Ralf Kinscherf

Involvement of growth differentiation factor-15/macrophage inhibitory cytokine-1 (GDF-15/MIC-1) in oxLDL-induced apoptosis of human macrophages in vitro and in arteriosclerotic lesions.

Growth differentiation factor-15/macrophage inhibitory cytokine-1 (GDF-15/MIC-1) is a new member of the transforming growth factor beta (TGF-β) superfamily, which has most recently been found in activated macrophages (MΦ). We have now investigated GDF-15/MIC-1 in human MΦ after exposure to oxidized low-density lipoproteins (oxLDL) related mediators in vitro and in arteriosclerotic carotid arteries. Using RT-PCR and Western blotting a pronounced induction of GDF-15/MIC-1 expression by oxLDL, C6-ceramide, tumor necrosis factor (TNFα) and hydrogen peroxide (H2O2) was found in cultured human MΦ. In 11 human arteriosclerotic carotid arteries, immunohistochemical analyses supported by computer-assisted morphometry and regression analyses demonstrated a significant colocalization of GDF-15/MIC-1 immunoreactivity (IR) with oxLDL IR and manganese superoxide dismutase (MnSOD) IR in CD68 immunoreactive (ir) MΦ, which were also expressing AIF-IR (apoptosis-inducing factor), caspase-3-IR (CPP32), PARP-IR, c-Jun/AP-1-IR and p53-IR. Our data suggest that GDF-15/MIC-1 is inducible in human MΦ by oxLDL and its mediators in vitro and is supposed to contribute to oxidative stress dependent consequences in arteriosclerotic plaques, e.g. modulating apoptosis and inflammatory processes in activated MΦ.

Apoptosis caused by oxidized LDL is manganese superoxide dismutase and p53 dependent

Oxidized low density lipoprotein (oxLDL) induces apoptosis in human macrophages (MΦ), a significant feature in atherogenesis. We found that induction of apoptosis in MΦ by oxLDL, C2‐ceramide, tumor necrosis factor α (TNF‐α), and hydrogen peroxide (H2O2) was associated with enhanced expression of manganese superoxide dis‐mutase (MnSOD) and p53. Treatment of cells with p53 or MnSOD antisense oligonucleotides prior to stimulation with oxLDL, C2‐ceramide, TNF‐α, or H2O2 caused an inhibition of the expression of the respective protein together with a marked reduction of apoptosis. Exposure to N‐acetylcysteine before treatment with oxLDL, C2‐ceramide, TNF‐α, or H2O2 reversed a decrease in cellular glutathione concentrations as well as the enhanced production of p53 and MnSOD mRNA and protein. In apoptotic macrophages of human atherosclerotic plaques, colocalization of MnSOD and p53 immunoreactivity was found. These results indicate that in oxLDL‐induced apoptosis, a concomitant induction of p53 and MnSOD is critical, and suggest that it is at least in part due to an enhancement of the sphingomyelin/ ceramide pathway.—Kinscherf, R., Claus, R., Wagner, M., Gehrke, C., Kamencic, H., Hou, D., Nauen, O., Schmiedt, W., Kovacs, G., Pill, J., Metz, J., Deigner, H.‐P. Apoptosis caused by oxidized LDL is manganese superoxide dismutase and p53 dependent. FASEB J. 12, 461–467 (1998)

Pure β-ParticleEmitting Stents Inhibit Neointima Formation in Rabbits

Background Considerable experimental evidence exists that neointimal hyperplasia after angioplasty is inhibited by γ-irradiation of the treated arteries. A β-particle radiation is absorbed in tissue within a shorter distance away from the source than γ-radiation and may be more suitable for localized vessel irradiation. This study outlines a method to implant a β-particle–emitting radioisotope ( 32 P; half-life, 14.3 days) into metallic stents. The effects of these stents on the inhibition of neointimal hyperplasia was compared with conventional stents in a rabbit model. Methods and Results 32 P was produced by irradiation of red amorphous phophorus ( 31 P) with neutrons and was implanted into Palmaz-Schatz stents (7.5 mm in length) after being kept apart from 31 P in a mass separator. The radioisotope was tightly fixed to the stents, and the ion implantation process did not alter the surface texture. Stent activity levels of 4 and 13 μCi were chosen for the study. Four and 12 weeks after placement of conventional stents and 32 P-implanted stents in rabbit iliac arteries, vascular injury and neointima formation were studied by histomorphometry. Immunostaining for smooth muscle cell (SMC) α-actin was performed to determine SMC cellularity in the neointima. SMCs were quantified by computer-assisted counting of α-actin immunoreactive cells. Endothelialization of the stents was evaluated by immunostaining for endothelial cell von Willebrand factor. No difference in vessel wall injury was found after placement of conventional and 32 P-implanted stents. Neointima formation was potently inhibited by 32 P-implanted stents only at an activity level of 13 μCi after 4 and 12 weeks. Neointimal SMC cellularity was reduced in 32 P-implanted stents compared with conventional stents. Radioactive stents were endothelialized after 4 weeks, but endothelialization was less dense than in conventional stents. Conclusions Neointima formation in rabbits is markedly suppressed by a β-particle–emitting stent incorporating the radioisotope 32 P. In this model, a dose-response relation with this type of radioactive stent was observed, indicating that a threshold radiation dose must be delivered to inhibit neointima formation after stent placement over the long term.

Complement C6 Deficiency Protects Against Diet-Induced Atherosclerosis in Rabbits

Low-density lipoprotein (LDL) can be transformed to an atherogenic moiety by nonoxidative, enzymatic degradation. Enzymatically degraded LDL induces macrophage foam cell formation, provokes release of cytokines, and also activates complement. To determine whether complement activation may contribute to atherogenesis, 6 pairs of homozygous C6-deficient rabbits and their non-C6-deficient heterozygous siblings were fed a cholesterol-rich diet for 14 weeks. Cholesterol levels and plasma lipoprotein profiles of the animals in the C6-competent and C6-deficient groups did not significantly differ, and the high density lipoprotein and LDL cholesterol ratios at the end of the experiment were 0.07+/-0.01 and 0.08+/-0.01 (SEM), respectively. However, differences in atherosclerotic plaque formation were discernible macroscopically, with extensive aortic lesions being visible in all C6-competent animals and absent in all C6-deficient animals. Aortas were sectioned from thorax to abdomen, and 10 sections were stained from each aorta. Quantification of atherosclerotic lesions and lumen stenosis with the use of computer-based morphometry documented a dramatic protective effect of C6 deficiency on the development of diet-induced atherosclerosis. We conclude that the terminal complement sequence is centrally involved in atherosclerotic lesion progression.

Apoptosis modulators in the therapy of neurodegenerative diseases

Apoptosis is a prerequisite to model the developing nervous system. However, an increased rate of cell death in the adult nervous system underlies neurodegenerative disease and is a hallmark of multiple sclerosis (MS) Alzheimer’s- (AD), Parkinson- (PD), or Huntington’s disease (HD). Cell surface receptors (e.g., CD95/APO-1/Fas; TNF receptor) and their ligands (CD95-L; TNF) as well as evolutionarily conserved mechanisms involving proteases, mitochondrial factors (e.g., Bcl-2-related proteins, reactive oxygen species, mitochondrial membrane potential, opening of the permeability transition pore) or p53 participate in the modulation and execution of cell death. Effectors comprise oxidative stress, inflammatory processes, calcium toxicity and survival factor deficiency. Therapeutic agents are being developed to interfere with these events, thus conferring the potential to be neuroprotective. In this context, drugs with anti-oxidative properties, e.g., flupirtine, N-acetylcysteine, idebenone, melatonin, but also novel dopamine agonists (ropinirole and pramipexole) have been shown to protect neuronal cells from apoptosis and thus have been suggested for treating neurodegenerative disorders like AD or PD. Other agents like non-steroidal anti-inflammatory drugs (NSAIDs) partly inhibit cyclooxygenase (COX) expression, as well as having a positive influence on the clinical expression of AD. Distinct cytokines, growth factors and related drug candidates, e.g., nerve growth factor (NGF), or members of the transforming growth factor-β (TGF-β ) superfamily, like growth and differentiation factor 5 (GDF-5), are shown to protect tyrosine hydroxylase or dopaminergic neurones from apoptosis. Furthermore, peptidergic cerebrolysin has been found to support the survival of neurones in vitro and in vivo. Treatment with protease inhibitors are suggested as potential targets to prevent DNA fragmentation in dopaminergic neurones of PD patients. Finally, CRIB (cellular replacement by immunoisolatory biocapsule) is an auspicious gene therapeutical approach for human NGF secretion, which has been shown to protect cholinergic neurones from cell death when implanted in the brain. This review summarises and evaluates novel aspects of anti-apoptotic concepts and pharmacological intervention including gene therapeutical approaches currently being proposed or utilised to treat neurodegenerative diseases.

Expression of Growth Differentiation Factor-15/ Macrophage Inhibitory Cytokine-1 (GDF-15/MIC-1) in the Perinatal, Adult, and Injured Rat Brain

We and others have recently cloned a new member of the transforming growth factor‐β superfamily, growth differentiation factor‐15/ macrophage inhibitory cytokine‐1 (GDF‐15/MIC‐1). Using in situ hybridization and immunohistochemistry, we determined the distribution of GDF‐15/MIC‐1 mRNA and protein in the perinatal and cryolesioned adult rat brain. The choroid plexus epithelium of all ventricles represents the site of strongest and almost exclusive mRNA expression in the normal perinatal and adult brain. The newborn rat brain reveals GDF‐15/MIC‐1 immunoreactivity (ir) in ependymal cells lining the ventricles, in the striatal subventricular zone, and in populations of nonneural cells of the thalamic/hippocampal lamina affixa, in addition to that in the choroid plexus. Unilateral cryogenic cortical lesioning induced a significant increase of GDF‐15/MIC‐1 mRNA expression and ir at the lesion site and expression in presumed neurons within the dorsal thalamic area. At the lesion site, GDF‐15/MIC‐1‐producing cells showed immuncytochemical features of neurons, macrophages, and activated microglial cells. Flourescent microscopy revealed both intra‐ and extracellular GDF‐15/MIC‐1 ir. Up‐regulation of GDF‐15/MIC‐1 in activated macrophages (Mϕ) is also supported by RT‐PCR, ICC, and Western blot experiments showing pronounced induction of GDF‐15/MIC‐1 expression (mRNA and protein) in retinoic acid/phorbol ester‐stimulated human Mϕ. Our data suggest that 1) GDF‐15/MIC‐1 is secreted into the cerebrospinal fluid and 2) in the newborn brain may penetrate through the ependymal lining and act on developing neurons and/or glial cells. As a constituent of cells in the lamina affixa, the protein might be involved in the regulation of mesenchyme–epithelial interactions. Finally, GDF‐15/MIC‐1 may also act within the antiinflammatory cytokine network activated in CNS lesions. J. Comp. Neurol. 439:32–45, 2001.

Induction of mitochondrial manganese superoxide dismutase in macrophages by oxidized LDL: its relevance in atherosclerosis of humans and heritable hyperlipidemic rabbits.

The objective of the study was to analyze the intracellular antioxidative response of macrophages (MΦ) exposed to increased levels of low density lipoprotein (LDL). We studied manganese superoxide dismutase (MnSOD) and, in part, GSH in cultured human and rabbit MΦ, and in atheromatous arterial tissue of humans and heritable hyperlipidemic (HHL) rabbits. Incubation of human MΦ with oxidized‐LDL (ox‐LDL) resulted in an induction of MnSOD mRNA production as shown by RT‐PCR. MnSOD immunoreactivity (IR) was found to be located in the mitochondria of MΦ. In HHL rabbits, MnSOD activity and GSH concentration were significantly increased in atherosclerotic intima compared to the media of the aorta, but significantly decreased (P<0.01) in larger plaques compared with smaller ones, resulting in a significant inverse correlation of MnSOD activity (r=–0.67, P<0.001) and GSH concentration (r=–0.57, P<0.01) with plaque size. Immunohistology of the atherosclerotic intima revealed MnSOD‐IR in Mac‐1 (CD 11b/CD 18)‐immunoreactive (ir) MΦ of human arteries and, similarly, in RAM‐11‐ir MΦ of rabbit ones. The relation of MnSOD‐ir MΦ decreased with plaque advancement, which is consistent with biochemical findings. Most MnSOD‐ir MΦ in atherosclerotic plaques revealed TUNEL‐positive nuclei, indicating DNA strand breaks, and p53‐IR. We conclude that mitochondrial antioxidants such as MnSOD are induced in MΦ in vitro and in atherosclerotic arteries as a reply to increased mitochondrial oxidation. As normal consequences of an increased oxidative stress due to the exposure to ox‐LDL nuclear DNA strand breaks occur, which are suggested to be a signal to increase p53 protein levels. Reactive oxygen species‐mediated mitochondrial‐dependent pathways are suggested as major contributing pathomechanisms to nuclear damage, which eventually may result in apoptosis. A common response to increased oxidative stress due to modified LDL is presumed in rabbit and human atherosclerotic plaques.—Kinscherf, R., Deigner, H.‐P., Usinger, C., Pill, J., Wagner, M., Kamencic, H., Hou, D., Chen, M., Schmiedt, W., Schrader, M., Kovacs, G., Kato, K., Metz, J. Induction of mitochondrial manganese superoxide dismutase in macrophages by oxidized LDL: its relevance in atherosclerosis of humans and heritable hyperlipidemic rabbits. FASEB J. 11, 1317–1328 (1997)

Effect of glutathione depletion and oral N-acetyl-cysteine treatment on CD4+ and CD8+ cells.

HIV‐infected individuals and SIV‐infected rhesus macaques have, on the average, decreased plasma cysteine and cystine concentrations and decreased intracellular glutathione levels. We show that the cysteine supply and the intracellular glutathione levels have a strong influence on the T cell system, A study of healthy human subjects revealed that persons with intracellular glutathione levels of 20‐30 nmol/mg protein had significantly higher numbers of CD4+ T cells than persons with either lower or higher glutathione levels. Persons who moved during a 4‐weck observation period from the optimal to the suboptimal range (10‐20 nmol/mg) experienced, on the average, a 30% decrease in CD4+ T cell numbers. This decrease was prevented by treatment with N‐acetyl‐cysteine (NAC). NAC caused this relative increase of CD4+ T cell numbers in spite of decreasing glutathione levels and not by increasing the glutathione level. Our studies suggest that the immune system may be exquisitely sensitive not only against a cysteine and glutathione deficiency but also against an excess of cysteine.—Kinscherf, R., Fischbach, T., Mihm, S., Roth, S., Hohenhaus‐Sievert, E., Weiss, C., Edler, L., Bärtsch, P., Dröge, W. Effect of glutathione depletion and oral N‐acetyl‐cysteine treatment on CD4+ and CD8+ cells. FASEB J. 8: 448‐451; 1994.

Role of increased sphingomyelinase activity in apoptosis and organ failure of patients with severe sepsis

Numerous studies support the notion that an activation of sphingomyelinases and a subsequent increase of the concentration of the bioactive lipid mediator ceramide are critical in the concert of inflammatory stimuli and to the induction of apoptosis during inflammation. Here we show that patients with severe sepsis exhibit an enhanced sphingolytic activity in comparison with controls [262 pmol/(mlxh) vs. 123.6 pmol/(mlxh), P<0.005]. During the clinical course, a further increase was paralleled by the severity of illness and by fatal outcome. Moreover, we show that oxidative stress may partially account for the increased activity through posttranslational modification of the enzyme. In a murine endotoxic shock model, administration of a low molecular weight inhibitor diminished the rise in enzymatic activity and improved the survival rate. In liver specimen, inhibition of activity correlated with a reduced rate of hepato‐cellular apoptosis. Our data support the concept that activation of the plasmatic isoform of sphingomyelinase may play a critical role in the development of apoptosis and organ failure in sepsis. An inhibition of the secreted isoform of sphingomyelinase should be explored further as a potential target in the complicated puzzle of sepsis.

Apoptosis After Stent Implantation Compared With Balloon Angioplasty in Rabbits: Role of Macrophages

Both cell proliferation and apoptosis (programmed cell death) are supposed to play a role in restenosis after angioplasty. We studied these processes in smooth muscle cells (SMCs) and macrophages 1, 4, and 12 weeks after balloon angioplasty or Palmaz-Schatz stent implantation in rabbit iliac arteries. Proliferating cells were visualized by immunostaining with antibodies directed against proliferating cell nuclear antigen. Apoptotic cells were detected using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling) technique, propidium iodide staining, and transmission electron microscopy. At all time points, the neointimal cross-sectional area of the arteries was twofold to fourfold greater after stent implantation than after balloon angioplasty. The total number of neointimal cells was similar 1 and 12 weeks after both interventions. The neointimal cell density, however, decreased by 58% between the 1st and the 12th week after stent implantation compared with a 20% decrease after balloon angioplasty (P < .01). Stent implantation induced more cell proliferation but also more apoptosis in the media than balloon angioplasty after 1 and 4 weeks. In addition, stent implantation caused more macrophage accumulation and apoptosis in the neointima, but cell proliferation rates did not differ significantly in comparison with balloon angioplasty. The higher rate of apoptosis in the neointima 1 week after stent implantation compared with balloon angioplasty is due to an increased rate of SMC and macrophage death. Macrophage accumulation and apoptosis in the early phase after stent implantation appear to play a role in extracellular matrix secretion, which increases neointima formation after 4 and 12 weeks compared with balloon angioplasty in this model.