Rolf Gräbner

Expanding expression of the 5-lipoxygenase pathway within the arterial wall during human atherogenesis

Oxidation products of low-density lipoproteins have been suggested to promote inflammation during atherogenesis, and reticulocyte-type 15-lipoxygenase has been implicated to mediate this oxidation. In addition, the 5-lipoxygenase cascade leads to formation of leukotrienes, which exhibit strong proinflammatory activities in cardiovascular tissues. Here, we studied both lipoxygenase pathways in human atherosclerosis. The 5-lipoxygenase pathway was abundantly expressed in arterial walls of patients afflicted with various lesion stages of atherosclerosis of the aorta and of coronary and carotid arteries. 5-lipoxygenase localized to macrophages, dendritic cells, foam cells, mast cells, and neutrophilic granulocytes, and the number of 5-lipoxygenase expressing cells markedly increased in advanced lesions. By contrast, reticulocyte-type 15-lipoxygenase was expressed at levels that were several orders of magnitude lower than 5-lipoxygenase in both normal and diseased arteries, and its expression could not be related to lesion pathology. Our data support a model of atherogenesis in which 5-lipoxygenase cascade-dependent inflammatory circuits consisting of several leukocyte lineages and arterial wall cells evolve within the blood vessel wall during critical stages of lesion development. They raise the possibility that antileukotriene drugs may be an effective treatment regimen in late-stage disease.

The 5-lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent aortic aneurysm

Activation of the 5-lipoxygenase (5-LO) pathway leads to the biosynthesis of proinflammatory leukotriene lipid mediators. Genetic studies have associated 5-LO and its accessory protein, 5-LO-activating protein, with cardiovascular disease, myocardial infarction and stroke. Here we show that 5-LO-positive macrophages localize to the adventitia of diseased mouse and human arteries in areas of neoangiogenesis and that these cells constitute a main component of aortic aneurysms induced by an atherogenic diet containing cholate in mice deficient in apolipoprotein E. 5-LO deficiency markedly attenuates the formation of these aneurysms and is associated with reduced matrix metalloproteinase-2 activity and diminished plasma macrophage inflammatory protein-1α (MIP-1α; also called CCL3), but only minimally affects the formation of lipid-rich lesions. The leukotriene LTD4 strongly stimulates expression of MIP-1α in macrophages and MIP-2 (also called CXCL2) in endothelial cells. These data link the 5-LO pathway to hyperlipidemia-dependent inflammation of the arterial wall and to pathogenesis of aortic aneurysms through a potential chemokine intermediary route.

Lymphotoxin β receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE−/− mice

Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE−/− mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin β receptor (LTβR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE−/− mice with LTβR-Ig to interrupt LTβR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTβR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

l-Ascorbic Acid Potentiates Nitric Oxide Synthesis in Endothelial Cells

Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether l-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1–100 μm ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 μm) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 μm and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor moleculel-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate l-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid.

Hypochlorite-modified Low Density Lipoprotein Inhibits Nitric Oxide Synthesis in Endothelial Cells via an Intracellular Dislocalization of Endothelial Nitric-oxide Synthase

Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H2O2/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.

Mouse Aorta Smooth Muscle Cells Differentiate Into Lymphoid Tissue Organizer-Like Cells on Combined Tumor Necrosis Factor Receptor-1/Lymphotoxin β-Receptor NF-κB Signaling

Objective—Mouse aorta smooth muscle cells (SMC) express tumor necrosis factor receptor superfamily member 1A (TNFR-1) and lymphotoxin &bgr;-receptor (LT&bgr;R). Circumstantial evidence has linked the SMC LT&bgr;R to tertiary lymphoid organogenesis in hyperlipidemic mice. Here, we explored TNFR-1 and LT&bgr;R signaling in cultured SMC. Methods and Results—TNFR-1 signaling activated the classical RelA NF-&kgr;B pathway, whereas LT&bgr;R signaling activated the classical RelA and alternative RelB NF-&kgr;B pathways, and both signaling pathways synergized to enhance p100 inhibitor processing to the p52 subunit of NF-&kgr;B. Microarrays showed that simultaneous TNFR-1/LT&bgr;R activation resulted in elevated mRNA encoding leukocyte homeostatic chemokines CCL2, CCL5, CXCL1, and CX3CL1. Importantly, SMC acquired features of lymphoid tissue organizers, which control tertiary lymphoid organogenesis in autoimmune diseases through hyperinduction of CCL7, CCL9, CXCL13, CCL19, CXCL16, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. TNFR-1/LT&bgr;R cross-talk resulted in augmented secretion of lymphorganogenic chemokine proteins. Supernatants of TNFR-1/LT&bgr;R–activated SMC markedly supported migration of splenic T cells, B cells, and macrophages/dendritic cells. Experiments with ltbr−/− SMC indicated that LT&bgr;R-RelB activation was obligatory to generate the lymphoid tissue organizer phenotype. Conclusion—SMC may participate in the formation of tertiary lymphoid tissue in atherosclerosis by upregulation of lymphorganogenic chemokines involved in T-lymphocyte, B-lymphocyte, and macrophage/dendritic cell attraction.

Flow cytometric determination of E‐selectin, vascular cell adhesion molecule‐1, and intercellular cell adhesion molecule‐1 in formaldehyde‐fixed endothelial cell monolayers

BACKGROUND Endothelial cell adhesion molecules are involved in initiation and progression of vascular diseases. The purpose of this study was to determine conditions of fixation and dissociation of human umbilical vein endothelial cell (HUVEC) monolayers that permit a reliable flow cytometric determination of intracellular and surface content of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). METHODS TNFalpha-treated HUVEC monolayers were fixed with 0.5% formaldehyde at the end of the experimental incubation. Subsequently, either the monolayer was trypsinized and thereafter the cells were subjected to indirect fluorescence labeling or the monolayer was first labeled and then dissociated by trypsinization. Cell integrity was assessed by vimentin staining. Total adhesion molecule content was detected in saponin-permeabilized cells. RESULTS HUVEC integrity was maintained when the fixation time of the monolayer did not exceed 5 min and trypsin/EDTA was used for dissociation. Surface adhesion molecules were partially hydrolyzed by trypsin when trypsinization preceded labeling but antibody binding protected adhesion molecules from degradation. VCAM-1 and E-selectin exhibited substantial trypsin-sensitive surface fractions but surface ICAM-1 was mainly trypsin resistant. Permeabilization with 0.06% saponin allowed the detection of considerable intracellular pools of the investigated adhesion molecules. CONCLUSIONS The described method permits the reliable determination of surface and intracellular fractions of adhesion molecules in formaldehyde-fixed HUVEC monolayers and may be used for studies on the regulation of adhesion molecule expression.

Genetic and pharmacological inhibition of the 5-lipoxygenase/leukotriene pathway in atherosclerotic lesion development in ApoE deficient mice

The 5-lipoxygenase (5-LO) catalyzed formation of leukotriene (LT) lipid mediators is a pathway contributing to inflammatory events in asthma and more recently has been associated with cardiovascular disease. However, the relative impact of this pathway in atherogenesis has been controversial and a variety of mixed results reported. The goal of these studies was to assess the importance of the 5-LO/LT pathway in mice with either genetic (5-LO(-/-)) or pharmacological (L-739,010) inhibition of the 5-LO pathway on an apolipoprotein E deficient (apoE(-/-)) background when subjected to either an 8-week (Paigen) or 6 months (Western) atherosclerotic diet regimen. Atherosclerotic lesion analysis at the aortic root, brachiocephalic artery and throughout the whole aorta by en face Sudan IV staining was determined, as well as blood lipid levels. Ex vivo calcium ionophore-stimulation of whole blood demonstrated a significant reduction in the capacity to form LTB(4) in 5-LO(-/-) and drug-treated 5-LO(+/+) mice. Quantitative analysis of atherosclerotic lesions did not differ between groups at all three sites. Moreover, the composition of advanced lesions in the brachiocephalic arteries did not indicate altered plaque disruption as a result of 5-LO gene inactivation. These results do not support a role for the 5-LO/LT pathway in intermediate to advanced atherosclerotic lesion development in mice.

Laser-Capture Microdissection of Hyperlipidemic/ApoE −/− Mouse Aorta Atherosclerosis

Atherosclerosis is a transmural chronic inflammatory condition of small and large arteries that is associated with adaptive immune responses at all disease stages. However, impacts of adaptive immune reactions on clinically apparent atherosclerosis such as intima lesion (plaque) rupture, thrombosis, myocardial infarction, and aneurysm largely remain to be identified. It is increasingly recognized that leukocyte infiltrates in plaque, media, and adventitia are distinct but that their specific roles have not been defined. To map these infiltrates, we employed laser-capture microdissection (LCM) to isolate the three arterial wall laminae using apoE⁻/⁻ mouse aorta as a model. RNA from LCM-separated tissues was extracted and large-scale, whole-genome expression microarrays were prepared. We observed that the quality of the resulting gene expression maps was compromised by tissue RNA carried over from adjacent laminae during LCM. To account for these flaws, we established quality controls and algorithms to improve the predictive power of LCM-derived microarray data. Our approach creates robust transcriptome atlases of normal and atherosclerotic aorta. Assessing LCM transcriptomes for immunity-related mRNAs indicated markedly distinctive gene expression patterns in the three laminae of the atherosclerotic aorta. These mouse mRNA expression data banks can now be mined to address a wide range of questions in cardiovascular biology.