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Impaired clearance of cerebral amyloid-β (Aβ) across the blood-brain barrier (BBB) may facilitate the onset and progression of Alzheimer's disease (AD). Additionally, experimental evidence suggests a central role for cellular cholesterol in amyloid-β protein precursor (AβPP) processing. The present study investigated whether brain capillary endothelial cells (BCEC; the anatomical basis of the BBB) are capable of endogenous AβPP synthesis and whether and to what extent AβPP synthesis and processing is under control of cellular cholesterol homeostasis. Intracellular cholesterol metabolism was pharmacologically manipulated by using natural and synthetic liver-X receptor (LXR) agonists. Using an in vitro model of the BBB consisting of primary porcine BCEC (pBCEC), we demonstrate that endogenous full-length AβPP synthesis by pBCEC is significantly increased while the amount of cell-associated, amyloidogenic Aβ oligomers is decreased in response to 24(S)-hydroxycholesterol (24OH-C) or 27OH-C, TO901317, cholesterol, or simvastatin treatment. Oxysterols, as well as simvastatin, enhanced the secretion of non-amyloidogenic sAβPPα up to 2.5-fold. In parallel, LXR agonists reduced cholesterol biosynthesis by 30-80% while stimulating esterification (up to 2.5-fold) and efflux (up to 2.5-fold) of cellular cholesterol by modifying hydroxymethylglutaryl-CoA reductase (HMGCR), sterol regulatory element-binding protein (SREBP-2), acyl-CoA: cholesterol acyltransferase 2 (ACAT-2), and ATP binding cassette transporter A1 (ABCA1) expression levels. In a polarized in vitro model mimicking the BBB, pBCEC secreted sAβPPα preferentially to the basolateral compartment. In summary endothelial cells of the BBB actively synthesize AβPP, Aβ oligomers, and secrete AβPPα in a polarized manner. AβPP processing by pBCEC is regulated by LXR agonists, which have been proven beneficial in experimental AD models. [ABSTRACT FROM AUTHOR]

The most serious consequence of sterol 27-hydroxylase deficiency in humans [cerebrotendinous xanthomatosis (CTX)] is the development of cholestanol-containing brain xanthomas. The cholestanol in the brain may be derived from the circulation or from 7α-hydroxylated intermediates in bile acid synthesis, present at 50- to 250-fold increased levels in plasma. Here, we demonstrate a transfer of 7α-hydroxy-4-cholesten-3-one across cultured porcine brain endothelial cells (a model for the blood-brain barrier) that is ∼100-fold more efficient than the transfer of cholestanol. Furthermore, there was an efficient conversion of 7α-hydroxy-4-cholesten-3-one to cholestanol in cultured neuronal and glial cells as well as in monocyte-derived macrophages of human origin. It is concluded that the continuous intracellular production of cholestanol from a bile acid precursor capable of rapidly passing biomembranes, including the blood-brain barrier, is likely to be of major importance for the accumulation of cholestanol in patients with CTX. Such a mechanism also fits well with the observation that treatment with chenodeoxycholic acid, which normalizes the level of the bile acid precursor, results in a reduction of cholestanol-containing xanthomas even in the brain.

Defective degradation and clearance of amyloid-β as well as inflammation per se are crucial players in the pathology of Alzheimer's disease (AD). A defective transport across the blood-brain barrier is causative for amyloid-β (Aβ) accumulation in the brain, provoking amyloid plaque formation. Using primary porcine brain capillary endothelial cells and murine organotypic hippocampal slice cultures as in vitro models of AD, we investigated the effects of the antioxidant astaxanthin (ASX) on Aβ clearance and neuroinflammation. We report that ASX enhanced the clearance of misfolded proteins in primary porcine brain capillary endothelial cells by inducing autophagy and altered the Aβ processing pathway. We observed a reduction in the expression levels of intracellular and secreted amyloid precursor protein/Aβ accompanied by an increase in ABC transporters ABCA1, ABCG1 as well as low density lipoprotein receptor-related protein 1 mRNA levels. Furthermore, ASX treatment increased autophagic flux as evidenced by increased lipidation of LC3B-II as well as reduced protein expression of phosphorylated S6 ribosomal protein and mTOR. In LPS-stimulated brain slices, ASX exerted anti-inflammatory effects by reducing the secretion of inflammatory cytokines while shifting microglia polarization from M1 to M2 phenotype. Our data suggest ASX as potential therapeutic compound ameliorating AD-related blood brain barrier impairment and inflammation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: I.I., D.L., T.L., I.S., S.F., M.P. and B.H.P. are presently or formerly affiliated with QPS Austria.., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Oxysterols are oxidized cholesterol derivatives whose systemic levels are found elevated in pregnancy disorders such as gestational diabetes mellitus (GDM). Oxysterols act through various cellular receptors and serve as a key metabolic signal, coordinating inflammation. GDM is a condition of low-grade chronic inflammation accompanied by altered inflammatory profiles in the mother, placenta and fetus. Higher levels of two oxysterols, namely 7-ketocholesterol (7-ketoC) and 7β-hydroxycholesterol (7β-OHC), were observed in fetoplacental endothelial cells (fpEC) and cord blood of GDM offspring. In this study, we tested the effects of 7-ketoC and 7β-OHC on inflammation and investigated the underlying mechanisms involved. Primary fpEC in culture treated with 7-ketoC or 7β-OHC, induced the activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) signaling, which resulted in the expression of pro-inflammatory cytokines (IL-6, IL-8) and intercellular cell adhesion molecule-1 (ICAM-1). Liver-X receptor (LXR) activation is known to repress inflammation. Treatment with LXR synthetic agonist T0901317 dampened oxysterol-induced inflammatory responses. Probucol, an inhibitor of LXR target gene ATP-binding cassette transporter A-1 (ABCA-1), antagonized the protective effects of T0901317, suggesting a potential involvement of ABCA-1 in LXR-mediated repression of inflammatory signaling in fpEC. TLR-4 inhibitor Tak-242 attenuated pro-inflammatory signaling induced by oxysterols downstream of the TLR-4 inflammatory signaling cascade. Taken together, our findings suggest that 7-ketoC and 7β-OHC contribute to placental inflammation through the activation of TLR-4. Pharmacologic activation of LXR in fpEC decelerates its shift to a pro-inflammatory phenotype in the presence of oxysterols.

Background and Purpose: The cerebrospinal fluid (CSF)/plasma albumin ratio (QAlb) is believed to reflect the integrity of the blood-brain barrier (BBB). Recently, we reported that QAlb is lower in females. This may be important for uptake of neurotoxic 27-hydroxycholesterol (27OH) by the brain in particular because plasma levels of 27OH are higher in males. We studied sex differences in the relation between CSF and plasma levels of 27OH and its major metabolite 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) with QAlb. We tested the possibility of sex differences in the brain metabolism of 27OH and if its flux into the brain disrupted integrity of the BBB., Experimental Approach: We have examined our earlier studies looking for sex differences in CSF levels of oxysterols and their relation to QAlb. We utilized an in vitro model for the BBB with primary cultured brain endothelial cells to test if 27OH has a disruptive effect on this barrier. We measured mRNA and protein levels of CYP7B1 in autopsy brain samples., Key Results: The correlation between CSF levels of 27OH and QAlb was higher in males whereas, with 7HOCA, the correlation was higher in females. No significant sex difference in the expression of CYP7B1 mRNA in brain autopsy samples. A correlation was found between plasma levels of 27OH and QAlb. No support was obtained for the hypothesis that plasma levels of 27OH have a disruptive effect on the BBB., Conclusions and Implications: The sex differences are discussed in relation to negative effects of 27OH on different brain functions., Linked Articles: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc., (© 2020 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Serotonin (5-HT) plays an extensive role during pregnancy in regulating both the placental physiology and embryonic/fetal development. The uptake of 5-HT into cells is central to the control of local concentrations of 5-HT near its molecular targets. Here, we investigated the mechanisms of 5-HT uptake into human primary placental cells and cord blood platelets, all isolated immediately after birth. Trophoblasts and cord blood platelets showed 5-HT uptake with similar Michaelis constant ( Km ) values (~0.6 μM), typical of the high-affinity serotonin transporter (SERT). The uptake of 5-HT into trophoblasts was efficiently inhibited by various SERT-targeting drugs. In contrast, the uptake of 5-HT into feto-placental endothelial cells was not inhibited by a SERT blocker and showed a Km value (~782 μM) in the low-affinity range. Consistent with this, SERT mRNAs were abundant in term trophoblasts but sparse in feto-placental endothelial cells, whereas the opposite was found for the low-affinity plasma membrane monoamine transporter ( PMAT ) mRNAs. Organic cation transporter ( OCT ) 1 , 2, and 3 mRNAs were absent or sparse in both cell types. In summary, the results demonstrate, for the first time, the presence of functional 5-HT uptake systems in feto-placental endothelial cells and fetal platelets, cells that are in direct contact with fetal blood plasma. The data also highlight the sensitivity to various psychotropic drugs of 5-HT transport into trophoblasts facing the maternal blood. The multiple, high-, and low-affinity systems present for the cellular uptake of 5-HT underscore the importance of 5-HT homeostasis at the maternal-fetal interface.

Aberrant insulin signaling constitutes an early change in Alzheimer's disease (AD). Insulin receptors (IR) and low-density lipoprotein receptor-related protein-1 (LRP-1) are expressed in brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). There, insulin may regulate the function of LRP-1 in Aβ clearance from the brain. Changes in IR-β and LRP-1 and insulin signaling at the BBB in AD are not well understood. Herein, we identified a reduction in cerebral and cerebrovascular IR-β levels in 9-month-old male and female 3XTg-AD (PS1 M146V , APP Swe , and tau P301L ) as compared to NTg mice, which is important in insulin mediated signaling responses. Reduced cerebral IR-β levels corresponded to impaired insulin signaling and LRP-1 levels in brain. Reduced cerebral and cerebrovascular IR-β and LRP-1 levels in 3XTg-AD mice correlated with elevated levels of autophagy marker LC3B. In both genotypes, high-fat diet (HFD) feeding decreased cerebral and hepatic LRP-1 expression and elevated cerebral Aβ burden without affecting cerebrovascular LRP-1 and IR-β levels. In vitro studies using primary porcine (p)BCEC revealed that Aβ peptides 1-40 or 1-42 (240 nM) reduced cellular levels and interaction of LRP-1 and IR-β thereby perturbing insulin-mediated signaling. Further mechanistic investigation revealed that Aβ treatment accelerated the autophagy-lysosomal degradation of IR-β and LRP-1 in pBCEC. LRP-1 silencing in pBCEC decreased IR-β levels through post-translational pathways further deteriorating insulin-mediated responses at the BBB. Our findings indicate that LRP-1 proves important for insulin signaling at the BBB. Cerebral Aβ burden in AD may accelerate LRP-1 and IR-β degradation in BCEC thereby contributing to impaired cerebral and cerebromicrovascular insulin effects., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The pathogenesis of Alzheimer's disease (AD) is characterized by overproduction, impaired clearance, and deposition of amyloid-β peptides (Aβ) and connected to cholesterol homeostasis. Since the blood-brain barrier (BBB) is involved in these processes, we investigated effects of the retinoid X receptor agonist, bexarotene (Bex), and the peroxisome proliferator-activated receptor α agonist and antioxidant, astaxanthin (Asx), on pathways of cellular cholesterol metabolism, amyloid precursor protein processing/Aβ production and transfer at the BBB in vitro using primary porcine brain capillary endothelial cells (pBCEC), and in 3xTg AD mice. Asx/Bex downregulated transcription/activity of amyloidogenic BACE1 and reduced Aβ oligomers and ~80 kDa intracellular 6E10-reactive APP/Aβ species, while upregulating non-amyloidogenic ADAM10 and soluble (s)APPα production in pBCEC. Asx/Bex enhanced Aβ clearance to the apical/plasma compartment of the in vitro BBB model. Asx/Bex increased expression levels of ABCA1, LRP1, and/or APOA-I. Asx/Bex promoted cholesterol efflux, partly via PPARα/RXR activation, while cholesterol biosynthesis/esterification was suppressed. Silencing of LRP-1 or inhibition of ABCA1 by probucol reversed Asx/Bex-mediated effects on levels of APP/Aβ species in pBCEC. Murine (m)BCEC isolated from 3xTg AD mice treated with Bex revealed elevated expression of APOE and ABCA1. Asx/Bex reduced BACE1 and increased LRP-1 expression in mBCEC from 3xTg AD mice when compared to vehicle-treated or non-Tg treated mice. In parallel, Asx/Bex reduced levels of Aβ oligomers in mBCEC and Aβ species in brain soluble and insoluble fractions of 3xTg AD mice. Our results suggest that both agonists exert beneficial effects at the BBB by balancing cholesterol homeostasis and enhancing clearance of Aβ from cerebrovascular endothelial cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Gestational diabetes mellitus (GDM) is associated with excessive oxidative stress which may affect placental vascular function. Cholesterol homeostasis is crucial for maintaining fetoplacental endothelial function. We aimed to investigate whether and how GDM affects cholesterol metabolism in human fetoplacental endothelial cells (HPEC). HPEC were isolated from fetal term placental arterial vessels of GDM or control subjects. Cellular reactive oxygen species (ROS) were detected by H 2 DCFDA fluorescent dye. Oxysterols were quantified by gas chromatography-mass spectrometry analysis. Genes and proteins involved in cholesterol homeostasis were detected by real-time PCR and immunoblotting, respectively. Cholesterol efflux was determined from [ 3 H]-cholesterol labeled HPEC and [ 14 C]-acetate was used as cholesterol precursor to measure cholesterol biosynthesis and esterification. We detected enhanced formation of ROS and of specific, ROS-derived oxysterols in HPEC isolated from GDM versus control pregnancies. ROS-generated oxysterols were simultaneously elevated in cord blood of GDM neonates. Liver-X receptor activation in control HPEC by synthetic agonist TO901319, 7-ketocholesterol, or 7β-hydroxycholesterol upregulated ATP-binding cassette transporters (ABC)A1 and ABCG1 expression, accompanied by increased cellular cholesterol efflux. Upregulation of ABCA1 and ABCG1 and increased cholesterol release to apoA-I and HDL 3 (78 ± 17%, 40 ± 9%, respectively) were also observed in GDM versus control HPEC. The LXR antagonist GGPP reversed ABCA1 and ABCG1 upregulation and reduced the increased cholesterol efflux in GDM HPEC. Similar total cellular cholesterol levels were detected in control and GDM HPEC, while GDM enhanced cholesterol biosynthesis along with upregulated 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and sterol O-acyltransferase 1 (SOAT1) mRNA and protein levels. Our results suggest that in GDM cellular cholesterol homeostasis in the fetoplacental endothelium is modulated via LXR activation and helps to maintain its proper functionality., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Transgenic mouse models are indispensable tools to mimic human diseases and analyze the effectiveness of related new drugs. For a long time amyotrophic lateral sclerosis (ALS) research depended on only a few mouse models that exhibit a very strong and early phenotype, e.g. SOD1 mice, resulting in a short treatment time window. By now, several models are available that need to be characterized to highlight characteristics of each model. Here we further characterized the mThy1-hTDP-43 transgenic mouse model TAR6/6 that overexpresses wild type human TARDBP, also called TDP-43, under control of the neuronal Thy-1 promoter presented by Wils and colleagues, 2010, by using biochemical, histological and behavioral readouts. Our results show that TAR6/6 mice exhibit a strong TDP-43 expression in the hippocampus, spinal cord, hypothalamus and medulla oblongata. Apart from prominent protein expression in the nucleus, TDP-43 protein was found at lower levels in the cytosol of transgenic mice. Additionally, we detected insoluble TDP-43 in the cortex, motoneuron loss, and increased neuroinflammation in the central nervous system of TAR6/6 animals. Behavioral analyses revealed early motor deficits in the clasping- and wire suspension test as well as decreased anxiety in the elevated plus maze. Further motor tests showed differences at later time points compared to non-transgenic littermates, thus allowing the observation of onset and severity of such deficits. Together, TAR6/6 mice are a valuable tool to test new ALS/FTLD drugs that target TDP-43 expression and insolubility, neuroinflammation, motoneuron loss or other TDP-43 related downstream signaling pathways since these mice exhibit a later pathology as previously used ALS/FTLD mouse models., Competing Interests: BS, RR, SF, JN, NT, MT, VN, BHP are employees of QPS Austria GmbH. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Anomalous neuronal accumulation of Aβ peptides was shown to affect synaptic transmission and contribute to neurodegeneration in Alzheimer's disease (AD) brain. Neuronal cells internalize amyloid beta (Aβ) peptides from the brain extracellular space even under normal physiological conditions, and these endocytotic pathways go awry during AD progression. We hypothesized that exposure to toxic Aβ species accumulating in AD brain contributes to perturbations in neuronal endocytosis. We have shown substantial down-regulation of KEGG endocytotic pathway genes in AD patient brain regions that accumulate Aβ compared to those in non-demented individuals. While both Aβ40 and Aβ42 perturbed endocytosis and intracellular trafficking in neuronal cells, Aβ40 had a greater effect than Aβ42. Moreover, Aβ40 decreased the neuronal uptake and lysosomal accumulation of Aβ42, which tends to oligomerize at low lysosomal pH. Hence, Aβ40 may reduce the prevalence of stable Aβ42 oligomers that are closely associated with neurodegeneration and are intercellularly propagated across the vulnerable brain regions to eventually nucleate as amyloid plaques. In conclusion, elevated brain Aβ levels and Aβ42:40 ratio apparent in the early stages of AD could perturb intraneuronal trafficking, augment the anomalous accumulation of amyloid peptides in AD brain, and drive AD pathogenesis., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Amyloid-β peptides (Aβ) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aβ metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aβ. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aβ metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aβ oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aβ clearance across the pBCEC monolayer. Treatment of pBCEC with Aβ (1-40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3×Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aβ oligomers and reduced Aβ uptake and cell-associated Aβ oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aβ processing and clearance at the BBB., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)-hydroxycholesterol and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL and their apolipoproteins are crucial for cerebral lipid transfer, and loss of ATP binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-β HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB, we examined expression, regulation, and functions of ABCG1, ABCG4, and apoM in primary porcine brain capillary endothelial cells (pBCEC). RT Q-PCR analyses and immunoblotting revealed that in addition to ABCA1 and scavenger receptor, class B, type I (SR-BI), pBCEC express high levels of ABCG1, which was up-regulated by LXR activation. Immunofluorescent staining, site-specific biotinylation and immunoprecipitation revealed that ABCG1 is localized both to early and late endosomes and on apical and basolateral plasma membranes. Using siRNA interference to silence ABCG1 (by 50%) reduced HDL-mediated [ 3 H]-cholesterol efflux (by 50%) but did not reduce [ 3 H]-24(S)-hydroxycholesterol efflux. In addition to apoA-I, pBCEC express and secrete apoM mainly to the basolateral (brain) compartment. HDL enhanced expression and secretion of apoM by pBCEC, apoM-enriched HDL promoted cellular cholesterol efflux more efficiently than apoM-free HDL, while apoM-silencing diminished cellular cholesterol release. We suggest that ABCG1 and apoM are centrally involved in regulation of cholesterol metabolism/turnover at the BBB., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Phospholipid transfer protein (PLTP) is a key protein involved in biogenesis and remodeling of plasma HDL. Several neuroprotective properties have been ascribed to HDL. We reported earlier that liver X receptor (LXR) activation promotes cellular cholesterol efflux and formation of HDL-like particles in an established in vitro model of the blood-brain barrier (BBB) consisting of primary porcine brain capillary endothelial cells (pBCEC). Here, we report PLTP synthesis, regulation, and its key role in HDL metabolism at the BBB. We demonstrate that PLTP is highly expressed and secreted by pBCEC. In a polarized in vitro model mimicking the BBB, pBCEC secreted phospholipid-transfer active PLTP preferentially to the basolateral ("brain parenchymal") compartment. PLTP expression levels and phospholipid transfer activity were enhanced (up to 2.5-fold) by LXR activation using 24(S)-hydroxycholesterol (a cerebral cholesterol metabolite) or TO901317 (a synthetic LXR agonist). TO901317 administration elevated PLTP activity in BCEC from C57/BL6 mice. Preincubation of HDL3 with human plasma-derived active PLTP resulted in the formation of smaller and larger HDL particles and enhanced the capacity of the generated HDL particles to remove cholesterol from pBCEC by up to 3-fold. Pre-β-HDL, detected by two-dimensional crossed immunoelectrophoresis, was generated from HDL3 in pBCEC-derived supernatants, and their generation was markedly enhanced (1.9-fold) upon LXR activation. Furthermore, RNA interference-mediated PLTP silencing (up to 75%) reduced both apoA-I-dependent (67%) and HDL3-dependent (30%) cholesterol efflux from pBCEC. Based on these findings, we propose that PLTP is actively involved in lipid transfer, cholesterol efflux, HDL genesis, and remodeling at the BBB.

Niemann-Pick type C disease (NPC) is an inherited disorder mainly caused by loss-of-function mutations in the NPC1 gene, that lead to intracellular cholesterol accumulation and disturbed cholesterol homeostasis. Similarly to Alzheimer's disease (AD), NPC is associated with progressive neurodegeneration and altered metabolism of amyloid precursor protein (APP). Liver X receptors (LXRs), the key transcriptional regulators of cholesterol homeostasis, were reported to play neuroprotective roles in NPC mice. We investigated the impacts of LXRs on APP metabolism in mutant CHO cells lacking the NPC1 gene (-NPC1 cells). Pharmacological activation of LXRs in -NPC1 cells tended to reduce the ratio of total secreted APP (sAPP) to full length APP (flAPP) levels and sAPPβ levels as well as to increase the ratio of APP Cterminal fragments to flAPP levels, resulting in decreased levels of amyloid β (Aβ) peptides. -NPC1 cells treated with LXR agonist TO901317 (TO90) displayed a modest increase in cholesterol efflux to apolipoprotein A-I (apoA-I) but not to HDL3, or in the absence of extracellular cholesterol acceptors. The observed similar reduction of Aβ levels upon TO90 treatment in the presence or in the absence of extracellular apoA-I indicated a cholesterol-efflux independent effect of TO90 on Aβ levels. Furthermore, TO90 had no effect on the cholesterol synthesis rate in -NPC1 cells, while it reduced the rate of cholesterol esterification. The obtained results indicate that LXR activation may decrease Aβ levels in NPC1- deficient conditions. The underlying mechanism of this action does not appear to be related to effects on cholesterol efflux or synthesis rates.

Context: Phospholipid (PL) transfer protein (PLTP) plays a crucial role in high-density lipoprotein (HDL) metabolism. In the fetal circulation, HDL particles are the main cholesterol carriers and are involved in maternal-fetal cholesterol transfer across human placental endothelial cells (HPEC)., Objective: The aim was to investigate local function(s) of PLTP at the fetoplacental endothelium. Because HPEC display morphological and functional diversity when isolated from arteries or veins, we hypothesized that PLTP activity may differ between arterial and venous HPEC., Design: We determined PLTP mRNA and activity levels from isolated HPEC and investigated PLTP-mediated remodeling of fetal HDL particles and their capacity in mediating cholesterol efflux from HPEC., Results: Incubation of fetal HDL with active human plasma PLTP resulted in increased particle size (12.6 vs. 13.2 nm, P < 0.05), with a concomitant increase (3.5-fold) in pre-β-mobile HDL particles. Arterial HPEC showed higher Pltp expression levels and secreted PL transfer activity (1.8-fold, P < 0.001) than venous HPEC. In contrast to adult HDL(3), [(3)H]cholesterol efflux to fetal HDL was 21% higher (P < 0.05) from arterial than from venous HPEC. PLTP-facilitated particle conversion increased the cholesterol efflux capacity of fetal HDL to similar extents (55 and 48%, P < 0.001) from arterial and venous HPEC, respectively., Conclusion: PLTP mediates PL transfer and participates in reverse cholesterol transport pathways at the fetoplacental barrier. Enhanced cellular cholesterol efflux from HPEC to fetal HDL remodeled by PLTP supports the idea of a local atheroprotective role of PLTP in the placental vasculature.

Context: Gestational diabetes mellitus (GDM) causes alterations in fetal high-density lipoproteins (HDL). Because phospholipid transfer protein (PLTP) is important for HDL (re)assembly and is expressed in the human placenta, we hypothesized that circulating fetal and/or placental PLTP expression and activity are altered in GDM., Design: PLTP levels and activity were determined in maternal and fetal sera from GDM and controls. Placental PLTP was immunolocalized, and its expression was measured in placental tissue. PLTP regulation by glucose/insulin was studied in human endothelial cells isolated from placental vessels (HPEC)., Results: Placental Pltp expression was up-regulated in GDM (1.8-fold, P < 0.05). PLTP protein (5-fold, P < 0.01) and activity (1.4- to 2.5-fold) were higher in fetal than in maternal serum. The placental endothelium was identified as a major PLTP location. Insulin treatment of HPEC significantly increased secreted PLTP levels and activity. In GDM, fetal cholesterol, HDL-triglycerides and phospholipids were elevated compared with controls. Fetal PLTP activity was higher than maternal but unaltered in GDM., Conclusion: HPEC contribute to the release of active PLTP into the fetal circulation. Pltp expression is increased in GDM with hyperglycemia and/or hyperinsulinemia contributing. High PLTP activity in fetal serum may enhance conversion of HDL into cholesterol-accepting particles, thereby increasing maternal-fetal cholesterol transfer.

Currently, little is known about the role of intracellular triacylglycerol (TAG) lipases in the brain. Adipose triglyceride lipase (ATGL) is encoded by the PNPLA2 gene and catalyzes the rate-limiting step of lipolysis. In this study, we investigated the effects of ATGL deficiency on brain lipid metabolism in vivo using an established knock-out mouse model (ATGL-ko). A moderate decrease in TAG hydrolase activity detected in ATGL-ko versus wild-type brain tissue was accompanied by a 14-fold increase in TAG levels and an altered composition of TAG-associated fatty acids in ATGL-ko brains. Oil Red O staining revealed a severe accumulation of neutral lipids associated to cerebrovascular cells and in distinct brain regions namely the ependymal cell layer and the choroid plexus along the ventricular system. In situ hybridization histochemistry identified ATGL mRNA expression in ependymal cells, the choroid plexus, pyramidal cells of the hippocampus, and the dentate gyrus. Our findings imply that ATGL is involved in brain fatty acid metabolism, particularly in regions mediating transport and exchange processes: the brain-CSF interface, the blood-CSF barrier, and the blood-brain barrier., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Proteins of the activator protein-1 family are known to have roles in many physiological processes such as proliferation, apoptosis, and inflammation. However, their role in fat metabolism has yet to be defined in more detail. Here we study the impact of JunB deficiency on the metabolic state of mice. JunB knockout (JunB-KO) mice show markedly decreased weight gain, reduced fat mass, and a low survival rate compared with control mice. If fed a high-fat diet, the weight gain of JunB-KO mice is comparable to control mice and the survival rate improves dramatically. Along with normal expression of adipogenic marker genes in white adipose tissue (WAT) of JunB-KO mice, this suggests that adipogenesis per se is not affected by JunB deficiency. This is supported by in vitro data, because neither JunB-silenced 3T3-L1 cells nor mouse embryonic fibroblasts from JunB-KO mice show a change in adipogenic potential. Interestingly, the key enzymes of lipolysis, adipose triglyceride lipase and hormone-sensitive lipase, were significantly increased in WAT of fasted JunB-KO mice. Concomitantly, the ratio of plasma free fatty acids per gram fat mass was increased, suggesting an elevated lipolytic rate under fasting conditions. Furthermore, up-regulation of TNFα and reduced expression of perilipin indicate that this pathway is also involved in increased lipolytic rate in these mice. Additionally, JunB-KO mice are more insulin sensitive than controls and show up-regulation of lipogenic genes in skeletal muscle, indicating a shuttling of energy substrates from WAT to skeletal muscle. In summary, this study provides valuable insights into the impact of JunB deficiency on the metabolic state of mice.

Although maternal-fetal cholesterol transfer may serve to compensate for insufficient fetal cholesterol biosynthesis under pathological conditions, it may have detrimental consequences under conditions of maternal hypercholesterolemia leading to preatherosclerotic lesion development in fetal aortas. Maternal cholesterol may enter fetal circulation by traversing syncytiotrophoblast and endothelial layers of the placenta. We hypothesized that endothelial cells (ECs) of the fetoplacental vasculature display a high and tightly regulated capacity for cholesterol release. Using ECs isolated from human term placenta (HPECs), we investigated cholesterol release capacity and examined transporters involved in cholesterol efflux pathways controlled by liver-X-receptors (LXRs). HPECs demonstrated 2.5-fold higher cholesterol release to lipid-free apolipoprotein (apo)A-I than human umbilical vein ECs (HUVECs), whereas both cell types showed similar cholesterol efflux to high-density lipoproteins (HDLs). Interestingly, treatment of HPECs with LXR activators increased cholesterol efflux to both types of acceptors, whereas no such response could be observed for HUVECs. In line with enhanced cholesterol efflux, LXR activation in HPECs increased expression of ATP-binding cassette transporters ABCA1 and ABCG1, while not altering expression of ABCG4 and scavenger receptor class B type I (SR-BI). Inhibition of ABCA1 or silencing of ABCG1 decreased cholesterol efflux to apoA-I (-70%) and HDL(3) (-57%), respectively. Immunohistochemistry localized both transporters predominantly to the apical membranes of placental ECs in situ. Thus, ECs of human term placenta exhibit unique, efficient and LXR-regulated cholesterol efflux mechanisms. We propose a sequential pathway mediated by ABCA1 and ABCG1, respectively, by which HPECs participate in forming mature HDL in the fetal blood.

Recently, we demonstrated a net blood-to-brain passage of the oxysterol 27-hydroxycholesterol corresponding to 4-5 mg/day. As the steady-state levels of this sterol are only 1-2 mug/g brain tissue, we hypothesized that it is metabolized and subsequently eliminated from the brain. To explore this concept, we first measured the capacity of in vitro systems representing the major cell populations found in the brain to metabolize 27-hydroxycholesterol. We show here that 27-hydroxycholesterol is metabolized into the known C(27) steroidal acid 7alpha-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7alpha-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7alpha-hydroxy-3-oxo-4-cholestenoic acid in plasma from the internal jugular vein and brachial artery of healthy volunteers. Calculation of the arteriovenous concentration difference revealed a significant in vivo flux of this steroid from the brain into the circulation in human. Together, these studies identify a novel metabolic route for the elimination of 27-hydroxylated sterols from the brain. Given the emerging connections between cholesterol and neurodegeneration, this pathway may be of importance for the development of these conditions.

Drug delivery to the brain is severely restricted by formation of tight junctions between adjacent brain capillary endothelial cells (BCEC). In the present study we have evaluated the effects of protamine-oligonucleotide nanoparticles (proticles) on the functional properties of primary porcine BCEC and characterized uptake and transcytosis of proticles by these cells. Proticles had no adverse effects on BCEC properties relevant to blood-brain barrier (BBB) function. Transcytosis of (125)I-labeled proticles across polarized BCEC cultures occurred in a time- and concentration-dependent manner. As apolipoproteins were suggested to enhance cellular proticle uptake, proticle coating was performed with apoA-I, the major apolipoprotein component of high density lipoproteins. Adsorption of apoA-I on the surface of proticles resulted in significantly improved uptake and transcytosis properties as compared to uncoated proticles. ApoA-I coating enhanced proticle delivery to astrocytes in an in vitro model of the BBB almost twofold. Blocking of scavenger receptor class B, type I (the prime receptor for high density lipoprotein/apoA-I that is expressed on BCEC) reduced transcytosis of apoA-I-coated proticles to levels observed for uncoated proticles. Our data indicate that apoA-I-coating of proticles could be a feasible targeting technology to improve delivery across the BBB.

Substantial evidence supports the notion that oxidative processes participate in the pathogenesis of atherosclerotic heart disease. Major evidence for myeloperoxidase (MPO) as enzymatic catalyst for oxidative modification of lipoproteins in the artery wall has been suggested in numerous studies performed with low-density lipoprotein. In contrast to low-density lipoprotein, plasma levels of high-density lipoprotein (HDL)-cholesterol and apoAI, the major apolipoprotein of HDL, inversely correlate with the risk of developing coronary artery disease. These antiatherosclerotic effects are attributed mainly to HDL's capacity to transport excess cholesterol from arterial wall cells to the liver during 'reverse cholesterol transport'. There is now strong evidence that HDL is a selective in vivo target for MPO-catalyzed oxidation impairing the cardioprotective and antiinflammatory capacity of this antiatherogenic lipoprotein. MPO is enzymatically active in human lesion material and was found to be associated with HDL extracted from human atheroma. MPO-catalyzed oxidation products are highly enriched in circulating HDL from individuals with cardiovascular disease where MPO concentrations are also increased. The oxidative potential of MPO involves an array of intermediate-generated reactive oxygen and reactive nitrogen species and the ability of MPO to generate chlorinating oxidants-in particular hypochlorous acid/hypochlorite-under physiological conditions is a unique and defining activity for this enzyme. All these MPO-generated reactive products may affect structure and function of HDL as well as the activity of HDL-associated enzymes involved in conversion and remodeling of the lipoprotein particle, and represent clinically useful markers for atherosclerosis.

The blood-brain barrier contributes to maintain brain cholesterol metabolism and protects this uniquely balanced system from exchange with plasma lipoprotein cholesterol. Brain capillary endothelial cells, representing a physiological barrier to the central nervous system, express apolipoprotein A-I (apoA-I, the major high-density lipoprotein (HDL)-associated apolipoprotein), ATP-binding cassette transporter A1 (ABCA1), and scavenger receptor, class B, type I (SR-BI), proteins that promote cellular cholesterol mobilization. Liver X receptors (LXRs) and peroxisome-proliferator activated receptors (PPARs) are regulators of cholesterol transport, and activation of LXRs and PPARs has potential therapeutic implications for lipid-related neurodegenerative diseases. To clarify the functional impact of LXR/PPAR activation, sterol transport along the: (i) ABCA1/apoA-I and (ii) SR-BI/HDL pathway was investigated in primary, polarized brain capillary endothelial cells, an in vitro model of the blood-brain barrier. Activation of LXR (24(S)OH-cholesterol, TO901317), PPARalpha (bezafibrate, fenofibrate), and PPARgamma (troglitazone, pioglitazone) modulated expression of apoA-I, ABCA1, and SR-BI on mRNA and/or protein levels without compromising transendothelial electrical resistance or tight junction protein expression. LXR-agonists and troglitazone enhanced basolateral-to-apical cholesterol mobilization in the absence of exogenous sterol acceptors. Along with the induction of cell surface-located ABCA1, several agonists enhanced cholesterol mobilization in the presence of exogenous apoA-I, while efflux of 24(S)OH-cholesterol (the major brain cholesterol metabolite) in the presence of exogenous HDL remained unaffected. Summarizing, in cerebrovascular endothelial cells apoA-I, ABCA1, and SR-BI represent drug targets for LXR and PPAR-agonists to interfere with cholesterol homeostasis at the periphery of the central nervous system.

Normal neurological function depends on a constant supply of polyunsaturated fatty acids to the brain. A considerable proportion of essential fatty acids originates from lipoprotein-associated lipids that undergo uptake and/or catabolism at the blood-brain barrier (BBB). This study aimed at identifying expression and regulation of endothelial lipase (EL) in brain capillary endothelial cells (BCEC), major constituents of the BBB. Our results revealed that BCEC are capable of EL synthesis and secretion. Overexpression of EL resulted in enhanced hydrolysis of extracellular high-density lipoprotein (HDL)-associated sn-2-labeled [(14)C]20 : 4 phosphatidylcholine. [(14)C]20 : 4 was recovered in cellular lipids, indicating re-uptake and intracellular re-esterification. To investigate local regulation of EL in the cerebrovasculature, BCEC were cultured in the presence of peroxisome-proliferator activated receptor (PPAR)- and liver X receptor (LXR)-agonists, known to regulate HDL levels. These experiments revealed that 24(S)OH-cholesterol (a LXR agonist), bezafibrate (a PPARalpha agonist), or pioglitazone (a PPARgamma agonist) resulted in down-regulation of EL mRNA and protein levels. Our findings implicate that EL could generate fatty acids at the BBB for transport to deeper regions of the brain as building blocks for membrane phospholipids. In addition PPAR and LXR agonists appear to contribute to HDL homeostasis at the BBB by regulating EL expression.

We previously reported that scavenger receptor class B, type I (SR-BI) mediates uptake of lipoprotein-associated cholesteryl ester and Vitamin E by porcine brain capillary endothelial cells (pBCECs). In the present study we investigated whether SR-BI is capable of mediating phosphatidylcholine (PC) uptake by pBCECs from low- and high density lipoproteins. SR-BI-overexpressing CHO cells and pBCECs showed significantly enhanced uptake rates of PC from both lipoprotein classes. In addition, preincubation of pBCECs in the presence of both lipoprotein species resulted in a significant increase of the cellular fatty acid content, particularly linoleic and arachidonic acid. Our results suggest that uptake of lipoprotein-associated PC by the cerebrovasculature via SR-BI could generate a pool of lipids containing polyunsaturated fatty acids available for transport into deeper regions of the brain.

The present study aimed to investigate pathways that contribute to uptake and transcytosis of high-density lipoproteins (HDLs) and HDL-associated alpha-tocopherol (alpha TocH) across an in vitro model of the blood-brain barrier (BBB). In primary porcine brain capillary endothelial cells HDL-associated alpha TocH was taken up in 10-fold excess of HDL holoparticles, indicating efficient selective uptake, a pathway mediated by scavenger receptor class B, type I (SR-BI). SR-BI was present in caveolae of brain capillary endothelial cells and expressed almost exclusively at the apical membrane. Disruption of caveolae with methyl-beta-cyclodextrin (CDX) resulted in (mis)sorting of SR-BI to the basolateral membrane. Immunohistochemistry of porcine brain cryosections revealed SR-BI expression on brain capillary endothelial cells and presumably astrocytic endfeet. HDL-associated [(14)C]alpha TocH taken up by brain capillary endothelial cells was recovered in sucrose gradient fractions containing the majority of cellular caveolin-1, the major caveolae-associated protein. During mass transfer studies using alpha TocH-enriched HDL, approximately 50% of cellular alpha TocH was recovered with the bulk of cellular caveolin-1 and SR-BI. Efflux experiments revealed that a substantial amount of cell-associated [(14)C]alpha TocH could be mobilized into the culture medium. In addition, apical-to-basolateral transport of HDL holoparticles and HDL-associated alpha TocH was saturable. Results from the present study suggest that part of cerebral apolipoprotein A-I and alpha TocH originates from plasma HDL transcytosed across the BBB and that caveolae-located SR-BI facilitates selective uptake of HDL-associated alpha TocH at the BBB.

The objective of the present study was to investigate the involvement of key players in reverse cholesterol/24(S)OH-cholesterol transport in primary porcine brain capillary endothelial cells (pBCEC) that constitute the BBB. We identified that, in addition to scavenger receptor class B, type I (SR-BI), pBCEC express ABCA1 and apolipoprotein A-I (apoA-I) mRNA and protein. Studies on the regulation of ABCA1 by the liver X receptor agonist 24(S)OH-cholesterol revealed increased ABCA1 expression and apoA-I-dependent [3H]cholesterol efflux from pBCEC. In unpolarized pBCEC, high density lipoprotein, subclass 3 (HDL3)-dependent [3H]cholesterol efflux, was unaffected by 24(S)OH-cholesterol treatment but was enhanced 5-fold in SR-BI overexpressing pBCEC. Efflux of cellular 24(S)-[3H]OH-cholesterol was highly efficient, independent of ABCA1, and correlated with SR-BI expression. Polarized pBCEC were cultured on porous membrane filters that allow separate access to the apical and the basolateral compartment. Addition of cholesterol acceptors to the apical compartment resulted in preferential [3H]cholesterol efflux to the basolateral compartment. HDL3 was a better promoter of basolateral [3H]cholesterol efflux than lipid-free apoA-I. Basolateral pretreatment with 24(S)OH-cholesterol enhanced apoA-I-dependent basolateral cholesterol efflux up to 2-fold along with the induction of ABCA1 at the basolateral membrane. Secretion of apoA-I also occurred preferentially to the basolateral compartment, where the majority of apoA-I was recovered in an HDL-like density range. In contrast, 24(S)-[3H]OH-cholesterol was mobilized efficiently to the apical compartment of the in vitro BBB by HDL3, low density lipoprotein, and serum. These results suggest the existence of an autoregulatory mechanism for removal of potentially neurotoxic 24(S)OH-cholesterol. In conclusion, the apoA-I/ABCA1- and HDL/SR-BI-dependent pathways modulate polarized sterol mobilization at the BBB.

During the present study the contribution of lipoprotein lipase (LPL) to low density lipoprotein (LDL) holoparticle and LDL-lipid (alpha-tocopherol (alphaTocH)) turnover in primary porcine brain capillary endothelial cells (BCECs) was investigated. The addition of increasing LPL concentrations to BCECs resulted in up to 11-fold higher LDL holoparticle cell association. LPL contributed to LDL holoparticle turnover, an effect that was substantially increased in response to LDL-receptor up-regulation. The addition of LPL increased selective uptake of LDL-associated alphaTocH in BCECs up to 5-fold. LPL-dependent selective alphaTocH uptake was unaffected by the lipase inhibitor tetrahydrolipstatin but was substantially inhibited in cells where proteoglycan sulfation was inhibited by treatment with NaClO(3). Thus, selective uptake of LDL-associated alphaTocH requires interaction of LPL with heparan-sulfate proteoglycans. Although high level adenoviral overexpression of scavenger receptor BI (SR-BI) in BCECs resulted in a 2-fold increase of selective LDL-alphaTocH uptake, SR-BI did not act in a cooperative manner with LPL. Although the addition of LPL to BCEC Transwell cultures significantly increased LDL holoparticle cell association and selective uptake of LDL-associated alphaTocH, holoparticle transcytosis across this porcine blood-brain barrier (BBB) model was unaffected by the presence of LPL. An important observation during transcytosis experiments was a substantial alphaTocH depletion of LDL particles that were resecreted into the basolateral compartment. The relevance of LPL-dependent alphaTocH uptake across the BBB was confirmed in LPL-deficient mice. The absence of LPL resulted in significantly lower cerebral alphaTocH concentrations than observed in control animals.

The structural integrity of apolipoprotein A-I (apo A-I) is critical to the physiological function of high-density lipoprotein (HDL). Oxidized lipoproteins are thought to be of central importance in atherogenesis, and oxidation products characteristic of myeloperoxidase, a heme protein secreted by activated phagocytes, have been detected in human atherosclerotic tissue. At plasma concentrations of halide ion, hypochlorous acid is a major product of the myeloperoxidase-hydrogen peroxide-chloride system. We therefore investigated the effects of activated human neutrophils, a potent source of myeloperoxidase and hydrogen peroxide, on the protein and lipid components of HDL. Both free and HDL-associated apo A-I exposed to activated human neutrophils underwent extensive degradation as monitored by RP-HPLC and Western blotting with a polyclonal antibody to apo A-I. Replacement of the neutrophils with reagent HOCl resulted in comparable damage (at molar oxidant : HDL subclass 3 ratio = 100) as observed in the presence of activated phagocytes. Apo A-I degradation by activated neutrophils was partially inhibited by the HOCl scavenger methionine, by the heme inhibitor azide, by chloride-free conditions, by the peroxide scavenger catalase, and by a combination of superoxide dismutase (SOD)/catalase, implicating HOCl in the cell-mediated reaction. The addition of a protease inhibitor (3,4-dichloroisocoumarin) further reduced the extent of apo A-I damage. In contrast to the protein moiety, there was little evidence for oxidation of unsaturated fatty acids or cholesterol in HDL3 exposed to activated neutrophils, suggesting that HOCl was selectively damaging apo A-I. Our observations indicate that HOCl generated by myeloperoxidase represents one pathway for protein degradation in HDL3 exposed to activated phagocytes.

alpha-Tocopherol (alphaTocH) is transported in association with lipoproteins in the aqueous milieu of the plasma. Although up to 50% of circulating alphaTocH is transported by high-density lipoproteins (HDLs), little is known about the mechanisms of uptake of HDL-associated alphaTocH. During the current study, human apolipoprotein (apo)E-free HDL subclass 3 (HDL3) labelled with [14C]alphaTocH was used to investigate uptake mechanisms of HDL3-associated alphaTocH by a permanent hepatoblastoma cell line (HepG2). HDL3-associated alphaTocH was taken up independently of HDL3 holoparticles in excess of apoA-I comparable with the non-endocytotic delivery of cholesteryl esters to cells termed the 'selective' cholesteryl ester uptake pathway. Experiments with unlabelled HDL3 demonstrated net mass transfer of alphaTocH to HepG2 cells. Time-dependent studies with [14C]alphaTocH-labelled HDL3 revealed tracer uptake in 80-fold excess of apoA-I and in 4-fold excess of cholesteryl linoleate. In addition to HLDs, low-density lipoprotein (LDL)-associated alphaTocH was also taken up in excess of holoparticles, although to a lesser extent. These findings were confirmed with unlabelled lipoprotein preparations, in which HDL3 displayed a 2- to 3-fold higher alphaTocH donor efficiency than LDLs (lipoproteins adjusted for equal amounts of alphaTocH). An important factor affecting particle-independent uptake of alphaTocH was the cellular cholesterol content (a 2-fold increase in cellular cholesterol levels resulted in a 2.3-fold decrease in uptake). Pulse-chase studies demonstrated that some of the HDL3-associated alphaTocH taken up independently of holoparticle uptake was resecreted along with a newly synthesized apoB-containing lipoprotein fraction.

A simple, accurate, and fast procedure for quantitative analysis of fatty acids (FA) in simple lipid subclasses from different biological specimens is presented. Lipid extracts of isolated plasma lipoproteins (very low, low, and high density lipoproteins; VLDL, LDL, and HDL, respectively) and permanent J774 mouse macrophages were fractionated into lipid subclasses by thin-layer chromatography (TLC) on silica gel 60 plates. Bands comigrating with authentic lipid standards were scraped off under argon and subjected to direct, in situ transesterification with BF3/MeOH in the presence of the TLC adsorbent. Fatty acid methyl esters were subsequently quantitated by capillary gas chromatography. A comparison of the FA content present in total lipid extracts and in lipid subclasses separated by TLC revealed recoveries ranging from 93 (J774 cell extracts) to 99.7% (LDL). The method described is applicable for the measurement of FA in individual lipid subclasses and was successfully applied to quantitatively analyze the FA composition of the phospholipid, triacylglycerol, and cholesteryl ester fraction derived from VLDL, LDL, and HDL. In J774 lipid extracts, the FA composition of the phospholipid-, monoacylglycerol-, diacylglycerol-, free fatty acid-, triacylglycerol-, and cholesteryl ester fraction was quantitated. In addition we have analyzed the time-dependent loss of the major HDL polyunsaturated fatty acids (18:2, 20:4) in the phospholipid and cholesteryl ester fraction during copper-dependent peroxidation of HDL. We have not encountered analytical problems concerning low FA recoveries from CE-rich lipid extracts as indicated by almost quantitative recoveries of FA in LDL, HDL, and J774 extracts.