Gabriele Plenz

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.

Vascular collagens: spotlight on the role of type VIII collagen in atherogenesis

Collagens play a central role in maintaining the integrity and stability of the undiseased as well as of the atherosclerotic vessel wall. An imbalanced metabolism may lead to uncontrolled collagen accumulation reducing vessel wall velocity, frequently resulting in arterial occlusion or thrombosis. A reduced production of collagen and its uncontrolled degradation may affect the stability of the vessel wall and especially of the atherosclerotic plaques by making them prone to rupture and aneurysm. This review presents an overview on the four groups of vascular collagens and on their role in atherogenesis. The major focus was to highlight the extraordinary role and importance of the short chain network forming type VIII collagen in the extracellular matrix of undiseased arteries and of atherosclerotic plaques. The molecular structure of type VIII collagen, its cellular origin, its implication in atherogenesis, its temporal and spatial expression patterns in human and experimental models of atherogenesis, the factors modulating its expression, and--not at least--its potential function is discussed.

Lipids partition caveolin-1 from ER membranes into lipid droplets: updating the model of lipid droplet biogenesis

Caveolin‐1, a putative mediator of intracellular cholesterol transport, is generally assumed to be integrated into the cytoplasmic leaflets of all cellular membranes. Lipid droplets form by budding at the endoplasmic reticulum (ER), and caveolin‐1 is thought to be transferred to the droplet surface along with the cytoplasmic leaflet of ER membranes and not to enter the droplet core. We explored how caveolin‐1 accesses lipid droplets from the ER by localizing caveolin‐1 in ER membranes and in lipid droplets in cultured smooth muscle cells using freeze‐fracture immunocytochemistry. We detected caveolin‐1 in endoplasmic leaflets of ER membranes but never in cytoplasmic leaflets. Caveolin‐1 was also present in lipid droplet cores. These findings are incompatible with the current hypothesis of lipid droplet biogenesis. We suggest that the inherent high affinity of caveolin‐1 for neutral lipids causes caveolin‐1 molecules to be extracted from the endoplasmic leaflets of ER membranes and to be transferred into the droplet core by inundating lipids during droplet formation.

Proinflammatory Cytokines Regulate LOX-1 Expression in Vascular Smooth Muscle Cells

Objective—Atherogenesis represents a type of chronic inflammation and involves elements of the immune response, eg, the expression of proinflammatory cytokines. In advanced atherosclerotic lesions, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is expressed in endothelial cells, macrophages, and smooth muscle cells (SMCs). In vitro, the expression of LOX-1 is induced by inflammatory cytokines like TNF-&agr; and transforming growth factor (TGF)-&bgr;. Therefore, LOX-1 is thought to be upregulated locally in response to cytokines in vivo. Methods and Results—We determined by reverse-transcription polymerase chain reaction (PCR) and Western blot analysis whether the mediators of the acute phase response in inflammation, IL-1&agr;, IL-1&bgr;, and TNF-&agr;, regulate LOX-1 expression in cultured SMC, and whether this regulation is influenced by peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;). We studied by immunohistochemistry whether these cytokines are spatially correlated with LOX-1 expression in advanced atherosclerotic lesions. We found upregulation of LOX-1 expression in SMC in a dose- and time-dependent manner after incubation with IL-1&agr;, IL-1&bgr;, and TNF-&agr;. Simultaneous incubation with these cytokines at saturated concentrations had an additive effect on LOX-1 expression. The PPAR&ggr; activator, 15d-PGJ2, however, inhibited IL-1&bgr;–induced upregulation of LOX-1. In the intima of atherosclerotic lesions regions of IL-1&agr;, IL-1&bgr;, and TNF-&agr; expression corresponded to regions of LOX-1 expression. Conclusion—We suppose that upregulated LOX-1 expression in SMC of advanced atherosclerotic lesions is a response to these proinflammatory cytokines. Moreover, the proinflammatory effects of these cytokines can be decreased by the antiinflammatory effect of PPAR&ggr;.

Paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation and prevention of apoptosis in collagen lattices

Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat fibroblasts. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and 20% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of fibroblasts, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) fibroblast-conditioned media; 3) exogenous decorin; or 4) treatment with individual growth factors known to be involved in angiogenesis. The mechanism(s) by which fibroblasts induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of fibroblasts for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.

The interleukin-6/interleukin-6-receptorsystem is activated in donor hearts

OBJECTIVES To assess the potential of the donor heart to respond to interleukin-6 (IL6), the present study investigated the expression of IL6 receptor components in the myocardium of donor hearts before transplantation. BACKGROUND Donor heart dysfunction early after transplantation has been associated with the cytokine storm after donor brain death. Proinflammatory cytokines are thought to play a central role in this process. Interleukin-6 is of specific interest because it has been associated with cardiac allograft dysfunction and is related to an impaired prognosis. Its action requires expression of the specific IL6 receptor (IL6R), and the common signal transducer of the IL6 family glycoprotein 130 (gp130) in the donor heart. METHODS The activation of IL6, IL6R and gp130 messenger ribonucleic acid (mRNA) and protein was studied via reverse transcription-polymerase chain reaction (RT-PCR) and immunohistology in donor hearts (n = 6) and compared with patients undergoing evaluation of ventricular arrhythmias (control, n = 9) or with advanced heart failure (n = 20). RESULTS Messenger RNA of IL6, IL6R and gp130 was strongly expressed in all chambers of donor hearts, whereas right ventricles of control patients did not show any expression (donor vs. control: p < 0.005). Right ventricles of failing hearts showed IL6, IL6R and gp130 mRNA levels comparable with those found in donor hearts. Immunohistochemistry paralleled the RT-PCR data on the protein level. While IL6 was mainly expressed by myocytes, both receptor components were preferentially found mainly on interstitial cells. CONCLUSIONS The expression of the IL6 receptor components in the donor heart before transplantation establishes the condition sine qua non for the response of the donor heart to circulating IL6. This mechanism may explain the close association of elevated IL6 serum levels to acute cardiac allograft dysfunction in the early perioperative period.

Expression of the Novel Scavenger Receptor SR-PSOX in Cultured Aortic Smooth Muscle Cells and Umbilical Endothelial Cells

To the Editor: In the November issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Minami et al1 demonstrated expression of the novel scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX) in lipid-laden macrophages accumulated in the intima of human atherosclerotic lesions. Because SR-PSOX seems to be identical to the membrane-anchored chemokine CXCL16,2,3⇓ which may play a dual role in inflammation and homeostasis, Minami et al1 discussed the potential regulation of SR-PSOX by pro-inflammatory cytokines. Although the authors did not detect SR-PSOX in smooth muscle cells (SMCs) and endothelial cells (ECs), they did discuss the possible expression of SR-PSOX in these cell types. Until now, only the expression of the scavenger receptors SR-AI/II,4 CD36,5 and LOX-16 in SMCs has been described. In our studies on the formation of SMC-derived foam cells during atherogenesis, we have focused on the expression of scavenger receptors,7 including SR-PSOX, in SMCs and ECs. We have also investigated the influence of cytokines on the expression of SR-PSOX in SMCs. Reverse transcriptase–polymerase chain reaction (PCR; primers for human SR-PSOX: 5′-TACACGAGGTTCCAGCTCCT-3′ and 5′-GGGGGCTGGT- AGGAAGTAAA-3′, porcine SR-PSOX: 5′-TATGTGGAGGCAGCAG- TGAC-3′ and …

Human macrophages synthesize type VIII collagen in vitro and in the atherosclerotic plaque

Type VIII collagen is a short‐chain collagen that is present in increased amounts in atherosclerotic lesions. Although the physiological function of this matrix protein is unclear, recent data suggest an important role in tissue remodeling. Type VIII collagen in the atherosclerotic lesion is mainly derived from smooth muscle cells. We now show that macrophages in the atherosclerotic vessel wall and monocytes in adjacent mural thrombi also express type VIII collagen. We demonstrated this using a novel combined fluorescence technique that simultaneously stains, within the same tissue section, specific RNAs by in situ hybridization and proteins by indirect immunofluorescence. In culture, human monocyte/macrophages expressed type VIII collagen at all time points from1 h to 3 wk after isolation. Western blotting and immunoprecipitation also revealed secretion of type VIII collagen into the medium of 14‐day‐old macrophages. Because this is the first report of secretion of a collagen by macrophages, we tested the effect of lipopolysaccharide (LPS) and interferon γ, substances that stimulate macrophages to secrete lytic enzymes, on macrophage expression of type VIII collagen. LPS and interferon γ decreased expression of type VIII collagen. By contrast, secretion of matrix metalloproteinase 1 (MMP 1) was increased, indicating a switch from a collagen‐producing to a degradative phenotype. Double in situ hybridization studies of expression of type VIII collagen and MMP 1 in human coronary arteries showed that in regions important for plaque stability, the ratio of MMP 1 RNA to macrophage type VIII collagen RNA varies widely, indicating that the transition from one phenotype to the other that we observed in vitro may also occur in vivo.—Weitkamp, B., Cullen, P., Plenz, G., Robenek, H., Rauterberg, J. Human macrophages synthesize type VIII collagen in vitro and in the atherosclerotic plaque. FASEB J. 13, 1445–1457 (1999)

Reversal of metallothionein expression is different throughout the human myocardium after prolonged left-ventricular mechanical support

OBJECTIVES We examined the distribution of metallothionein (MT), a stress-inducible protein, and the cardiomyocyte diameter in human hearts after left-ventricular assist device (LVAD) support. BACKGROUND Remodeling in end-stage heart failure is characterized by myocyte hypertrophy and alterations of several inducible proteins. LVADs used as a bridge to cardiac transplantation unload the left ventricle and may lead to a reversal of the remodeling, but little is known about the pathophysiology of this process. METHODS The immunoreactivity for MT and the cardiomyocyte diameter was analyzed in left-ventricular tissue specimens of 17 patients with end-stage heart failure before and after LVAD support. RESULTS MT positive cells were mainly located sub-endocardially in vacuolized cardiomyocytes and in small vessels throughout the myocardium. During LVAD support, MT-positive myocytes decreased in the sub-endocardial (p < 0.008) and sub-epicardial region (p < 0.003), MT-positive vessels decreased similarly (p < 0.003). Cardiomyocyte diameter decreased significantly only in the sub-endocardium (p < 0.03). Hearts of patients supported longer than 88 days (= median) showed substantially lower MT reactivity at the time of LVAD explantation as compared to patients supported less than 88 days. CONCLUSION Our results suggest that unloading of the left ventricle during prolonged LVAD support leads to regression of cellular hypertrophy and a decrease of MT expression. The preferential reduction of MT-positive vacuolized cardiomyocytes in the sub-endocardium is comparable with the concept of greatest reduction of wall stress in this area of the myocardium and may be due to the improvement of myocardial blood flow and the energy balance.

Differential Expression of Connexin43 and Desmin Defines Two Subpopulations of Medial Smooth Muscle Cells in the Human Internal Mammary Artery

Upregulation of connexin43-gap junctions is associated with transition of contractile vascular smooth muscle cells (SMCs) to the synthetic state. To determine whether phenotypically distinct subpopulations of medial SMCs differentially express connexin43, we investigated the human distal internal mammary artery, a structurally heterogeneous vessel with features ranging from elastic to elastomuscular to muscular. Immunoconfocal microscopy combined with quantitative analysis and complemented by in situ hybridization showed that SMCs in the elastic medial regions expressed high levels of connexin43 but low levels of desmin, whereas those of muscular medial regions expressed low levels of connexin43 but high levels of desmin. Ultrastructurally, SMCs of both regions were of the contractile phenotype, but the former cells were irregular in shape with relatively prominent synthetic organelles whereas the latter were spindle shaped with fewer synthetic organelles. Vimentin, smooth muscle alpha-actin, calponin, h-caldesmon, and myosin heavy chains (SM1 and SM2) were equally highly expressed by most cells in both subpopulations. The connexin43/desmin expression pattern of SMCs in regions of intimal thickening resembled those of elastic medial regions. These findings refine the view suggested from previous studies that high levels of connexin43 expression are associated with SMCs of a less contractile/more synthetic phenotype. In the internal mammary artery, the 2 subpopulations of SMCs with markedly different connexin43 expression levels both represent a differentiated contractile phenotype, but the subpopulation showing high levels of connexin43-gap junctions is characterized by low levels of desmin and structural features that reflect a more synthetic tendency.