J. Metz

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)

Morphological alterations and functional changes of interhepatocellular junctions induced by bile duct ligation

SummaryThe effect of bile duct ligation on the intercellular junctions of hepatocytes was investigated. The features and the arrangement of the bile canaliculi and the zonulae occludentes alter concomitant to the increase of the intracanalicular pressure. The lumen of the bile canaliculi enlarges and the microvilli disappear. The array of the zonulae occludentes becomes irregularly shaped, the number of strands diminishes and interruptions of the strands occur. With peroxidase a leakage in the bile-blood barrier is detected. Furthermore a disappearance of gap junctions between the hepatocytes after bile duct ligation is observed. The present investigation shows that the zonulae occludentes are mobile structures which are changed by increased unilateral pressure. Due to their ultrastructural alterations, a leakage of the permeability barrier between physiological compartments is found.We acknowledge the helpful criticism and discussion of Prof. H.D. Fahimi. We are indebted to Mrs. B. Brühl, M. Bürkle and Ch. Walenta for technical assistance, and to stud. med. Jon Greenberg for preparing the manuscript

The right auricle of the heart is an endocrine organ

SummaryA new polypeptide hormone candidate regulating vascular smooth muscle function was extracted from porcine atrial tissue. The purification steps were followed by a bioassay. The hormonally active substance has been analyzed and found to be a small polypeptide exhibiting a molecular weight of about 7500 and is named “cardiodilatin” (CDD). Further chemical data on this new hormone will be published elsewhere. A partial amino acid sequence of cardiodilatin is offered and shows that among the well known hormones or neuropeptides, none exhibit a homologue partial sequence.

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)

The auricular myocardiocytes of the heart constitute an endocrine organ characterization of a porcine cardiac peptide hormone, cardiodilatin-126

SummaryA peptide hormone was extracted from the porcine right atrium following a bioassay for differential vaso-relaxant effects on smooth muscle strips from aorta and renal and inferior mesenteric arteries. The isolation procedure included several steps of gel-permeation and ion-exchange chromatography, and high performance liquid chromatography. During the isolation procedure, other peptides of smaller molecular weight were also found, which, in relation to cardiodilatin-126 (CDD-126), are shorter at their N-terminal. Among these, CDD-88 has also been isolated and characterizied, and has been established as a prominent member of the cardiac hormone family. The N-terminal and C-terminal segments of the 126 amino acid-containing molecule were synthesized and used to raise region-specific antibodies. The natural peptide was then localized within myoendocrine cells of the right atrium where specific atrial granules are located. Renal effects of cardiodilation were studied in conscious dogs and showed strong diuretic and natriuretic activities. According to our functional studies, cardiodilatin-126 and cardiodilatin-88 possess qualities of a significant hormone family regarding the regulation of extracellular fluid volume and blood pressure.

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.

In vivo intravascular electric impedance spectroscopy using a new catheter with integrated microelectrodes.

Abstract Interventional techniques are necessary, which allow the characterization of intravascular pathological processes. Electric impedance spectroscopy (EIS) can provide cellular information of biological tissue. We tested the feasibility of intravascular EIS by using a new impedance catheter system with integrated microelectrodes in an experimental animal model. Eighteen stents were implanted into the iliac arteries of female New Zealand White rabbits (n = 11) to induce intimal proliferation. After 14, 28 and 56 days the electric impedance was measured inside and outside of the stented arterial segments by using a balloon catheter with four integrated microelectrodes. The impedance was recorded at a frequency ranging from 1 Hz to 1 MHz. After the measurements, the stents were explanted and histomorphometry was performed. The impedance inside and outside the stent was analysed and compared with the histomorphometric data.Fourteen (n = 6), 28 (n = 5) and 56 (n = 6) days after stent implantation the difference of the electrical impedance between the native and the stented iliac artery segment increased from –924 ± 715 Ohm to 3689 ± 1385 Ohm (14 days vs. 28 days; p < 0.05) and 8637 ± 2881 Ohm (14 days vs. 56 days; p < 0.05), respectively. The increase of the electrical impedance corresponded to an increased neointimal proliferation in the stented arterial segment of 3.6% ± 0.7% after 14 days, 8.4% ± 4.8% after 28 days (14 days vs. 28 days; p < 0.05) and 10.0% ± 4.1% after 56 days (14 days vs. 56 days; p < 0.01).Intravascular EIS can be performed by a balloon catheter with integrated microelectrodes and allows the detection of neointimal proliferation after stent implantation.

Intravascular electric impedance spectroscopy of atherosclerotic lesions using a new impedance catheter system

Newer techniques are required to identify atherosclerotic lesions that are prone to rupture. Electric impedance spectroscopy (EIS) can characterize biological tissues by measuring the electrical impedance over a frequency range. We tested a newly designed intravascular impedance catheter (IC) by measuring the impedance of different stages of atherosclerosis induced in an animal rabbit model. Six female New Zealand White rabbits were fed for 17 weeks with a 5% cholesterol–enriched diet to induce early forms of atherosclerotic plaques. All aortas were prepared from the aortic arch to the renal arteries and segments of 5–10 mm were marked by ink spots. A balloon catheter system with an integrated polyimide–based microelectrode structure was introduced into the aorta and the impedance was measured at each spot by using an impedance analyzer. The impedance was measured at frequencies of 1 kHz and 10 kHz and compared with the corresponding histomorphometric data of each aortic segment.Forty–four aortic segments without plaques and 48 segments with evolving atherosclerotic lesions could be exactly matched by the histomorphometric analysis. In normal aortic segments (P0) the change of the magnitude of impedance at 1 kHz and at 10 kHz (|Z|1 kHz – |Z|10 kHz, = ICF) was 208.5 ± 357.6 Ω. In the area of aortic segments with a plaque smaller than that of the aortic wall diameter (PI), the ICF was 137.7 ± 192.8 Ω. (P 0 vs. P I; p = 0.52), whereas in aortic segments with plaque formations larger than the aortic wall (PII) the ICF was significantly lower –22.2 ± 259.9 Ω. (P0 vs. PII; p = 0.002). Intravascular EIS could be successfully performed by using a newly designed microelectrode integrated onto a conventional coronary balloon catheter. In this experimental animal model atherosclerotic aortic lesions showed significantly higher ICF in comparison to the normal aortic tissue.

Ceramide induces aSMase expression: implications for oxLDL-induced apoptosis

Sphingomyelinase (SMase) stimulation and subsequent ceramide generation are suggested to be involved in signal transduction of stress‐induced apoptosis. We now show that apoptosis of human macrophages (MФ) and fibroblasts initiated by oxi¬dized low density lipoproteins (minimally modified LDL, mmLDL) is associated with an increase in acid SMase (aSMase, E.C. 3.1.4.12) expression and ceramide concentration. Application of a novel, potent, and specific inhibitor of aSMase expression (NB6) diminished the effects of mmLDL and C6‐ceramide treatment by inhibiting transcription via Sp1 and AP‐2. Moreover, apoptosis was abolished after mmLDL and C6‐ceramide treatment of hereditary aSMase‐deficient fibroblasts (from Niemann‐Pick patients). We suggest that in mmLDL‐initiated apoptosis 1) enhanced ceramide generation via aSMase appears to be required as well as 2) a positive feedback control of aSMase expression by the increase in intracellular ceramide concentration.—Deigner, H.‐P., Claus, R., Bonaterra, G. A., Gehrke, C., Bibak, N., Blaess, M., Cantz, M., Metz, J., Kinscherf, R. Ceramide induces aSMase ex¬pression: implications for oxLDL‐induced apoptosis. FASEBJ. 15, 807–814 (2001)