Wolfgang Sattler

Myeloperoxidase: a target for new drug development?

Myeloperoxidase (MPO), a member of the haem peroxidase‐cyclooxygenase superfamily, is abundantly expressed in neutrophils and to a lesser extent in monocytes and certain type of macrophages. MPO participates in innate immune defence mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity of MPO is its ability to use chloride as a cosubstrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent. However, evidence has emerged that MPO‐derived oxidants contribute to tissue damage and the initiation and propagation of acute and chronic vascular inflammatory disease. The fact that circulating levels of MPO have been shown to predict risks for major adverse cardiac events and that levels of MPO‐derived chlorinated compounds are specific biomarkers for disease progression, has attracted considerable interest in the development of therapeutically useful MPO inhibitors. Today, detailed information on the structure of ferric MPO and its complexes with low‐ and high‐spin ligands is available. This, together with a thorough understanding of reaction mechanisms including redox properties of intermediates, enables a rationale attempt in developing specific MPO inhibitors that still maintain MPO activity during host defence and bacterial killing but interfere with pathophysiologically persistent activation of MPO. The various approaches to inhibit enzyme activity of MPO and to ameliorate adverse effects of MPO‐derived oxidants will be discussed. Emphasis will be put on mechanism‐based inhibitors and high‐throughput screening of compounds as well as the discussion of physiologically useful HOCl scavengers.

Uptake and transport of high‐density lipoprotein (HDL) and HDL‐associated α‐tocopherol by an in vitro blood–brain barrier model

The present study aimed to investigate pathways that contribute to uptake and transcytosis of high‐density lipoproteins (HDLs) and HDL‐associated α‐tocopherol (αTocH) across an in vitro model of the blood–brain barrier (BBB). In primary porcine brain capillary endothelial cells HDL‐associated αTocH 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‐β‐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 [14C]αTocH 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 αTocH‐enriched HDL, approximately 50% of cellular αTocH was recovered with the bulk of cellular caveolin‐1 and SR‐BI. Efflux experiments revealed that a substantial amount of cell‐associated [14C]αTocH could be mobilized into the culture medium. In addition, apical‐to‐basolateral transport of HDL holoparticles and HDL‐associated αTocH was saturable. Results from the present study suggest that part of cerebral apolipoprotein A‐I and αTocH originates from plasma HDL transcytosed across the BBB and that caveolae‐located SR‐BI facilitates selective uptake of HDL‐associated αTocH at the BBB.

Alterations in Lipid Metabolism Mediate Inflammation, Fibrosis, and Proliferation in a Mouse Model of Chronic Cholestatic Liver Injury

BACKGROUND & AIMSnThe liver controls central processes of lipid and bile acid homeostasis. We aimed to investigate whether alterations in lipid metabolism contribute to the pathogenesis of chronic cholestatic liver disease in mice.nnnMETHODSnWe used microarray and metabolic profiling analyses to identify alterations in systemic and hepatic lipid metabolism in mice with disruption of the gene ATP-binding cassette sub-family B member 4 (Abcb4(-/-) mice), a model of inflammation-induced cholestatic liver injury, fibrosis, and cancer.nnnRESULTSnAlterations in Abcb4(-/-) mice, compared with wild-type mice, included deregulation of genes that control lipid synthesis, storage, and oxidation; decreased serum levels of cholesterol and phospholipids; and reduced hepatic long-chain fatty acyl-CoAs (LCA-CoA). Feeding Abcb4(-/-) mice the side chain-modified bile acid 24-norursodeoxycholic acid (norUDCA) reversed their liver injury and fibrosis, increased serum levels of lipids, lowered phospholipase and triglyceride hydrolase activities, and restored hepatic LCA-CoA and triglyceride levels. Additional genetic and nutritional studies indicated that lipid metabolism contributed to chronic cholestatic liver injury; crossing peroxisome proliferator-activated receptor (PPAR)-α-deficient mice with Abcb4(-/-) mice (to create double knockouts) or placing Abcb4(-/-) mice on a high-fat diet protected against liver injury, with features similar to those involved in the response to norUDCA. Placing pregnant Abcb4(-/-) mice on high-fat diets prevented liver injury in their offspring. However, fenofibrate, an activator of PPARα, aggravated liver injury in Abcb4(-/-) mice.nnnCONCLUSIONSnAlterations in lipid metabolism contribute to the pathogenesis and progression of cholestatic liver disease in mice.

2-Chlorohexadecanal Derived From Hypochlorite-Modified High-Density Lipoprotein–Associated Plasmalogen Is a Natural Inhibitor of Endothelial Nitric Oxide Biosynthesis

Objective—Myeloperoxidase, a heme enzyme that is present and active in human atherosclerotic lesions, provides a source for the generation of proinflammatory chlorinated reactants contributing to endothelial dysfunction. Modification of high-density lipoprotein (HDL) by hypochlorous acid/hypochlorite (HOCl/Oce−)—generated in vivo by the myeloperoxidase-hydrogen peroxide-chloride system of activated phagocytes—forms a proatherogenic lipoprotein particle that binds to and is internalized by endothelial cells. Methods and Results—Here we show that HDL, modified with physiologically relevant HOCl concentrations, attenuates the expression and activity of vasculoprotective endothelial nitric oxide synthase. HOCl-HDL promotes dislocalization of endothelial nitric oxide synthase from the plasma membrane and perinuclear location of human umbilical venous endothelial cells. We could identify 2-chlorohexadecanal as the active component mediating this inhibitory activity. This chlorinated fatty aldehyde is formed during HOCl-mediated oxidative cleavage of HDL-associated plasmalogen. Conclusion—2-Chlorohexadecanal, produced by the myeloperoxidase-hydrogen peroxide-chloride system of activated phagocytes may act as a mediator of vascular injury associated with ischemia-reperfusion injury, glomerulosclerosis, and atherosclerosis.

Novel route for elimination of brain oxysterols across the blood-brain barrier: Conversion into 7α-hydroxy-3-oxo-4-cholestenoic acid

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 μg/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 C27 steroidal acid 7α-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7α-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7α-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.

Lipoprotein-associated α-tocopheryl-succinate inhibits cell growth and induces apoptosis in human MCF-7 and HBL-100 breast cancer cells

K-Tocopheryl succinate (K-TS) is a potent inhibitor of tumor cell proliferation. The goal of the present study was to investigate whether and to what extent K-TS associates with plasma lipoproteins and if K-TS-enriched lipoproteins inhibit breast cancer cell growth in a manner comparable to the free drug. In vitro enrichment of human plasma revealed that K-TS readily associated with the main lipoprotein classes, findings confirmed in vivo in mice. At the highest K-TS concentrations, lipoproteins carrying 50 000 (VLDL), 5000 (LDL) and 700 (HDL) K-TS molecules per lipoprotein particle were generated. KTS enrichment generated lipoprotein particles with slightly decreased density and increased particle radius. To study whether the level of LDL-receptor (LDL-R) expression affects K-TS uptake from apoB/E containing lipoprotein particles human breast cancer cells with low (MCF-7) and normal (HBL-100) LDL-R expression were used. The uptake of free, VLDL- and (apoE-free) HDL3-associated K-TS was nearly identical for both cell lines. In contrast, uptake of LDL-associated K-TS by HBL-100 cells (normal LDL-R expression) was about twice as high as compared to MCF-7 cells (low LDL-R expression). VLDL and LDL-associated K-TS inhibited proliferation most effectively at the highest concentration of K-TS used (100% inhibition of MCF-7 growth with 20 Wg/ml of lipoprotein-associated K-TS). However, also K-TS-free VLDL and LDL inhibited HBL-100 cell proliferation up to 55%. In both cell lines, K-TS-enriched HDL3 inhibited cell growth by 40^60%. Incubation of both cell lines in the presence of free or lipoprotein-associated K-TS resulted in DNA fragmentation indicative of apoptosis. Collectively, the present findings demonstrate that: (1) K-TS readily associates with lipoproteins in vitro and in vivo; (2) the lipoprotein-enrichment efficacy was dependent on the particle size and/or the triglyceride content of the lipoprotein; (3) uptake of LDL-associated K-TS was apparently dependent on the level of LDL-R expression ; and (4) lipoproteins were efficient K-TS carriers inducing reduced cell proliferation rates and apoptosis in human breast cancer cells as observed for the free drug. fl 2000 Elsevier Science B.V. All rights reserved.

Cholesterylester hydroperoxide reducing activity associated with isolated high- and low-density lipoproteins

Exposure of isolated high-(HDL) and low-density lipoproteins (LDL) to aqueous peroxyl radicals generated from a thermo-labile azo-compound resulted in immediate formation of cholesteryllinoleate hydroxide (Ch18:2-OH) in addition to hydroperoxides of cholesteryllinoleate (Ch18:2-OOH) and phospholipids. Ch18:2-OH was also formed in peroxyl radical-oxidizing human plasma devoid of ascorbate or low molecular weight compounds or isolated lipoproteins in the presence of desferrioxamine. In contrast, peroxyl radical-mediated oxidation of HDL or LDL lipid extracts or detergent-solubilized lipoproteins resulted in the formation of Ch18:2-OOH without concomitant formation of Ch18:2-OH. Heat treatment of the isolated lipoproteins prior to oxidation greatly reduced Ch18:2-OH formation. Compared to the concentrations of Ch18:2-OOH accumulating, formation of Ch18:2-OH was more pronounced in oxidizing HDL than LDL isolated from the same blood donor. The levels of Ch18:2-OH detected after prolonged oxidation periods were independent of the radical flux to which the lipoproteins were exposed. In the absence of peroxyl radical generator, [3H]Ch18:2-OOH associated with HDL was converted readily and in a biphasic manner into [3H]Ch18:2-OH upon incubation at 37 but not 4 degrees C. LDL-associated [3H]Ch18:2-OOH were also reduced, albeit with an initial reaction rate approximately 10 times slower than that observed with labelled HDL. Together, the results show that cholesterylester hydroxides are formed during (peroxyl) radical-mediated oxidation of isolated intact HDL and LDL under transition metal-free conditions. The findings suggest the presence of a hydroperoxide reducing activity in isolated human lipoproteins, particularly HDL.

Sequential Synthesis and Methylation of Phosphatidylethanolamine Promote Lipid Droplet Biosynthesis and Stability in Tissue Culture and in Vivo

Triacylglycerols are stored in eukaryotic cells within lipid droplets (LD). The LD core is enwrapped by a phospholipid monolayer with phosphatidylcholine (PC), the major phospholipid, and phosphatidylethanolamine (PE), a minor component. We demonstrate that the onset of LD formation is characterized by a change in cellular PC, PE, and phosphatidylserine (PS). With induction of differentiation of 3T3-L1 fibroblasts into adipocytes, the cellular PC/PE ratio decreased concomitant with LD formation, with the most pronounced decline between confluency and day 5. The mRNA for PS synthase-1 (forms PS from PC) and PS decarboxylase (forms PE from PS) increased after day 5. Activity and protein of PE N-methyltransferase (PEMT), which produces PC by methylation of PE, are absent in 3T3-L1 fibroblasts but were induced at day 5. High fat challenge induced PEMT expression in mouse adipose tissue. PE, produced via PS decarboxylase, was the preferred substrate for methylation to PC. A PEMT-GFP fusion protein decorated the periphery of LD. PEMT knockdown in 3T3-L1 adipocytes correlated with increased basal triacylglycerol hydrolysis. Pemt−/− mice developed desensitization against adenosine-mediated inhibition of basal hydrolysis in adipose tissue, and adipocyte hypotrophy was observed in Pemt−/− animals on a high fat diet. Knock-out of PEMT in adipose tissue down-regulated PS synthase-1 mRNA, suggesting coordination between PE supply and converting pathways during LD biosynthesis. We conclude that two consecutive processes not previously related to LD biogenesis, (i) PE production via PS and (ii) PE conversion via PEMT, are implicated in LD formation and stability.

Uptake of Lipoprotein-Associated α-Tocopherol by Primary Porcine Brain Capillary Endothelial Cells

Abstract: From the severe neurological syndromes resulting from vitamin E deficiency, it is evident that an adequate supply of the brain with α‐tocopherol (αTocH), the biologically most active member of the vitamin E family, is of utmost importance. However, uptake mechanisms of αTocH in cells constituting the blood‐brain barrier are obscure. Therefore, we studied the interaction of low (LDL) and high (HDL) density lipoproteins (the major carriers of αTocH in the circulation) with monolayers of primary porcine brain capillary endothelial cells (pBCECs) and compared the ability of these two lipoprotein classes to transfer lipoprotein‐associated αTocH to pBCECs. With regard to potential binding proteins, we could identify the presence of the LDL receptor and a putative HDL3 binding protein with an apparent molecular mass of 100 kDa. At 4°C, pBCECs bound LDL with high affinity (KD = 6 nM) and apolipoprotein E‐free HDL3 with low affinity (98 nM). The binding capacity was 20,000 (LDL) and 200,000 (HDL3) lipoprotein particles per cell. αTocH uptake was approximately threefold higher from HDL3 than from LDL when [14C]αTocH‐labeled lipoprotein preparations were used. The majority of HDL3‐associated αTocH was taken up in a lipoprotein particle‐independent manner, exceeding HDL3 holoparticle uptake 8‐ to 20‐fold. This uptake route is less important for LDL‐associated αTocH (αTocH uptake ∼1.5‐fold higher than holoparticle uptake). In line with tracer experiments, mass transfer studies with unlabeled lipoproteins revealed that αTocH uptake from HDL3 was almost fivefold more efficient than from LDL. Biodiscrimination studies indicated that uptake efficacy for the eight different stereoisomers of synthetic αTocH is nearly identical. Our findings indicate that HDL could play a major role in supplying the central nervous system with αTocH in vivo.

Hypochlorite-modified albumin colocalizes with RAGE in the artery wall and promotes MCP-1 expression via the RAGE-Erk1/2 MAP-kinase pathway

Signal transduction via the endothelial receptor for advanced glycation end products (RAGE) plays a key role in vascular inflammation. Recent observations have shown that the myeloperoxidase‐H2O2‐chloride system of activated phagocytes is highly up‐regulated under inflammatory conditions where hypochlorous acid (HOCl) is formed as the major oxidant. Albumin, an in vivo carrier for myeloperoxi‐dase is highly vulnerable to oxidation and a major representative of circulating advanced oxidized proteins during inflammatory diseases. Immunohistochem‐ical studies performed in the present study revealed marked colocalization of HOCl‐modified epitopes with RAGE and albumin in sections of human atheroma, mainly at the endothelial lining. We show that albumin modified with physiologically relevant concentrations of HOCl, added as reagent or generated by the myelo‐peroxidase‐H2O2‐chloride system, is a high affinity li‐gand for RAGE. Albumin, modified by HOCl in the absence of free amino acids/carbohydrates/lipids to exclude formation of AGE‐like structures, induced a rapid, RAGE‐dependent activation of extracellular signal‐regulated kinase 1/2 and up‐regulation of the proin‐flammatory mediator monocyte chemoattractant pro‐tein‐1. Cellular activation could be blocked either by a specific polyclonal anti‐RAGE IgG and/or a specific mitogen‐activated protein‐kinase kinase inhibitor. The present study demonstrates that HOCl‐modified albumin acts as a ligand for RAGE and promotes RAGEmediated inflammatory complications.—Marsche, G., Semlitsch, M., Hammer, A., Frank, S., Weigle, B., Demling, N., Schmidt, K., Windischhofer, W., Waeg, G., Sattler, W., Malle, E. Hypochlorite‐modified albumin colocalizes with RAGE in the artery wall and promotes MCP‐1 expression via the RAGE‐Erk1/2 MAP‐kinase pathway. FASEB J. 21, 1145–1152 (2007)