High-density Lipoprotein Transport Research Articles

PON1 has palmitoyl-protein thioesterase (PPT) activity, and can affect the presence of SR-B1 on the endothelial cell membrane.

The high-density lipoprotein (HDL)-associated enzyme paraoxonase 1 (PON1) is expressed almost exclusively in the liver and is then transported by HDL to the peripheral tissues. The lipophilic nature of PON1 enables its easy exchange between the lipoprotein and cell membranes in a process that is dependent on the HDL receptor scavenger receptor class B, type 1 (SR-B1). In endothelial cells, PON1 binding to the cell membrane leads to its internalization by endocytosis and subsequent lysosomal degradation. PON1 is a "promiscuous" enzyme with unusually broad substrate specificity in vitro, but its actual function and substrate are still unknown. The enzyme requires a lipid environment and becomes completely inactive upon delipidation. However, when PON1 binds HDL, its active site faces the lipoprotein's core and is inaccessible to external substrates. Hence, the HDL-bound PON1 is inactive toward substrates outside the particle's lipid core and is rapidly degraded and becomes inactive upon internalization. Consequently, the enzyme is only active in the cell membrane during its transit from HDL to the cytoplasm. To assign a function to PON1, we investigated whether it is a palmitoyl-protein thioesterase (PPT) that can hydrolyze the palmitoyl moieties of membrane proteins involved in HDL and cholesterol transport, such as SR-B1, ABCA1, or their neighboring caveola proteins to facilitate the release of HDL or trigger its endocytosis. This study shows that PON1 can hydrolyze palmitoyl-cysteine thioester bonds in vitro, has direct or indirect PPT activity in vivo, and can significantly decrease the presence of SR-B1 in the endothelial membrane.

Decoding Functional High-Density Lipoprotein Particle Surfaceome Interactions.

High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport (RCT) and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the molecular level. During HDL’s journey throughout the body, its functions are mediated through interactions with cell surface receptors on different cell types. To characterize and better understand the functional interplay between HDL particles and tissue, we analyzed the surfaceome-residing receptor neighborhoods with which HDL potentially interacts. We applied a combination of chemoproteomic technologies including automated cell surface capturing (auto-CSC) and HATRIC-based ligand–receptor capturing (HATRIC-LRC) on four different cellular model systems mimicking tissues relevant for RCT. The surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAECs) revealed the main currently known HDL receptor scavenger receptor B1 (SCRB1), as well as 155 shared cell surface receptors representing potential HDL interaction candidates. Since vascular endothelial growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF-modulated surfaceome of HAEC using the auto-CSC technology. VEGF-A treatment led to the remodeling of the surfaceome of HAEC cells, including the previously reported higher surfaceome abundance of SCRB1. In total, 165 additional receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated receptors potentially involved in HDL function. Using the HATRIC-LRC technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor tyrosine-protein kinase Mer (MERTK). Through RNA interference, we revealed its contribution to endothelial HDL binding and uptake. Furthermore, subsequent proximity ligation assays (PLAs) demonstrated the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence binding and uptake of HDL.

Apolipoprotein M and Sphingosine-1-Phosphate Receptor 1 Promote the Transendothelial Transport of High-Density Lipoprotein.

Objective: ApoM enriches S1P (sphingosine-1-phosphate) within HDL (high-density lipoproteins) and facilitates the activation of the S1P1 (S1P receptor type 1) by S1P, thereby preserving endothelial barrier function. Many protective functions exerted by HDL in extravascular tissues raise the question of how S1P regulates transendothelial HDL transport. Approach and Results: HDL were isolated from plasma of wild-type mice, Apom knockout mice, human apoM transgenic mice or humans and radioiodinated to trace its binding, association, and transport by bovine or human aortic endothelial cells. We also compared the transport of fluorescently-labeled HDL or Evans Blue, which labels albumin, from the tail vein into the peritoneal cavity of apoE-haploinsufficient mice with (apoE-haploinsufficient mice with endothelium-specific knockin of S1P1) or without (control mice, ie, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1) endothelium-specific knockin of S1P1. The binding, association, and transport of HDL from Apom knockout mice and human apoM-depleted HDL by bovine aortic endothelial cells was significantly lower than that of HDL from wild-type mice and human apoM-containing HDL, respectively. The binding, uptake, and transport of 125I-HDL by human aortic endothelial cells was increased by an S1P1 agonist but decreased by an S1P1 inhibitor. Silencing of SR-BI (scavenger receptor BI) abrogated the stimulation of 125I-HDL transport by the S1P1 agonist. Compared with control mice, that is, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1, apoE-haploinsufficient mice with endothelium-specific knockin of S1P1 showed decreased transport of Evans Blue but increased transport of HDL from blood into the peritoneal cavity and SR-BI expression in the aortal endothelium. Conclusions: ApoM and S1P1 promote transendothelial HDL transport. Their opposite effect on transendothelial transport of albumin and HDL indicates that HDL passes endothelial barriers by specific mechanisms rather than passive filtration.

The Endothelium Is Both a Target and a Barrier of HDL's Protective Functions.

The vascular endothelium serves as a barrier between the intravascular and extravascular compartments. High-density lipoproteins (HDL) have two kinds of interactions with this barrier. First, bloodborne HDL must pass the endothelium to access extravascular tissues, for example the arterial wall or the brain, to mediate cholesterol efflux from macrophages and other cells or exert other functions. To complete reverse cholesterol transport, HDL must even pass the endothelium a second time to re-enter circulation via the lymphatics. Transendothelial HDL transport is a regulated process involving scavenger receptor SR-BI, endothelial lipase, and ATP binding cassette transporters A1 and G1. Second, HDL helps to maintain the integrity of the endothelial barrier by (i) promoting junction closure as well as (ii) repair by stimulating the proliferation and migration of endothelial cells and their progenitor cells, and by preventing (iii) loss of glycocalix, (iv) apoptosis, as well as (v) transmigration of inflammatory cells. Additional vasoprotective functions of HDL include (vi) the induction of nitric oxide (NO) production and (vii) the inhibition of reactive oxygen species (ROS) production. These vasoprotective functions are exerted by the interactions of HDL particles with SR-BI as well as specific agonists carried by HDL, notably sphingosine-1-phophate (S1P), with their specific cellular counterparts, e.g., S1P receptors. Various diseases modify the protein and lipid composition and thereby the endothelial functionality of HDL. Thorough understanding of the structure–function relationships underlying the multiple interactions of HDL with endothelial cells is expected to elucidate new targets and strategies for the treatment or prevention of various diseases.

Organ-specific lymphatics play distinct roles in regulating HDL trafficking and composition.

Recently, peripheral lymphatic vessels were found to transport high-density lipoprotein (HDL) from interstitial tissues to the blood circulation during reverse cholesterol transport. This function is thought to be critical to the clearance of cholesterol from atherosclerotic plaques. The role of organ-specific lymphatics in modulating HDL transport and composition is, however, incompletely understood. This study aimed to 1) determine the contribution of the lymphatics draining the intestine and liver (which are major sites of HDL synthesis) to total (thoracic) lymph HDL transport and 2) verify whether the HDLs in lymph are derived from specific organs and are modified during trafficking in lymph. The mesenteric, hepatic, or thoracic lymph duct was cannulated in nonfasted Sprague-Dawley rats, and lymph was collected over 5 h under anesthesia. Whole lymph and specific lymph lipoproteins (isolated by ultracentrifugation) were analyzed for protein and lipid composition. The majority of thoracic lymph fluid, protein, and lipid mass was sourced from the mesenteric, and to a lesser extent, hepatic lymph. Mesenteric and thoracic lymph were both rich in chylomicrons and very low-density lipoprotein, whereas hepatic lymph and plasma were HDL-rich. The protein and lipid mass in thoracic lymph HDL was mostly sourced from mesenteric lymph, whereas the cholesterol mass was equally sourced from mesenteric and hepatic lymph. HDLs were compositionally distinct across the lymph sources and plasma. The composition of HDL also appeared to be modified during passage from the mesenteric and hepatic to the thoracic lymph duct. Overall, this study demonstrates that the lipoproteins in lymph are organ specific in composition, and the intestine and liver appear to be the main source of HDL in the lymph.NEW & NOTEWORTHY High-density lipoprotein in lymph are organ-specific in composition and derive mostly from the intestine and liver. High-density lipoprotein also appears to be remodeled during transport through the lymphatics. These findings have implications to cardiometabolic diseases that involve perturbations in lipoprotein distribution and metabolism.

Sex Bias in Cytokines Transported by High‐Density Lipoproteins in Patients with Coronary Artery Disease

Inflammation plays an important role in the induction of Coronary Artery Disease (CAD) and the progression to deadly coronary syndromes. Cytokines are key mediators of inflammation, both in pro‐ and anti‐inflammatory capacities. We have recently discovered that cytokines are transported by high‐density lipoproteins (HDL), suggesting a novel mechanism contributing to the anti‐inflammatory effects of HDL. We examined the HDL‐associated cytokine profiles of male and female patients with CAD to better understand the differences in coronary disease presentation and therapeutic response between men and women.MethodsPatients with confirmed CAD, defined by history of myocardial infarction, coronary artery bypass surgery, or angiography, with controlled serum lipid levels were included in the study. Native HDL species were isolated from plasma samples by selected‐affinity immunosorption. Plasma and isolated HDL samples were assayed for cytokine content by a multiplex array detecting the quantity of 35 cytokines.ResultsThe majority of cytokines assayed were measurable in HDL samples. The distribution of HDL‐associated cytokines varied significantly between males and females. Female HDL samples indicate a much greater degree of remodeling of cytokine cargo than males. Cluster analysis of cytokine expression patterns suggests a difference in inflammatory pathway activation and response in females with CAD.ConclusionsThe cytokines transported by HDL in CAD patients differs greatly between men and women. The pattern of HDL‐associated cytokines provide new insights into the inflammatory processes contributing to CAD in a sex‐dependent manner. The amount of cytokine‐HDL remodeling is exhibited to a greater degree in females; the physiological relevance of this phenomenon is unknown. We aim to elucidate the mechanism of HDL transport of these cytokines and the clinical implications of sex differences in inflammatory response in CAD in future studies.Support or Funding InformationThis work is supported by the Read Family Foundation and the Campini Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Charge-transfer interactions induce surface dependent conformational changes in apolipoprotein biocorona.

Upon introduction into a biological system, engineered nanomaterials (ENMs) rapidly associate with a variety of biomolecules such as proteins and lipids to form a biocorona. The presence of "biocorona" influences nano-bio interactions considerably, and could ultimately result in altered biological responses. Apolipoprotein A-I (ApoA-I), the major constituent of high-density lipoprotein (HDL), is one of the most prevalent proteins found in ENM-biocorona irrespective of ENM nature, size, and shape. Given the importance of ApoA-I in HDL and cholesterol transport, it is necessary to understand the mechanisms of ApoA-I adsorption and the associated structural changes for assessing consequences of ENM exposure. Here, the authors used a comprehensive array of microscopic and spectroscopic tools to elucidate the interactions between ApoA-I and 100 nm Ag nanoparticles (AgNPs) with four different surface functional groups. The authors found that the protein adsorption and secondary structural changes are highly dependent on the surface functionality. Our electrochemical studies provided new evidence for charge transfer interactions that influence ApoA-I unfolding. While the unfolding of ApoA-I on AgNPs did not significantly change their uptake and short-term cytotoxicity, the authors observed that it strongly altered the ability of only some AgNPs to generate of reactive oxygen species. Our results shed new light on the importance of surface functionality and charge transfer interactions in biocorona formation.

A high-throughput screening of kinase inhibitors identifies a regulatory role of VEGF signaling for transendothelial transport of high-density lipoproteins

A high-throughput screening of kinase inhibitors identifies a regulatory role of VEGF signaling for transendothelial transport of high-density lipoproteins

Abstract 565: Vascular Endothelial Growth Factor - An Important Factor in HDL Binding and Transcytosis Through Aortic Endothelial Cells

In the reverse cholesterol transport pathway cholesterol acceptors like high density lipoproteins (HDL) and apolipoprotein A-I (apoA-I), must cross the endothelium to get access to the donor cells in the arterial initima. However, it is unknown how they get translocated from the blood stream into the vascular wall. Previously, we showed that transendothelial HDL transport is modulated by scavenger receptor B I (SR-BI), ATP binding cassette transporter G1, endothelial lipase (EL), and the ectopic beta-ATPase/purinergic receptor axis. None of these proteins is a good candidate to directly mediate holoparticle uptake. They appear rather to indirectly modulate transendothelial transport of HDL by signalling or altering the structure of HDL. To unravel HDL transcytosis, a high-throughput screening was performed, incubating aortic endothelial cells (ECs) with kinase inhibiting drugs and fluorescent labelled HDL. Fixed cells were imaged using fluorescence wide-field microscope. Processing of microscopy acquired data using cell profiler and principle component analysis yielded, only inhibitors of the vascular endothelial growth factor receptor (VEGFR) and its down-stream signaling kinases. To verify and further characterize the screening hits towards their impact on binding, internalization and transport of 125I-HDL, we applied RNA interference and pharmacological means to ECs. Binding, internalization and transport of 125I-HDL through ECs were increased by VEGF treatment but decreased by inhibitors of VEGFR2 as well as knock-down of VEGFR2. RNA interference with VEGFR1 or VEGFR3 had no effect. The stimulatory effect of VEGF on 125I-HDL binding and internalization was decreased by knocking-down SR-BI or PDZK1. The identification of VEGF as a regulatory factor of transendothelial transport supports the concept that the endothelium is a specific and hence druggable barrier for the entry of HDL into the vascular wall.

Cholesterol crystal induced arterial inflammation and destabilization of atherosclerotic plaque.

Evolution of plaque that is prone to rupture is characterized by inflammation and physical changes. Accumulation of low-density lipoprotein in the sub-intima provides esterified cholesterol (ESC) to macrophages and smooth muscle cells that convert it into free cholesterol (FRC) by cholesteryl ester hydrolases (CEHs). Membrane-bound cholesterol carriers transport FRC to high-density lipoprotein (HDL). Impaired HDL transport function and altered composition can lead to extracellular accumulation of FRC, whereas impaired membrane carrier activity can lead to intracellular FRC accumulation. Saturation of FRC can result in cholesterol crystallization with cell death and intimal injury. Disequilibrium between ESC and FRC can impact foam cell and cholesterol crystal (CC) formation. Cholesterol crystals initiate inflammation via NLRP3 inflammasome leading to interleukin-1β (IL-1β) production inducing C-reactive protein. Eventually, crystals growing from within the plaque and associated inflammation destabilize the plaque. Thus, inhibition of inflammation by antagonists to IL-1β or agents that dissolve or prevent CC formation may stabilize vulnerable plaques.

Abstract 31: Functional Impact of Inflammatory Cell-Originating HDL-miR-223 Communication in vivo

Atherosclerosis is associated with endothelial dysfunction and inflammation, and high-density lipoproteins (HDL) play critical roles in both. HDL transport a wide-variety of proteins, nucleic acids, and small molecules, which likely confer many of HDL’s alternative functions, including its anti-inflammatory and anti-atherogenic properties. Using in vitro studies, we demonstrated that macrophages export miR-223 to HDL and subsequently, HDL delivers functional miR-223 to human coronary artery endothelial cells (HCAEC). Moreover, intracellular adhesion molecule 1 (ICAM-1), a miR-223 target, was found to be down-regulated by HDL-miR-223 at a specific target site within ICAM-1’s 3’ untranslated region. These in vitro findings support the potential of a HDL-miRNA communication pathway that may contribute to atherogenesis. To validate HDL-miRNA communication in vivo, bone-marrow transplant (BMT) studies were completed with Mir223-/- and C57B/6J (WT) mice. To demonstrate that functional HDL-miR-223 is delivered to recipient cells/tissues in vivo, BM from WT mice was transplanted into irradiated Mir223-/- mice. Strikingly, we found that HDL-miR-223 communication was restored after BMT, as evidenced by significantly increased miR-223 levels on HDL and in the liver, white adipose, aortic endothelium, and skeletal muscle. Conversely, HDL-miR-223 intercellular communication was depleted by transplanting BM from Mir223-/- into WT mice, and resulted in significant loss of miR-223 on HDL and in many tissues. In both BMT studies, miR-223 changes led to altered expression of many target genes in multiple tissues. Most importantly, these studies identified a novel miR-223 target gene which we experimentally validated, vesicle transport through interaction with t-SNAREs homolog 1A (VTI1A), which was decreased and increased in the aortic endothelium from each BMT study, respectively. VTI1A has been previously characterized as a SNARE protein that contributes to cytokine secretion and membrane lipid dynamics. As such, HDL likely mediates an inflammatory cell-originating miR-223 communication pathway that suppresses inflammation and cytokine secretion in the aortic endothelium, thus antagonizing atherosclerosis.

Transport of high density lipoproteins though endothelial cells (855.7)

Atherosclerosis is a progressive accumulation of lipids in the arterial intima leading to plaque formation. Epidemiological studies show an inverse association of high density lipoprotein (HDL) cholesterol with cardiovascular events. HDL has multiple anti‐atherogenic functions, some of which take place in the vessel wall. To get access to the intima and the lipid‐laden macrophages, HDL has to pass the endothelial barrier.To elucidate the itinerary of HDL through endothelial cells (ECs), we investigated localisation and distribution of fluorescently and 1.4 nm Nanogold labelled HDL in fluorescent (FL) as well as electron microscopy (EM). For EM, we used high pressure freezing (HPF) and to avoid lipid extraction we developed a fast method for silver enhancement of the nanogold‐HDL after fixation in OsO4.HDL is detectable in ECs in a concentration and time dependent manner. Colocalisations of HDL with dextran were negative excluding fluid phase uptake and transferrin and albumin show only slight colocalisation indicating an atypical uptake mechanism.Further experiments showed that HDL is neither targeted to lysosomes nor to the Golgi or the endoplasmatic reticulum. Only small amounts can be detected in early endosomes (Rab5) and endosome to trans‐golgi network (Rab9), but not at all with late endosomes (Rab7), the recycling endosomes (Rab11a) or vesicles involved in trans‐Golgi network sorting (Stx6). EM ultrastructure shows HDL to be located mainly in multivesicular bodies.We corroborated our findings in aortic artery endothelium. Live microscopy experiments as well as biochemical data help us to understand the itinerary of HDL through aortic ECs.

HDL-transferred microRNA-223 regulates ICAM-1 expression in endothelial cells

High-density lipoproteins (HDL) have many biological functions, including reducing endothelial activation and adhesion molecule expression. We recently reported that HDL transport and deliver functional microRNAs (miRNA). Here we show that HDL suppresses expression of intercellular adhesion molecule 1 (ICAM-1) through the transfer of miR-223 to endothelial cells. After incubation of endothelial cells with HDL, mature miR-223 levels are significantly increased in endothelial cells and decreased on HDL. However, miR-223 is not transcribed in endothelial cells and is not increased in cells treated with HDL from miR-223(-/-) mice. HDL inhibit ICAM-1 protein levels, but not in cells pretreated with miR-223 inhibitors. ICAM-1 is a direct target of HDL-transferred miR-223 and this is the first example of an extracellular miRNA regulating gene expression in cells where it is not transcribed. Collectively, we demonstrate that HDL's anti-inflammatory properties are conferred, in part, through HDL-miR-223 delivery and translational repression of ICAM-1 in endothelial cells.

Association of HDL-Related Loci with Age-Related Macular Degeneration and Plasma Lutein and Zeaxanthin: the Alienor Study

BackgroundSeveral genes implicated in high-density lipoprotein (HDL) metabolism have been reported to be associated with age-related macular degeneration (AMD). Furthermore, HDL transport the two carotenoids, lutein and zeaxanthin, which are highly suspected to play a key-role in the protection against AMD. The objective is to confirm the associations of HDL-related loci with AMD and to assess their associations with plasma lutein and zeaxanthin concentrations.MethodsAlienor study is a prospective population-based study on nutrition and age-related eye diseases performed in 963 elderly residents of Bordeaux, France. AMD was graded according to the international classification, from non-mydriatic colour retinal photographs. Plasma lutein and zeaxanthin were determined by normal-phase high-performance liquid chromatography. The following polymorphisms were studied: rs493258 and rs10468017 (LIPC), rs3764261 (CETP), rs12678919 (LPL) and rs1883025 (ABCA1).ResultsAfter multivariate adjustment, the TT genotype of the LIPC rs493258 variant was significantly associated with a reduced risk for early and late AMD (OR=0.64, 95%CI: 0.41-0.99; p=0.049 and OR=0.26, 95%CI: 0.08-0.85; p=0.03, respectively), and with higher plasma zeaxanthin concentrations (p=0.03), while plasma lipids were not significantly different according to this SNP. Besides, the LPL variant was associated with early AMD (OR=0.67, 95%CI: 0.45-1.00; p=0.05) and both with plasma lipids and plasma lutein (p=0.047). Associations of LIPC rs10468017, CETP and ABCA1 polymorphisms with AMD did not reach statistical significance.ConclusionThese findings suggest that LIPC and LPL genes could both modify the risk for AMD and the metabolism of lutein and zeaxanthin.

Interleukin 6 Stimulates Endothelial Binding and Transport of High-Density Lipoprotein Through Induction of Endothelial Lipase

In the reverse cholesterol transport pathway, high-density lipoprotein (HDL) passes the endothelial cell barrier by mechanisms involving the scavenger receptor class B type I and the ATP-binding cassette G1. However, little is known on how inflammation influences this transendothelial transport. On stimulation with interleukin-6, cultivated primary endothelial cells showed increased binding and transport of (125)I-HDL without changing the expression of scavenger receptor class B type I and ATP-binding cassette G1. Therefore, we analyzed the involvement of endothelial lipase (EL), a known HDL-binding protein expressed by endothelial cells. Here, we show an increased EL expression after interleukin-6 stimulation. Moreover, using pharmacological inhibitors or RNA interference against EL, we demonstrated its participation in HDL binding and transport through the endothelium. Furthermore, adenovirus-mediated transfection of endothelial cells with either catalytically active or nonactive EL revealed that EL facilitates the endothelial binding and transport by both bridging and lipolysis of HDL. EL was also found responsible for the reduction of HDL particle size occurring during the specific transport through a monolayer of endothelial cells. Finally, pharmacological inhibition of EL reversed the inducing effect of interleukin-6 on HDL binding and transport. Interleukin-6 stimulates the translocation of HDL through the endothelium, the first step in reverse cholesterol transport pathway, by enhancing EL expression. In addition, we demonstrated the role of EL in the transendothelial transport of HDL.

Maternal Lipids and Small for Gestational Age Birth at Term

To compare maternal lipid and lipoprotein concentrations between small for gestational age (SGA) infants and infants with normal growth born at term. This was a case-control study nested within a large (n = 5337) prospective multicenter cohort of pregnant women followed to delivery. SGA cases (n = 323) were all term infants with birth weight below the 10th percentile for their gestational age and sex. Controls (n = 671) were selected at random from term infants with birth weight between the 25th and 75th percentiles. Plasma samples obtained at 24-26 weeks were analyzed for lipoproteins using a recently developed nuclear magnetic resonance-based procedure that distinguishes high-density lipoprotein (HDL) and low-density lipoprotein particles of different sizes. Apolipoprotein A-1 and C-II levels were analyzed using turbidimetric methods. Compared with controls, mothers of SGA cases had significantly higher mean concentrations of total HDL particles, medium and small HDL particles, and apolipoprotein A-1, with evidence of a dose-response relationship across quartiles of the control distribution. aORs for the highest quartiles were 2.8 (95% CI, 1.7-4.5) for total HDL particles and 3.1 (95% CI, 1.9-5.0) for apolipoprotein A-1. Our results suggest that the higher HDL particle and apolipoprotein A-1 concentrations in mothers of SGA cases may reflect defective placental transport of HDL, which could compromise cholesterol uptake by the developing fetus.

Lymphatics as a New Active Player in Reverse Cholesterol Transport

Reverse cholesterol transport (RCT), a key function of high-density lipoproteins (HDL), prevents excess cholesterol in tissues. A study in this issue (Lim etal., 2013) suggests that lymphatic vessels are critical for normal RCT and mediate the active trans-endothelial transport of HDL via the HDL receptor SR-BI.

The Fusion Protein of IFN-α and Apolipoprotein A-I Crosses the Blood–Brain Barrier by a Saturable Transport Mechanism

IFN-α is widely used for the treatment of chronic viral hepatitis and malignancies. However, systemic IFN-α treatment causes severe neuropsychiatric complications in humans, including depression, anxiety, and cognitive impairments. We have previously reported that the fusion protein formed by IFN-α and apolipoprotein A-I (IA) circulates bound to high-density lipoproteins (HDLs) and exhibits liver targeting, increased half-life, enhanced immunostimulatory activity, and reduced cytotoxicity. As the transport of HDLs across the blood-brain barrier is a highly complex and regulated process, in this study, we examine the effects of IA on the brain. Determination of IFN-α in brain and serum after hydrodynamic administration of different doses of a plasmid encoding IFN-α or IA showed that IA penetrated into the brain by a saturable transport mechanism. Thus, at high serum levels of the transgenes, the induction of IFN-sensitive genes and the number of phospho-STAT1(+) cell nuclei in the brain were substantially higher with IFN-α than with IA. This was associated with attenuation of neurodepression in mice given IA, as manifested by shorter immobility time in the tail suspension test. However, when given low doses of rIFN-α or the same antiviral units of HDLs containing IA, the induction of IFN-stimulated genes in the brain was significantly greater with the latter. In conclusion, IA crosses the blood-brain barrier not by diffusion, as is the case of IFN-α, but by a facilitated saturable transport mechanism. Thus, linkage to apolipoprotein A-I may serve to modulate the effects of IFN-α on the CNS.

Plasma PLTP (phospholipid-transfer protein): an emerging role in ‘reverse lipopolysaccharide transport’ and innate immunity

Plasma PLTP (phospholipid-transfer protein) is a member of the lipid transfer/LBP [LPS (lipopolysaccharide)-binding protein] family, which constitutes a superfamily of genes together with the short and long PLUNC (palate, lung and nasal epithelium clone) proteins. Although PLTP was studied initially for its involvement in the metabolism of HDL (high-density lipoproteins) and reverse cholesterol transport (i.e. the metabolic pathway through which cholesterol excess can be transported from peripheral tissues back to the liver for excretion in the bile), it displays a number of additional biological properties. In particular, PLTP can modulate the lipoprotein association and metabolism of LPS that are major components of Gram-negative bacteria. The delayed association of LPS with lipoproteins in PLTP-deficient mice results in a prolonged residence time, in a higher toxicity of LPS aggregates and in a significant increase in LPS-induced mortality as compared with wild-type mice. It suggests that PLTP may play a pivotal role in inflammation and innate immunity through its ability to accelerate the 'reverse LPS transport' pathway.

Carboxyl Terminus of Apolipoprotein A-I (ApoA-I) Is Necessary for the Transport of Lipid-free ApoA-I but Not Prelipidated ApoA-I Particles through Aortic Endothelial Cells

High density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) must leave the circulation and pass the endothelium to exert their atheroprotective actions in the arterial wall. We previously demonstrated that the transendothelial transport of apoA-I involves ATP-binding cassette transporter (ABC) A1 and re-secretion of lipidated particles. Transendothelial transport of HDL is modulated by ABCG1 and the scavenger receptor BI (SR-BI). We hypothesize that apoA-I transport is started by the ABCA1-mediated generation of a lipidated particle which is then transported by ABCA1-independent pathways. To test this hypothesis we analyzed the endothelial binding and transport properties of initially lipid-free as well as prelipidated apoA-I mutants. Lipid-free apoA-I mutants with a defective carboxyl-terminal domain showed an 80% decreased specific binding and 90% decreased specific transport by aortic endothelial cells. After prior cell-free lipidation of the mutants, the resulting HDL-like particles were transported through endothelial cells by an ABCG1- and SR-BI-dependent process. ApoA-I mutants with deletions of either the amino terminus or both the amino and carboxyl termini showed dramatic increases in nonspecific binding but no specific binding or transport. Prior cell-free lipidation did not rescue these anomalies. Our findings of stringent structure-function relationships underline the specificity of transendothelial apoA-I transport and suggest that lipidation of initially lipid-free apoA-I is necessary but not sufficient for specific transendothelial transport. Our data also support the model of a two-step process for the transendothelial transport of apoA-I in which apoA-I is initially lipidated by ABCA1 and then further processed by ABCA1-independent mechanisms.