification of HDL with aldehydes impairs HDL’s athero-protective functions in macrophages, we first measured the ability of modified HDL to protect against foam cell formation. Cholesterol-loaded peritoneal macrophages isolated from wild-type C57Bl/J mice were incubated with native HDL, acrolein-modified HDL (acro-HDL), HNE-modified HDL (HNE-HDL) or MDA-modified HDL (MDA-HDL) for 24 h. Contrary to native HDL, oxidized forms of HDL were unable to prevent foam cell formation as shown by increased Oil red-O staining. Next, using a Boyden chamber assay, we demonstrated that acro- and MDA-HDL had impaired abilities to promote macrophage migration (64% and 67% of native HDL cell migration, respectively). Finally, using a secreted alkaline phosphatase reporter THP-1 cell-based assay, we determined that acro-HDL promotes activation of the pro-inflammatory NFkappaB pathway. Interestingly, immunoblot and quantitative RT-PCR analyses revealed that incubation of macrophages with acro- and MDA-HDL leads to increased expression of the pro-atherogenic receptor, cluster of differentiation 36 (CD36). Therefore, we repeated the foam cell formation and migration experiments using similar ligands, but this time, in CD36-null peritoneal macrophages. We found that both of these functions were dependent on CD36; however, the extent of the functional changes varied based on the type of oxidative modification present on HDL. In conclusion, modification of HDL with reactive aldehydes generates a particle that has pro-atherogenic effects in macrophages, many of which are dependent on CD36.

Computational and Experimental Studies of Gold Nanoparticle Templated HDL-Like Nanoparticles for Cholesterol Metabolism

High-density lipoprotein (HDL) is involved in the transport and metabolism of phospholipids, triglycerides, and cholesterol. Mimics of HDL are being explored as potential therapeutic agents for removing excess cholesterol from arterial plaques. Gold nanoparticles (AuNPs) functionalized with apolipoprotein A-I (apoA-I), and with the lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (PDP PE) have been demonstrated to be acceptors of cellular cholesterol. However, detailed structural properties of this functionalized HDL AuNP are not well understood. We combined coarse-grained and all-atom MD simulations to simulate the self-assembly of lipids and cholesteryl ester on the AuNP/apoA-I construct to gain insights into its structural properties, and further make comparisons with experimental results. We find that lipids are oriented differently in regions with and without apoA-I. We also find that in this functionalized HDL AuNP, the distribution of cholesteryl ester maintains a reverse concentration gradient that is similar to the gradient found in native HDL. Incubating the HDL AuNP with cholesterol and lecithin:cholesterol acyltransferase (LCAT) demonstrate the HDL AuNP is able to efficiently activate LCAT and form esterified cholesterol from free cholesterol and phospholipid.

Myeloperoxidase-oxidized high density lipoprotein impairs atherosclerotic plaque stability by inhibiting smooth muscle cell migration

BackgroundHigh density lipoprotein (HDL) has been proved to be a protective factor for coronary heart disease. Notably, HDL in atherosclerotic plaques can be nitrated (NO2-oxHDL) and chlorinated (Cl-oxHDL) by myeloperoxidase (MPO), likely compromising its cardiovascular protective effects.MethodHere we determined the effects of NO2-oxHDL and Cl-oxHDL on SMC migration using wound healing and transwell assays, proliferation using MTT and BrdU assays, and apoptosis using Annexin-V assay in vitro, as well as on atherosclerotic plaque stability in vivo using a coratid artery collar implantation mice model.ResultsOur results showed that native HDL promoted SMC proliferation and migration, whereas NO2-oxHDL and Cl-oxHDL inhibited SMC migration and reduced capacity of stimulating SMC proliferation as well as migration, respectively. OxHDL had no significant influence on SMC apoptosis. In addition, we found that ERK1/2-phosphorylation was significantly lower when SMCs were incubated with NO2-oxHDL and Cl-oxHDL. Furthermore, transwell experiments showed that differences between native HDL, NO2-oxHDL and Cl-oxHDL was abolished after PD98059 (MAPK kinase inhibitor) treatment. In aortic SMCs from scavenger receptor BI (SR-BI) deficient mice, differences between migration of native HDL, NO2-oxHDL and Cl-oxHDL treated SMCs vanished, indicating SR-BI’s possible role in HDL-associated SMC migration. Importantly, NO2-oxHDL and Cl-oxHDL induced neointima formation and reduced SMC positive staining cells in atherosclerotic plaque, resulting in elevated vulnerable index of atherosclerotic plaque.ConclusionThese findings implicate MPO-catalyzed oxidization of HDL may contribute to atherosclerotic plaque instability by inhibiting SMC proliferation and migration through MAPK-ERK pathway which was dependent on SR-BI.

Molecular Dynamics Simulation and Experimental Studies of Gold Nanoparticle Templated HDL-like Nanoparticles for Cholesterol Metabolism Therapeutics

High-density lipoprotein (HDL) plays an important role in the transport and metabolism of cholesterol. Mimics of HDL are being explored as potentially powerful therapeutic agents for removing excess cholesterol from arterial plaques. Gold nanoparticles (AuNPs) functionalized with apolipoprotein A-I and with the lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] have been demonstrated to be robust acceptors of cellular cholesterol. However, detailed structural information about this functionalized HDL AuNP is still lacking. In this study, we have used X-ray photoelectron spectroscopy and lecithin/cholesterol acyltransferase activation experiments together with coarse-grained and all-atom molecular dynamics simulations to model the structure and cholesterol uptake properties of the HDL AuNP construct. By simulating different apolipoprotein-loaded AuNPs, we find that lipids are oriented differently in regions with and without apoA-I. We also show that in this functionalized HDL AuNP, the distribution of cholesteryl ester maintains a reverse concentration gradient that is similar to the gradient found in native HDL.

Pericoronary Adipose Tissue as Possible Supply Site and Storage for Lipoproteins in Human Coronary Artery

It is a general belief that low-density lipoprotein (LDL) enters from the lumen into the vessel wall and oxidized (oxLDL) and acts as an important pro-atherogenic role in atherosclerosis and the anti-atherogenic substances such as high-density lipoprotein (HDL) and its component apolipoprotein A1 (ApoA1), also enters from the lumen. However, definite in vivo clinical evidence is lacking. We have demonstrated immunohistochemically that native oxLDL, HDL and ApoA1 co-saved in adipocytes in the majority of human pericoronary adipose tissue (PCAT) samples, and obtained marks that they are conveyed by either CD68(+) macrophages or vasa vasorum to the adjacent coronary. These results recommended the existence of before unrecognized storing and supply site of these proteins and that therapies directing the PCAT could be active in stopping human coronary atherosclerosis.

Human Pericoronary Adipose Tissue as Storage and Possible Supply Site for Lipoproteins in Atherosclerotic Coronary Plaques

Aim of Review: It is generally believed that low-density lipoprotein (LDL) and monocytes enters the vascular wall from the lumen, and the former becomes oxidized LDL (oxLDL) and the latter macrophages and they participate in atherosclerosis However, definite in vivo clinical evidence is lacking. This review article summarizes our findings on the possible roles of oxLDL and other lipoproteins stored in pericoronary adipose tissue (PCAT), which, when thickened, becomes a risk factor for coronary artery disease.Findings: Immunohistochemical staining of PCAT and its adjacent coronary arteries obtained from autopsy subjects revealed that oxLDL, high-density lipoprotein (HDL) and apolipoprotein A1 (ApoA1) co-deposited in adipocytes in the majority of PCAT samples. LDL did not deposit in PCAT. The incidence of oxLDL was low in the intima of normal coronary segments, but increased during the growth stage and decreased during the mature stage of plaques, whereas that of HDL and ApoA1 was increased inthe growth stage and increased further in the mature stage. The incidence of LDL in plaques was low and showed no obvious relation to plaque morphology. OxLDL and ApoA1 deposited in either a dotted or diffuse pattern whereas HDL showed a diffuse pattern in both the PCAT and intima. The dotted pattern occurred when oxLDL or ApoA1 was contained in CD68(+) -macrophages that were observed in the PCAT, adventitia, media and intima. A certain group of CD68(+)-macrophages contained both oxLDL and ApoA1. The CD68(+) -macrophages across the border of the PCAT, external and internal elastic laminae were frequently observed, suggesting their traverse from the PCAT toward the intima. The localization of diffusely deposited oxLDL, HDL and ApoA1 coincided with that of intimal vasa vasorum. LDL was deposited diffusely in the intima but its localization did not necessarily coincide with that of vasa vasorum.Summary: Contrary to the generally believed mechanism, immunohistochemical findings suggested that native oxLDL, HDL and ApoA1 are stored in PCAT and conveyed either by CD68(+) - macrophages or vasa vasorum to coronary plaques. Therefore, therapies targeting the PCAT could be effective in preventing human coronary atherosclerosis.

Oxidized high density lipoprotein induces macrophage apoptosis via toll-like receptor 4-dependent CHOP pathway

Oxidized HDL (ox-HDL), unlike native HDL that exerts antiatherogenic effects, plays a proatherogenic role. However, the underlying mechanisms are not completely understood. This study was designed to explore the inductive effect of ox-HDL on endoplasmic reticulum (ER) stress-CCAAT-enhancer-binding protein homologous protein (CHOP)-mediated macrophage apoptosis and its upstream mechanisms. Our results showed that ox-HDL could be ingested by macrophages, causing intracellular lipid accumulation. As with tunicamycin (an ER stress inducer), ox-HDL induced macrophage apoptosis with concomitant activation of ER stress pathway, including nuclear translocation of activating transcription factor 6, phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α, and upregulation of glucose-regulated protein 78 and CHOP, which were inhibited by 4-phenylbutyric acid (PBA, an ER stress inhibitor) and CHOP gene silencing. Additionally, diphenyleneiodonium (DPI, an oxidative stress inhibitor), probucol (a reactive oxygen species scavenger), and toll-like receptor 4 (TLR4) silencing reduced ox-HDL-induced macrophage apoptosis, oxidative stress, and CHOP upregulation. Moreover, HDL isolated from patients with metabolic syndrome induced macrophage apoptosis, oxidative stress, and CHOP upregulation, which were blocked by PBA and DPI. These data indicate that ox-HDL may activate ER stress-CHOP-induced apoptotic pathway in macrophages via enhanced oxidative stress and that this pathway may be mediated by TLR4.

High-Density Lipoproteins Exert Pro-inflammatory Effects on Macrophages via Passive Cholesterol Depletion and PKC-NF-κB/STAT1-IRF1 Signaling

Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages invitro. These pro-inflammatory effects were confirmed invivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line withthese findings, the innate immune responses required for clearance of P.aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-κB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.

Abstract 13218: Remodeling of High Density Lipoprotein Induced by CSL112: Structure and Function of the Remodeled Lipoproteins

Introduction: CSL112 is a novel formulation of human apolipoprotein A-I (apoA-I) suitable for intravenous infusion, currently in development for reducing the high rate of early recurrent events after acute myocardial infarction. Infusion of CSL112 into healthy volunteers causes HDL remodeling, with an immediate rise in lipid-poor apoA-I and a strong and immediate increase in the ABCA1-dependent cholesterol efflux capacity of plasma. Interaction of CSL112 with whole plasma or purified HDL gives rise to three new remodeled HDL species: large (L-HDL rem ) and small (S-HDL rem ) species, and lipid-poor apoA-I. Here we characterized the composition and morphology of remodeled HDL species and compared their ability to efflux cholesterol via different transporters. Methods and Results: CSL112 and HDL3, fluorescently labeled on protein or lipid, were incubated together for varying periods of time at 37°C. Resultant HDL particle size distribution was assessed by native gel electrophoresis followed by fluorescence imaging. Morphology of the individual purified products of HDL remodeling was examined by negative stain electron microscopy. We show that L-HDL rem is a large, spherical species composed of apoA-I and phospholipid from both native HDL and CSL112. S-HDL rem is a small, disc-shaped species composed of apoA-I from CSL112 that is smaller due to the loss of phospholipids. The smallest species, lipid-poor apoA-I, is composed of apoA-I from HDL and CSL112. Lipid-poor apoA-I and S-HDL rem were found to have the greatest ability to efflux cholesterol via ABCA1. While S-HDL rem and L-HDL rem showed comparable ability to efflux cholesterol via ABCG1, L-HDL rem mediated additional efflux via SR-B1 and by an aqueous diffusion process. Conclusion: The generation of these remodeled HDL species can account for the strong and immediate increase in cholesterol efflux capacity observed upon infusion of CSL112, and make CSL112 a promising approach for rapidly removing cholesterol from atherosclerotic plaque.

Properties of Native High-Density Lipoproteins Inspire Synthesis of Actively Targeted In Vivo siRNA Delivery Vehicles.

Efficient systemic administration of therapeutic short interfering RNA (siRNA) is challenging. High-density lipoproteins (HDL) are natural in vivo RNA delivery vehicles. Specifically, native HDLs: 1) Load single-stranded RNA; 2) Are anionic, which requires charge reconciliation between the RNA and HDL, and 3) Actively target scavenger receptor type B-1 (SR-B1) to deliver RNA. Emphasizing these particular parameters, we employed templated lipoprotein particles (TLP), mimics of spherical HDLs, and self-assembled them with single-stranded complements of, presumably, any highly unmodified siRNA duplex pair after formulation with a cationic lipid. Resulting siRNA templated lipoprotein particles (siRNA-TLP) are anionic and tunable with regard to RNA assembly and function. Data demonstrate that the siRNA-TLPs actively target SR-B1 to potently reduce androgen receptor (AR) and enhancer of zeste homolog 2 (EZH2) proteins in multiple cancer cell lines. Systemic administration of siRNA-TLPs demonstrated no off-target toxicity and significantly reduced the growth of prostate cancer xenografts. Thus, native HDLs inspired the synthesis of a hybrid siRNA delivery vehicle that can modularly load single-stranded RNA complements after charge reconciliation with a cationic lipid, and that function due to active targeting of SR-B1.

Enhanced HDL Functionality in Small HDL Species Produced Upon Remodeling of HDL by Reconstituted HDL, CSL112

Rationale: CSL112, human apolipoprotein A-I (apoA-I) reconstituted with phosphatidylcholine, is known to cause a dramatic rise in small high-density lipoprotein (HDL). Objective: To explore the mechanisms by which the formation of small HDL particles is induced by CSL112. Methods and Results: Infusion of CSL112 into humans caused elevation of 2 small diameter HDL fractions and 1 large diameter fraction. Ex vivo studies showed that this remodeling does not depend on lipid transfer proteins or lipases. Rather, interaction of CSL112 with purified HDL spontaneously gave rise to 3 HDL species: a large, spherical species composed of apoA-I from native HDL and CSL112; a small, disc-shaped species composed of apoA-I from CSL112, but smaller because of the loss of phospholipids; and the smallest species, lipid-poor apoA-I composed of apoA-I from HDL and CSL112. Time-course studies suggest that remodeling occurs by an initial fusion of CSL112 with HDL and subsequent fission leading to the smaller forms. Functional studies showed that ATP-binding cassette transporter 1–dependent cholesterol efflux and anti-inflammatory effects in whole blood were carried by the 2 small species with little activity in the large species. In contrast, the ability to inactivate lipid hydroperoxides in oxidized low-density lipoprotein was carried predominantly by the 2 largest species and was low in lipid-poor apoA-I. Conclusions: We have described a mechanism for the formation of small, highly functional HDL species involving spontaneous fusion of discoidal HDL with spherical HDL and subsequent fission. Similar remodeling is likely to occur during the life cycle of apoA-I in vivo.

Impact of Oxidised Very Low-Density Lipoproteins on Endothelial Cell Viability and Migration: Implications for Vascular Repair

Background: Population studies consistently demonstrate a direct relationship between triglyceride levels and cardiovascular risk. The influence of triglyceride rich lipoproteins on the artery wall have not been well characterised. Here, we investigated the ex vivo effects of very low-density lipoproteins (VLDL) on vascular endothelial cells. Methods: VLDL was extracted from plasma of healthy human subjects by ultracentrifugation and modified by copper induced oxidation or glycation. The impact of co-incubation of native, oxidised and glycated VLDL (0-100 μg/ml) with human umbilical vein endothelial cells (HUVECs) for 12 hours on cellular viability was assessed using a colorimetric WST-1 assay and cellular migration using a scratch assay (50 μg/ml). These experiments were repeated following co-incubation of HUVECs with high-density lipoproteins isolated from healthy human subjects. Results: Oxidised, but not native or glycated VLDL, inhibited HUVECs viability by 15% (p<0.05) at concentrations greater than 50 μg/ml. In a similar fashion, oxidised, but not native or glycated VLDL, reduced HUVECs migration by 28% (p<0.05) compared with untreated HUVECs. Co-incubation of HUVECs with native HDL reduced the impact of oxidised VLDL on HUVEC viability by 21% (p<0.05). Conclusions: Oxidation of VLDL results in impairment of endothelial cell viability and migration, providing an important adverse mechanistic role of triglyceride rich lipoproteins in vascular repair. The ability of co-incubation with HDL to inhibit this effect provides another potential mechanism by which HDL exerts atheroprotective effects.

Biology of High-Density Lipoproteins: An Update49High density lipoproteins exert pro-inflammatory effects on macrophages via passive cholesterol depletion and PKC-NF-kB/STAT1-IRF1 signaling50Homocysteine accelerated the formation of THP-1 macrophages-derived foam cells and cholesterol disorder via regulating the expressions of LXRa, ABCA1 and ABCG151Protein components of HDL as markers of cardiovascular damage in patients with arterial hypertension

Biology of High-Density Lipoproteins: An Update49High density lipoproteins exert pro-inflammatory effects on macrophages via passive cholesterol depletion and PKC-NF-kB/STAT1-IRF1 signaling50Homocysteine accelerated the formation of THP-1 macrophages-derived foam cells and cholesterol disorder via regulating the expressions of LXRa, ABCA1 and ABCG151Protein components of HDL as markers of cardiovascular damage in patients with arterial hypertension

Impact of individual acute phase serum amyloid A isoforms on HDL metabolism in mice

The acute phase (AP) reactant serum amyloid A (SAA), an HDL apolipoprotein, exhibits pro-inflammatory activities, but its physiological function(s) are poorly understood. Functional differences between SAA1.1 and SAA2.1, the two major SAA isoforms, are unclear. Mice deficient in either isoform were used to investigate plasma isoform effects on HDL structure, composition, and apolipoprotein catabolism. Lack of either isoform did not affect the size of HDL, normally enlarged in the AP, and did not significantly change HDL composition. Plasma clearance rates of HDL apolipoproteins were determined using native HDL particles. The fractional clearance rates (FCRs) of apoA-I, apoA-II, and SAA were distinct, indicating that HDL is not cleared as intact particles. The FCRs of SAA1.1 and SAA2.1 in AP mice were similar, suggesting that the selective deposition of SAA1.1 in amyloid plaques is not associated with a difference in the rates of plasma clearance of the isoforms. Although the clearance rate of SAA was reduced in the absence of the HDL receptor, scavenger receptor class B type I (SR-BI), it remained significantly faster compared with that of apoA-I and apoA-II, indicating a relatively minor role of SR-BI in SAA's rapid clearance. These studies enhance our understanding of SAA metabolism and SAA's effects on AP-HDL composition and catabolism.