Ernst Malle

The matrix component biglycan is proinflammatory and signals through Toll-like receptors 4 and 2 in macrophages

Biglycan, a small leucine-rich proteoglycan, is a ubiquitous ECM component; however, its biological role has not been elucidated in detail. Here we show that biglycan acts in macrophages as an endogenous ligand of TLR4 and TLR2, which mediate innate immunity, leading to rapid activation of p38, ERK, and NF-kappaB and thereby stimulating the expression of TNF-alpha and macrophage inflammatory protein-2 (MIP-2). In agreement, the stimulatory effects of biglycan are significantly reduced in TLR4-mutant (TLR4-M), TLR2-/-, and myeloid differentiation factor 88-/- (MyD88-/-) macrophages and completely abolished in TLR2-/-/TLR4-M macrophages. Biglycan-null mice have a considerable survival benefit in LPS- or zymosan-induced sepsis due to lower levels of circulating TNF-alpha and reduced infiltration of mononuclear cells in the lung, which cause less end-organ damage. Importantly, when stimulated by LPS-induced proinflammatory factors, macrophages themselves are able to synthesize biglycan. Thus, biglycan, upon release from the ECM or from macrophages, can boost inflammation by signaling through TLR4 and TLR2, thereby enhancing the synthesis of TNF-alpha and MIP-2. Our results provide evidence for what is, to our knowledge, a novel role of the matrix component biglycan as a signaling molecule and a crucial proinflammatory factor. These findings are potentially relevant for the development of new strategies in the treatment of sepsis.

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.

Serum amyloid A (SAA): an acute phase protein and apolipoprotein.

Serum amyloid A (SAA) proteins comprise a family of apolipoproteins coded for by at least three genes with allelic variation and a high degree of homology between species. The synthesis of certain members of the family is greatly increased in inflammation. However, SAA is not often used as an acute-phase marker despite being at least as sensitive as C-reactive protein. SAA proteins can be considered as apolipoproteins since they associate with plasma lipoproteins mainly within the high density range, perhaps through amphipathic alpha-helical structure. It is not known why certain subjects expressing SAA develop secondary systemic amyloidosis. There is still no specific function attributed to SAA; however, a popular hypothesis suggests that SAA may modulate metabolism of high density lipoproteins (HDL). This may impede the protective function of HDL against the development of atherosclerosis. The potential significance of the association between SAA and lipoproteins needs further evaluation.

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.

Increased Hepatic Myeloperoxidase Activity in Obese Subjects with Nonalcoholic Steatohepatitis

Inflammation and oxidative stress are considered critical factors in the progression of nonalcoholic fatty liver disease. Myeloperoxidase (MPO) is an important neutrophil enzyme that can generate aggressive oxidants; therefore, we studied the association between MPO and nonalcoholic fatty liver disease. The distribution of inflammatory cells containing MPO in liver biopsies of 40 severely obese subjects with either nonalcoholic steatohepatitis (NASH) (n = 22) or simple steatosis (n = 18) was investigated by immunohistochemistry. MPO-derived oxidative protein modifications were identified by immunohistochemistry and correlated to hepatic gene expression of CXC chemokines and M1/M2 macrophage markers as determined by quantitative PCR. MPO plasma levels were determined by ELISA. The number of hepatic neutrophils and MPO-positive Kupffer cells was increased in NASH and was accompanied by accumulation of hypochlorite-modified and nitrated proteins, which can be generated by the MPO-H2O2 system. Liver CXC chemokine expression was higher in patients with accumulation of MPO-mediated oxidation products and correlated with hepatic neutrophil sequestration. Plasma MPO levels were elevated in NASH patients. Interestingly, neutrophils frequently surrounded steatotic hepatocytes, resembling the crown-like structures found in obese adipose tissue. Furthermore, hepatic M2 macrophage marker gene expression was increased in NASH. Our data indicate that accumulation of MPO-mediated oxidation products, partly derived from Kupffer cell MPO, is associated with induction of CXC chemokines and hepatic neutrophil infiltration and may contribute to the development of NASH.

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.

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.

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)

Influence of native and hypochlorite-modified low-density lipoprotein on gene expression in human proximal tubular epithelium

Inflammatory infiltrates can modify (lipo)proteins via hypochlorous acid/hypochlorite (HOCl/OCl(-)) an oxidant formed by the myeloperoxidase-H(2)O(2)-halide system. These oxidatively modified proteins emerge in tubuli in some proteinuric and interstitial diseases. Human proximal tubular cells (HK-2) were used to confirm the hypothesis of detrimental and differential impact of HOCl-modified low density lipoprotein (HOCl-LDL), an in vivo occurring lipoprotein modification exerting proatherogenic and proinflammatory capacity. HOCl-LDL showed dose-dependent antiproliferative effects in HK-2 cells. Small dedicated cDNA macroarrays were used to identify differentially regulated genes. A rapid increase in the expression of genes involved in reactive oxygen species metabolism and cell stress, eg, heme oxygenase-1, thioredoxin reductase, cytochrome b5 reductase, Gadd 153, amino acid transporter E16, and HSP70 was found after HOCl-LDL treatment of HK-2 cells. In parallel, genes involved in tissue remodeling and inflammation eg, CTGF, VCAM-1, IL-1beta, MMP7, and VEGF were up-regulated. Quantitative RT-PCR verified differential expression of a subset of these genes in microdissected tubulointerstitia from patients with acute tubular damage, progressive proteinuric renal disease, and membranous glomerulonephritis (with declining renal function), but not in stable patients with proteinuria caused by minimal change disease. The demonstration of selective up-regulation of a subgroup of genes if proteinuria is accompanied by the presence of HOCl-modified (lipo)proteins support the potential pathophysiological role of the myeloperoxidase-H(2)O(2)-halide system and HOCl-LDL in renal disease.