Ralf A. Claus

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)

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.

Anti-platelet drugs and outcome in severe infection: Clinical impact and underlying mechanisms

Platelet activation contributes to microvascular thrombosis and organ failure in systemic inflammation. We tested the hypothesis whether anti-platelet drugs might favourably affect outcome in patients at risk for organ failure as well as in a mouse model of endotoxin shock. Two hundred twenty-four consecutive patients who were admitted for community acquired pneumonia over a time period of 5 years to a University Hospital were enrolled; about 20% of whom received anti-platelet drugs (acetylsalicylic acid, thienopyridines) for secondary prevention of cardiovascular disease. Patients with anti-platelet drugs were about 12 years old but did not differ in SOFA score and routine laboratory parameters at admission. Logistic regression and 2 × 2 table analysis in age-matched subgroups indicated that anti-platelet drugs may reduce the need of intensive care treatment (odds ratio (OR) 0.32 [95% confidential interval: 0.10–1.00] and 0.19 [0.04–0.87], respectively). In age-matched subgroups, the use of anti-platelet drugs was also associated with a shorter stay in hospital (13.9 ± 6.2 vs. 18.2 ± 10.2 days; p < 0.02). In the animal model Balb/c mice were pre-treated with clopidogrel (added to drinking water) for 4 days prior to intraperitoneal (i.p.) administration of endotoxin (lipopolsaccharide (LPS) from Escherichia coli 0111:B4). Within the first 48 hours after LPS there were no differences between clopidogrel and control animals (n = 26 each) in macro-haemodynamics. However, clopidogrel abolished the LPS-induced drop in platelet count and reduced fibrin deposition in lung tissue. Using DNA microarray technology, we could show that clopidogrel suppressed endotoxin-induced up-regulation of inflammation-relevant genes, including arachidonate-5-lipoxygenase activating protein and leukotriene B4 receptor 1. According to our data a possible benefit of anti-platelet drugs in patients on risk for systemic inflammation and organ failure should be tested in a prospective trial.

Inflammation-associated ADAMTS13 deficiency promotes formation of ultra-large von Willebrand factor

The findings of this study suggest that systemic inflammation results in ADAMTS13 deficiency, and thereby activates hemostasis. In a prospective, longitudinal study, we investigated the association between decreased ADAMTS13 activity and impaired hemostasis, as well as organ dysfunctions in patients with systemic inflammation due to extracorporeal cardiopulmonary circuit or with severe sepsis. Similar to negative acute phase proteins, ADAMTS13 activity declined stepwise according to the extent of inflammatory responses. A marked imbalance between ADAMTS13 activity and VWF antigen level was associated with the appearance of ultra-large VWF multimers in plasma, with organ dysfunction and lethality. Our data support the view that systemic inflammation results in an ADAMTS13 deficiency which activates hemostasis.

Liver Dysfunction and Phosphatidylinositol-3-Kinase Signalling in Early Sepsis: Experimental Studies in Rodent Models of Peritonitis

Experimental studies in a rat model of fecal peritonitis conducted by Michael Bauer and colleagues show that in this model, changes in liver function occur early in the development of sepsis, with potential implications for prognosis and development of new therapeutic approaches.

Platelet-derived microvesicles transfer tissue factor to monocytes but not to neutrophils.

Activated platelets are known to adhere to both blood monocytes and neutrophils, and this adhesion is mainly mediated by the surface exposure of the platelet granule protein CD62P. Platelets as well as platelet-derived microvesicles (PMV) have also been shown to contain and to transfer tissue factor (TF), the most important initiator of intravascular thrombin and fibrin formation, to monocytes. However, the role of neutrophils for gathering platelet-derived TF is controversial. Here we studied the interaction of PMV with monocytes and neutrophils using a whole blood system. Platelet-rich plasma (PRP) obtained from citrated human blood was incubated with collagen (5 μg/ml, 15 min) and the platelets were removed by centrifugation (5 min at 5000 × g). After incubating the PMV-containing plasma for further 30 min with a sediment of red and white bloods cells that had been obtained after PRP preparation, monocytes and neutrophils were analysed by flow cytometry for the surface exposure of the platelet-specific antigen CD42a and TF. Compared to a control with non-activated PRP, there was a significant increase in the number of both CD42a-positive monocytes and neutrophils. In contrast, there was no change in the number of TF-positive neutrophils, but a more than 2-fold increase in the number of TF-positive monocytes. The changes in CD42a on monocytes and neutrophils as well as the changes in TF on monocytes could be significantly reduced by an anti-CD62P antibody or by removal of PMV from the plasma samples. The data indicate that the transfer of TF to monocytes is not simply an CD62P-mediated adhesion of platelets or PMV to monocytes, but may involve other not yet identified mechanisms.

Impact of plasma histones in human sepsis and their contribution to cellular injury and inflammation.

IntroductionCirculating histones have been identified as mediators of damage in animal models of sepsis and in patients with trauma-associated lung injury. Despite existing controversies on actual histone concentrations, clinical implications and mechanism of action in various disease conditions, histone levels in human sepsis, association with disease progression and mediated effects on endothelial and immune cells remain unreported. This study aimed to determine histone levels and its clinical implication in septic patients and to elucidate histone-mediated effects ex-vivo.MethodsHistone levels, endogenous activated protein C (APC) levels and clinical data from two independent cohorts of septic patients were obtained. Histone levels were compared with various control groups including healthy individuals, intensive care unit (ICU) patients without sepsis, ICU patients with multiple organ failure and patients with minor or multiple trauma, all without infection. Endothelial and monocytic cells were stimulated with histones. Cellular integrity and sepsis prototypical cytokines were evaluated. The mechanism of action of histones via Toll-like receptor 4 (TLR4) was evaluated using a function blocking antibody. Histone degradation in plasma was studied by immunoblotting.ResultsHistone H4 levels were significantly elevated in patients with sepsis (cohort I; n = 15 and cohort II; n = 19) versus ICU controls (n = 12), patients with multiple organ failure (n = 12) or minor trauma (n = 7), associated with need for renal replacement therapy and decrease in platelet count during disease progression, and remarkably were significantly associated with increased mortality rates in septic patients (ICU-, 28 day- and 90 day mortality rates). There was an inverse correlation between plasma histones and endogenous APC levels. Histone stimulation induced the release of sepsis prototypic cytokines and decreased cell integrity indicated by a significant increase of lactate dehydrogenase (LDH) and propidium iodide (PI) staining. Blocking of TLR4 decreased cellular cytotoxicity on endothelial cells. The calculated half-life of histones in spiked plasma was 4.6 minutes.ConclusionsHistone levels in septic patients are significantly increased and might mediate disease aggravation by cellular injury and inflammation via TLR4 signaling, which potentially results in multiple organ failure and fatal outcome.

Inhibition of platelet aggregation by grafting RGD and KGD sequences on the structural scaffold of small disulfide-rich proteins

Disintegrins represent a group of disulfide-rich peptides ranging in size from 41 to over 80 residues and are antagonists of several integrin receptors. Disintegrins containing an RGD or KGD sequence are potent inhibitors of platelet aggregation as they block the binding of fibrinogen to αIIbβ3 integrin. The high affinity binding to αIIbβ3 in comparison to short linear peptides has been attributed to the localisation of the RGD or KGD sequence within a defined three-dimensional structure. Cystine knot microproteins are members of another family of small disulfide-rich peptides that consist of only 28–40 amino acid residues. They display numerous biological activities depending on the peptide sequence of loop regions that are fixed on a structural scaffold that is stabilised by three knot-forming disulfide bonds. In the present study we grafted RGD and KGD containing peptide sequences with seven and 11 amino acids, respectively, into two cystine knot microproteins, the trypsin inhibitor EETI-II and the melanocortin receptor binding domain of the human agouti-related protein AGRP, as well as into the small disintegrin obtustatin. The engineered proteins were much more potent to inhibit the fibrinogen binding, αIIbβ3 activation and platelet aggregation when compared to the grafted peptides. Differences that were observed between the engineered proteins indicate the importance of the structural scaffold and the amino acids neighbouring the grafted peptide sequences.

Expression profiling: Toward an application in sepsis diagnostics

Sepsis is a common and serious health problem whereby improvements in diagnosis are crucial in increasing survival rates. To test whether profiling transcription is applicable to sepsis diagnosis, we analyzed whole blood using a microarray containing probes for 340 genes relevant to inflammation. The patient’s gene expression pattern was highly homogenous, resulting in 69% of differentially expressed genes. With a positive predictive value of 98%, a list of 50 differentially expressed genes was compiled, and randomly chosen transcripts were confirmed by PCR. Here, we present the first evidence that microarrays can identify typical gene expression profiles in the blood of patients with severe sepsis. Regardless of the heterogeneity of the patients, we observed a striking correlation between the conventional diagnostic classification and our approach. The unity of responses suggests that the principle of this multiparameter approach can be adapted to early stage sepsis diagnosis.

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)