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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 28, 2005 as doi:10.1096/fj.04-2842fje. |
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,1
* Department of Anaesthesiology and Intensive Care Medicine, Friedrich Schiller University Jena, Jena, Germany;
Department of Anatomy and Cell Biology III, University of Heidelberg, Heidelberg, Germany; and
School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, UK
1 Correspondence: R. A. C., Department of Anesthesiology and Intensive Care Medicine, Division for Experimental Anesthesiology, Erlanger Allee 101, Jena 07747, Germany. E-mail: ralf.claus{at}med.uni-jena.de; H.-P. D., School of Chemical Sciences and Pharmacy, University of East Anglia UEA, Norwich NR4 7TJ, UK. E-mail: h-p.deigner{at}uea.ac.uk
SPECIFIC AIMS
Sphingolipid hydrolysis and ceramide generation have been suggested to play a pivotal role in immune modulation and the development of multi-organ dysfunction. Using a novel test system we evaluated whether the activity of a plasma-secreted sphingomyelinase (pSMPD1) is altered in critically ill patients and whether its modulation by a novel, low molecular weight inhibitor in a murine endotoxic mice shock model improves survival rate and hepato-cellular apoptosis. Posttranslational modification of pSMPD1 by oxidative stress is elucidated as a putative mechanism for its activation.
PRINCIPAL FINDINGS
1. SMPD1 activity and its inhibition in a murine endotoxic shock model
SMPD1 is a product of the acid sphingomyelinase gene and an enzyme present in both plasma (pSMPD1) and intracellular lysosomes, converting sphingomyelin into ceramide. Among the known types of sphingomyelinases, the secretory variant, pSMPD1 is the only sphingomyelinase held responsible for an extracellular hydrolysis of sphingomyelin. In the present study, we elaborated a fluorescence based method for the determination of zinc-dependent acid sphingomyelinase in human and murine plasma.
To obtain further information on variations of sphingolytic activities in the course of murine endotoxic shock model, we analyzed pSMPD1 activities in plasma samples from mice (n=9) 24 h after endotoxin challenge. Average activity in the saline group was raised from 2458 to 5036 pmol/(mL · h). Next, an inhibitor of SMPD1-biosynthesis (NB6) was administered to reduce ceramide formation. Preadministration of NB6 prevented the increase of pSMPD1 activity as compared with endotoxin [median value of 2797 pmol/(mL · h) (P<0.05)], but had no effect on the enzyme activity in animals without endotoxin challenge [2875 pmol/(mL · h)] (Fig. 1
A). Absolute changes in SMPD1 activity in mice treated with endotoxin without NB6 pretreatment in comparison to basal levels are illustrated in Fig. 1B
(P<0.05).
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Previous studies have indicated that the liver is a major organ responsible for initiation of MOF. To further characterize a potential antiapoptotic effect of NB6 in a nonlethal experimental design, analysis of hepatic DNA fragmentation using the TUNEL method and determination of morphological changes during apoptosis after triggering of endotoxic shock showed a significantly diminished number of stained cells in the NB6 treated group (43 vs. 25%, P<0.0001) 7 h after endotoxin administration.
2. Plasmatic SMPD1 activity is increased in critically ill patients
Significantly elevated levels of pSMPD1 activity were found in patients in comparison to healthy controls: the median in the control group was 123.6 pmol/(mL · h) [Q1, Q3: 76.7, 181.9] as opposed to a median of 262.3 pmol/(mL · h) [234.4, 370.4] for the pSMPD1 activity in patients with sepsis at baseline (P<0.005). The comparison between nonsurvivors and survivors revealed no significant difference for median pSMPD1 activities at baseline (247.6 pmol/(mL · h) [185.0, 320.1] vs. 264.7 pmol/(mL · h) [259.9, 376.8].
In nonsurvivors (n=7) sphingolytic activity increased during the study period (+77.4 pmol/(mL · h), [–2.7, 365.8] ) compared with a decrease in survivors (–252.1 pmol/(mL · h), [–254.8, –108.2]). Considering intra-individual changes over the study period, there were significant differences between survivors and nonsurvivors: we found a further increase among nonsurvivors by 
24% (2.4, 143.9), but a decrease among survivors by 65% (–66.9, –41.6) was observed (P<0.02).
For association of pSMPD1 activity with clinical parameters and to obtain further insight into factors influencing pSMPD1 activity, we compared pSMPD1 activities with parameters relevant for clinical evaluation. We found that SOFA-scores were significantly higher on days with maximal pSMPD1 activity than on days with minimal pSMPD1 activity (median difference: 4 points [1.5, 5.5), P (one-sided) <0.005]. The same relationship was observed for procalcitonin [median difference: 1.84 ng/mL (0.3, 13.6), P (one-sided) <0.05, data not shown].
3. Effects of oxidative stress on enzyme activity
Based on the fact of an increase in enzymatic activity after oxidation of a C-terminally located cysteine group in recombinant SMPD1, we addressed the question whether pro-oxidative conditions affect activity of pSMPD1 in human plasma. More specifically, we assessed the immediate effect of oxidative stress known to be increased under inflammatory conditions, such as observed during sepsis. Basal pSMPD1 activity was determined as 121.7 ± 21.5 pmol/(mL · h). As expected, addition of ascorbic acid did not result in a significant change in sphingolytic activity [117.8±19.9 pmol/(mL · h)]. However, addition of the oxidant 2,2’-azobis-2-amidinopropane (AAPH) to the plasma was followed by a substantial rise in pSMPD1 activity by 87% to a value of 207.2 ± 26.1 pmol/(mL · h). This increase was significantly attenuated by parallel incubation with ascorbic acid.
CONCLUSIONS AND SIGNIFICANCE
This study provides the first evidence, that circulating pSMPD1 activity is markedly elevated in critically ill patients. In fact, plasma activities in both septic patients and healthy controls clearly exceed the enzymatic activities reported to induce biological effects in cell culture systems. The patient cohort selected for our study was included prospectively and are deliberately heterogeneous to evaluate the general value of plasma SMPD1 activity. Patients enrolled featured diverse degrees of concomitant diseases, variable sources of infection, a broad range of sepsis severity and various plasma levels of inflammatory markers. Despite this heterogeneity, a broad increase in the activity of the sphingomyelin-cleaving activity of SMPD1 was observed.
Patients with fatal outcome showed a marked increase in sphingolytic activity over time. The absolute changes of pSMPD1 activity during the observation period on ICU allowed significant discrimination between surviving and nonsurviving patients displaying an inverse trend of pSMPD1 activities. Our data suggest that this increase is paralleled by changes of established severity markers. In keeping with these findings, our animal studies support the concept for a crucial role of SMPD1 in the pathogenesis of sepsis and subsequent organ failure, with impact on survival rate and apoptosis of end organs such as the liver.
Various mechanisms may be involved in the observed increase of SMPD1 activity in plasma of septic patients. Up to now, strong evidence has been provided for a cytokine- and endotoxin-stimulated secretion of pSMPD1 from endothelial cells. Our data further suggest that posttranslational modification is also of relevance to the secreted sphingomyelinase in human plasma. Hence, oxidative stress induced by systemic inflammation might well have contributed to the increase in pSMPD1 activity that we observed in septic patients as well as in the endotoxic animal model (Fig. 2
).
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Ceramide, the product of sphingomyelin hydrolysis, is known to induce the production of reactive oxygen species in endothelial cells. Under oxidative conditions it may thus be part of a self-perpetuating positive feedback mechanism based on the posttranslational modification of pSMPD1. A similar positive feedback mechanism that implicates increased ceramide levels in the transcriptional up-regulation of SMPD1-activity, was previously suggested by our group. Macrophages show a marked increase of SMPD1 synthesis when exposed to hexanoyl-ceramide in cell culture experiments. Drobnik et al. recently described an increased ceramide to sphingomyelin ratio in septic patients. While the altered ratio may in itself be a consequence of the elevated pSMPD1 levels, ceramide could possibly function as a stimulus for a transcriptional up-regulation of the pSMPD1 synthesis (Fig. 3).
The actual sites of action and mediated effects of pSMPD1 continue to be somewhat elusive. Consistent with our findings and the concept of an extracellular sphingolipid metabolism, Lightle et al. provided evidence that probably both an increased pSMPD1 and elevated hepatic de novo synthesis of ceramide may account for increased plasma ceramide levels observed in the acute phase response and endotoxic shock. Elevated pSMPD1 levels may also influence the intracellular sphingolipid metabolism directly and lead to in situ generation of ceramide. The incubation of cells with exogenous sphingomyelinases is a commonly used in vitro model to illustrate the biological effects of intracellular ceramide generation. Garzia-Ruiz, et al. claim that with regard to the mediated biological effects there is a selective sensitivity to different types of exogenously added sphingomyelinases. In keeping with our results showing a decreased hepatic apoptosis rate after inhibition of the transcription of the smpd1, they demonstrated that hepatocytes are selectively induced to enter apoptosis by an acidic isoform of sphingomyelinases.
In a recent report, using an identical methodology (TUNEL-staining), Yu, et al. found a similar amount of apoptotic cells in murine liver biopsy specimens (40%) 8 h after intravenous application of Escherichia coli.
Our findings of improved long term survival after inhibition of SMPD1 synthesis by NB6 the deleterious effects of activation of sphingomyelin hydrolysis after injection of endotoxin in lethal dosages are abrogated in SMPD1–/– mice. Monitoring of biophysical interactions using immobilized anionic liposomes indicated a dissociation of the liposomal surface, the SMPD1-protein and its substrate sphingomyeline induced by a cationic amphiphilic compound, desipramine. Due to structural similarities of desipramine and NB6, comparable effects on enzyme-substrate interactions can be assumed beyond effects of NB6 on SMPD1-expression.
Inhibiting the hydrolyzing activity may have some therapeutic potential in preventing immunosuppression and MODS in sepsis. In line with that, Goggel et al. provided the first promising evidence that inhibition of acid sphingomyelinase constitutes a potential therapeutic target in the treatment of PAF-induced pulmonary edema and acute lung injury.
We conclude that synthesis and secretion of SMPD1 into plasma is increased in sepsis. Together with data from in vitro and ex vivo experiments as well as from the endotoxemic shock model, these results provide the first report for a potential role of increased pSMPD1 activity in sepsis. The uniform increase of pSMPD1 activity despite the heterogenity of our patients’ cohort suggests that molecular responses during the course of systemic inflammation may converge to a common pathway stimulating sphingomyelin hydrolysis. Changes in hydrolytic activity might be helpful in clinical diagnosis and therapeutic monitoring. Inhibition of pSMPD1 activity could be a potential target and can be achieved by the novel inhibitor NB6.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2842fje; doi: 10.1096/fj.04-2842fje
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