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• W1657909877 abstract "Objective In malaria and sepsis, apoptotic endothelial damage is preventable in vitro by antioxidants and protease inhibitors. Activated protein C, which has anti-apoptotic effects, improves survival in sepsis. Therefore, we studied whether activated protein C prevents endothelial cell apoptosis, induced by serum from patients with malaria or sepsis. Methods Endothelial cells were incubated with patient sera (Plasmodium falciparum malaria, Escherichia coli sepsis, Staphylococcus aureus sepsis) or culture supernatants of the respective organisms, with or without neutrophils. Activated protein C was used to reduce endothelial cell apoptosis in vitro. The proportion of apoptotic endothelial cells was determined by TUNEL staining. Results The apoptosis-inducing effect of patient sera or culture supernatants (P. falciparum, E. coli, S. aureus) on endothelial cells was augmented by neutrophils and reduced by activated protein C in the presence of neutrophils. Pre-incubating either endothelial cells or neutrophils with activated protein C also reduced the endothelial cell apoptosis rate. The pro-apoptotic effect of P. falciparum supernatant was reduced by pan-caspase inhibitor and caspase 8 inhibitor, but not by caspase 9 inhibitor. The pro-apoptotic effect of E. coli and S. aureus supernatants was also reduced by caspase 9 inhibitor. Conclusions Activated protein C protects vascular endothelial cells from apoptosis triggered by patient sera or culture supernatants in combination with neutrophils. It seems to act both on neutrophils and on endothelial cells. Activated protein C blocks caspase-8-dependent apoptosis, which accounts for endothelial damage in sepsis and malaria. Therefore, activated protein C might offer clinical benefit not only in sepsis but also in malaria. Objectif: Dans la malaria et le sepsis, des lésions endothéliales apoptotiques sont évitables in vitro par des antioxydants et des inhibiteurs de la protéase. La protéine C activée qui a des effets anti-apoptotiques, améliore la survie dans le sepsis. Dès lors, nous avons investigué si la protéine C activée empêche l’apoptose des cellules endothéliales, induite par le sérum de patients atteints de malaria ou de septicémie. Méthodes: Les cellules endothéliales ont été incubées avec du sérum de patients (avec malaria àPlasmodium falciparum, sepsis àEscherichia coli ou sepsis àStaphylococcus aureus) ou avec le surnageant de culture des organismes respectifs, avec ou sans neutrophiles. La protéine C activée a été utilisée pour réduire l’apoptose des cellules endothéliales in vitro. La proportion de cellules endothéliales apoptotiques a été déterminée par la coloration de TUNEL. Résultats: L’effet inducteur d’apoptose des sérums de patients ou des surnageants de culture (P. falciparum, E. coli, S. aureus) sur les cellules endothéliales est augmentée par les neutrophiles et réduit par la protéine C activée en présence de neutrophiles. La pré-incubation des cellules endothéliales ou des neutrophiles avec la protéine C activée a également réduit le taux d’apoptose des cellules endothéliales. L’effet pro-apoptotique du surnageant de P. falciparum a été réduite par l’inhibiteur de la pancaspase et l’inhibiteur de la caspase 8, mais pas par l’inhibiteur de la caspase 9. L’effet pro-apoptotique des surnageants de E. coli et S. aureus a également été réduit par l’inhibiteur de la caspase 9. Conclusions: La protéine C activée protège les cellules vasculaires endothéliales de l’apoptose déclenchée par du sérum de patients ou des surnageants de cultures en combinaison avec des neutrophiles. Elle semble agir à la fois sur les neutrophiles et les cellules endothéliales. Elle bloque l’apoptose liée à la caspase 8, responsable des lésions endothéliales dans le sepsis et la malaria. Par conséquent, la protéine C activée pourrait offrir des avantages cliniques non seulement dans le sepsis, mais aussi dans la malaria. Damage to the vascular endothelium, caused by apoptosis of endothelial cells, leads to fatal organ failure in severe malaria and sepsis (Hotchkiss et al. 2002; Hemmer et al. 2005, 2008). Potential triggers of endothelial apoptosis in these diseases include Plasmodium falciparum-parasitized erythrocytes (Pino et al. 2003), neutrophils and their secretory products (Westlin & Gimbrone 1993; Hemmer et al. 2005), lipopolysaccharides (LPS), lipoteichoic acid (LTA) (Aliprantis et al. 1999, 2000, 2001; Wang et al. 2001; Bannerman & Goldblum 2002) and antibodies against Intercellular Adhesion Molecule 1 (ICAM-1) (Hemmer et al. 2008). Bacterial and malarial triggers of apoptosis may synergize in severe P. falciparum malaria, which is often complicated by systemic bacterial infections (Bassat et al. 2009). In vitro, endothelial cell apoptosis can be prevented by ascorbic acid and tocopherol (antioxidants), ulinastatin (inhibitor of neutrophil elastase) and anti-LFA-1 antibodies (Hemmer et al. 2005, 2008). In vivo, tocopherol and anti-LFA-1 antibodies improve survival in murine malaria, and ulinastatin improves survival in canine sepsis (Thumwood et al. 1989; Grau et al. 1991; Tani et al. 1993). In humans, severe P. falciparum malaria tends to be associated with low serum concentrations of the antioxidant vitamins thiamine and ascorbic acid (Hassan et al. 2004; Mayxay et al. 2007). Because the mechanisms of endothelial damage in severe malaria and in severe bacterial sepsis seem to be similar, the same substances might be expected to offer benefit in both diseases. Reduced protein C levels and increased neutrophil activation are observed both in human malaria and in sepsis (Egbring et al. 1977; Hemmer et al. 1991, 1994; Yan et al. 2001; Vogetseder et al. 2004). Activated protein C specifically binds to endothelial cells (Esmon 2003) and protects them from apoptosis (Cheng et al. 2003; Mosnier & Griffin 2003). It also binds specifically to neutrophils (Sturn et al. 2003), inhibits neutrophil adhesion to endothelial cells (Grinnell et al. 1994) and reduces endotoxin-induced neutrophil-dependent vascular injury in rats (Murakami et al. 1996). In human sepsis, activated protein C is the only supportive therapy that is known to improve survival (Bernard et al. 2001), while in human malaria, several case reports strongly suggest a beneficial effect for activated protein C (Kapadia & Shirwadkar 2006; Kendrick et al. 2006; Nau et al. 2006; Bruneel et al. 2007; Rankin & Austin 2007; Robak et al. 2010). We aimed to determine whether endothelial cell apoptosis, induced by serum from patients with P. falciparum malaria, Staphylococcus aureus sepsis and Escherichia coli sepsis, can be prevented by activated protein C. Serum samples from five patients with severe P. falciparum malaria, five patients with E. coli sepsis, three patients with S. aureus sepsis and three healthy control subjects were used for the experiments. Informed written consent was obtained from all participants. Approval had been granted by the Ethics Committees of the State Medical Boards of Hamburg and Mecklenburg-Vorpommern. Plasmodium falciparum (clone D10) was maintained in continuous culture according to Trager and Jensen (1976). The parasites were grown in human red blood cells (blood group O+), RPMI 1640 medium supplemented with 25 nm HEPES, 20 nm sodium bicarbonate and 0.5% AlbuMAX (Invitrogen, Karlsruhe, Germany) at a haematocrit of 5% and a temperature of 37 °C in an atmosphere containing 90% N2, 5% O2 and 5% CO2 (Das Gupta et al. 2005). After 48 h of incubation at a maximum parasitemia of 10%, the medium was harvested for the experiments. Control medium was obtained by the same procedure, using non-parasitized erythrocytes. Escherichia coli (ATCC number 25922) and S. aureus (ATCC number 29213) were purchased from American Type Culture Collection (Promochem, Wesel, Germany) and cultured in Mueller–Hinton broth (Merck Life Science, Darmstadt, Germany) for 48 h, which resulted in a McFarland density of 5. Bacteria were sedimented in a centrifuge at 1000 g and 25 °C for 20 min. The supernatant was sterile filtrated with 0.2-μm filters (Millipore, Eschborn, Germany). For controls, fresh sterile Mueller–Hinton broth was used. Chemicals (analytical grade) were purchased from Sigma (Munich, Germany), unless otherwise indicated. Endothelial cell growth supplement was obtained from Intracel Co. (Rockville, MD, USA); injectable preparations of activated protein C (Drotrecogin alpha) from Lilly (Bad Homburg, Germany); ascorbic acid from Jenapharm (Jena, Germany); and ulinastatin from Mochida Co. (Tokyo, Japan). TUNEL kits (In Situ Cell Death Detection kit) were obtained from Roche Diagnostics (Mannheim, Germany). Endothelial cells were obtained from umbilical veins by mild collagenase digestion and seeded on gelatin-coated tissue culture flasks. Second- to third-passage endothelial cells were grown to confluence in 96-well plates, using RPMI medium with 20% foetal bovine serum and 35 mg/l endothelial cell growth supplement. EDTA blood from healthy donors was centrifuged at 500 g and 4 °C for 10 min. Neutrophils were isolated by discontinuous Percoll® gradient (Sigma), collected at the interface between the densities 1.098 and 1.090 and washed three times in ice-cold PBS (Dooley et al. 1982; Jackson et al. 1989). To test whether activated protein C protects endothelial cells from apoptosis by interacting directly with endothelial cells, endothelial cells were pre-incubated with endothelial cell medium containing activated protein C (final concentration of 2 × 10−6 or 2 × 10−7 m) for 24 h. To test whether activated protein C protects endothelial cells from apoptosis indirectly by interacting with neutrophils, neutrophils were pre-incubated with PBS containing activated protein C (final concentration of 2 × 10−6 or 2 × 10−7 m) for 1 h before the experiments and then washed three times with fresh PBS. Before incubation with endothelial cells, sera from patients with P. falciparum malaria, S. aureus or E. coli sepsis, or from healthy controls were diluted 1:10 in endothelial cell medium without endothelial cell growth supplement and without foetal bovine serum. Culture supernatants from S. aureus or E. coli, and fresh Mueller–Hinton medium, were diluted 1:10 in endothelial cell medium without endothelial cell growth supplement but with foetal bovine serum (2%), to avoid induction of apoptosis through starvation in the absence of serum. Culture supernatants from P. falciparum-parasitized erythrocytes or from non-parasitized control erythrocytes were not diluted. Freshly isolated neutrophil granulocytes from a healthy volunteer were added to diluted serum or culture supernatants at a final cell count of 1000 neutrophils per microlitre and then incubated with cultured endothelial cells at 37 °C for 1 h. The cells were then rinsed three times with HEPES-buffered saline at pH 7.4, incubated with RPMI medium containing 20% foetal bovine serum and endothelial cell growth supplement (35 mg/ml) for 4.5 h and again rinsed three times with HEPES-buffered saline. The cell cultures were then examined microscopically to ascertain that all neutrophils had been removed. Endothelial cell nuclei were stained for apoptosis by the TUNEL method as described elsewhere (2). Briefly, after fixation and permeabilization of endothelial cells, double-strand breaks of nuclear DNA were labelled with FITC-conjugated UDP by terminal deoxynucleotidyl transferase. The resulting fluorescence was stabilized with 1,4-diazabicyclo(2.2.2)octane (DABCO) in phosphate-buffered saline/glycerol (50:50 v/v). Fluorescent nuclei were counted in 100 cells in each well with an inversion fluorescence microscope (Zeiss Axiovert, Jena, Germany). The results were expressed as per cent fluorescent (i.e. apoptotic) nuclei in relation to all counted nuclei. In this model, the results of TUNEL staining agreed with the results of Annexin V staining and with morphological criteria of apoptosis, which confirmed TUNEL staining as a valid indicator of apoptosis (Hemmer et al. 2008). All experiments were carried out in duplicate and replicated on two other occasions. Results are given as arithmetic means of these duplicate experiments. To prevent apoptotic damage, activated protein C (2 × 10−8 to 2 × 10−6 m), ascorbic acid (10−6 to 10−4 m) or ulinastatin (10−9 to 10−7 m) were added to patient sera or culture supernatants immediately before incubation with neutrophils and endothelial cells. These concentration ranges were chosen because they are attainable in humans. To prevent caspase-dependent apoptotic damage, the pan-caspase inhibitor boc-Asp-OME-FMK was added to culture supernatants. To block the extrinsic and intrinsic apoptosis pathways selectively as far as they are caspase dependent, caspase-8 inhibitor z-Ile-Glu-Thr-Asp-OME-FMK and caspase-9 inhibitor z-Leu-Glu-OME-His-Asp-OME-FMK were used, respectively. All caspase inhibitors were purchased from Sigma-Aldrich (Munich, Germany). Caspase inhibitors were first dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10−2 m and applied at a final concentration of 5 × 10−6 m. To avoid toxic effects of DMSO towards endothelial cells, the final DMSO concentrations were kept at or below 0.05% (w/v). Sera from malaria or sepsis patients, P. falciparum culture supernatants, and S. aureus and E. coli culture supernatants served as positive controls. Sera from healthy volunteers, culture supernatants from non-parasitized erythrocytes and sterile Mueller–Hinton medium served as negative controls. The Wilcoxon test for connected samples (two-sided) was used to compare the apoptosis rates of endothelial cells in the absence and presence of activated protein C. In the presence of neutrophils, activated protein C reduced the pro-apoptotic effect of sera from patients with severe P. falciparum malaria, E. coli sepsis and S. aureus sepsis and of culture supernatants of P. falciparum, E. coli and S. aureus (Figure 1). Figure 2 shows that the anti-apoptotic effect of activated protein C was dose dependent. Reduction of the percentage of apoptotic endothelial cells by activated protein C (2 × 10−6 m) in the presence of neutrophils and patient sera. Bars indicate median endothelial cell apoptosis rates and whiskers indicate ranges. P values refer to the differences between incubation without and with activated protein C. Dose dependency of the effect of activated protein C on endothelial cell apoptosis in the presence of patient sera and neutrophils: Percentage of apoptotic cells (means of duplicate experiments). Activated protein C also reduced the apoptosis rate when either neutrophils or endothelial cells were pre-incubated with this substance. Without activated protein C, serum from a patient with severe P. falciparum malaria led to an endothelial cell apoptosis rate of 38%. After pre-incubation of neutrophils with activated protein C, the apoptosis rate was 23% (2 × 10−6 m) and 29% (2 × 10−7 m). After pre-incubation of endothelial cells with activated protein C, the apoptosis rate was 22% (2 × 10−6 m) and 35% (2 × 10−7 m). As activated protein C alone was unable to completely suppress apoptotic endothelial cell damage even in the highest concentration tested (2 × 10−6 m), activated protein C was combined with either ascorbic acid (10−4 m) or ulinastatin (10−6 m). In these combination experiments, activated protein C alone lowered the endothelial cell apoptosis rate from 48% to 38% in the presence of patient serum (P. falciparum malaria). Either ascorbic acid or ulinastatin alone led to apoptosis rates of 29% and 34%, respectively. In combination with ascorbic acid, activated protein C lowered the apoptosis rate to 25% and in combination with ulinastatin to 23%. Thus, in combination with either ascorbic acid or ulinastatin, activated protein C reduced the endothelial apoptosis level seen with serum from patients almost to the level seen with serum from healthy controls (16%). Without activated protein C, the apoptosis rate after incubation with P. falciparum culture supernatant in the presence of neutrophils was 44%. With activated protein C (2 × 10−6 m), the apoptosis rate was 33%. Without activated protein C, the endothelial cell apoptosis rates after incubation with E. coli and S. aureus culture supernatant in the presence of neutrophils were 46% and 42%. With activated protein C (2 × 10−6 m), the apoptosis rates were 28% (E. coli supernatant) and 35% (S. aureus supernatant). With P. falciparum supernatant, the endothelial cell apoptosis rate in the presence of neutrophils was reduced from 45% to 24% by pan-caspase inhibitor and to 22% by caspase 8 inhibitor (Table 1). It was not affected by caspase 9 inhibitor (42%). With E. coli supernatant, the endothelial cell apoptosis rate in the presence of neutrophils was reduced from 49% to 34% by pan-caspase inhibitor, to 32% by caspase 8 inhibitor and to 30% by caspase 9 inhibitor. With S. aureus supernatant, the endothelial cell apoptosis rate in the presence of neutrophils was reduced from 52% to 24% by pan-caspase inhibitor, to 19% by caspase 8 inhibitor and to 31% by caspase 9 inhibitor. In the absence of neutrophils, activated protein C did not consistently reduce the pro-apoptotic effect of sera from patients with severe P. falciparum malaria, E. coli sepsis or S. aureus sepsis, and culture supernatants of P. falciparum, E. coli or S. aureus (data not shown). In earlier studies, we had shown that sera from patients with malaria or bacterial sepsis promote apoptosis of endothelial cells, in combination with neutrophils (Hemmer et al. 2005, 2008). The contribution of neutrophils to endothelial damage is indicated by the observations that neutralization of toxic neutrophil secretion products and prevention of direct contact between neutrophils and endothelial cells reduce endothelial apoptosis. In human P. falciparum malaria, the plasma levels of neutrophil elastase and thrombomodulin, a parameter of endothelial damage in vivo, are correlated with each other (Hemmer et al. 1994). The present study shows that activated protein C protects vascular endothelial cells from apoptosis that is triggered by patient sera, or by culture supernatants, in the presence of neutrophils. It also shows that interaction of activated protein C with either neutrophils or endothelial cells reduces endothelial apoptosis. Activated protein C, which is best known for its anticoagulant properties, specifically binds to the endothelial protein C receptor (EPCR) (Fukudome & Esmon 1994). Its anti-apoptotic and anti-inflammatory effects are independent of its anticoagulant activities (Mosnier et al. 2004). In endothelial cells, activated protein C downregulates pro-apoptotic calreticulin and endoplasmatic reticulum luminal protein and upregulates anti-apoptotic Bcl2-analogue protein (A1), inhibitor of apoptosis protein (IAP) and cell-cycle dependent Gu helicase (Joyce et al. 2001). Another anti-apoptotic effect of activated protein C is mediated by downregulation of tumour suppression factor p53 and by reduction of the ratio of pro-apoptotic Bax-to-anti-apoptotic Bcl-2 (Cheng et al. 2003). Anti-inflammatory effects of activated protein C are mediated by inhibition of TNF-alpha-dependent expression of several leucocyte adhesion molecules like ICAM-1 on endothelial cells (Joyce et al. 2001; Franscini et al. 2004). Anti-apoptotic and anti-inflammatory actions of activated protein C may be closely related, because ICAM-1-associated functions also seem to contribute to endothelial cell apoptosis (Guo et al. 2004; Hemmer et al. 2008). Activated protein C inhibits the receptor-ligand-dependent (i.e. extrinsic) apoptosis pathway by blocking the caspase-8-dependent activation of caspase 3 in endothelial cells (Guo et al. 2004; Liu et al. 2004, 2005). We found that the pan-caspase inhibitor and the inhibitor of caspase 8, which blocks the extrinsic apoptosis pathway, reduce endothelial apoptosis induced by culture supernatants from P. falciparum, E. coli and S. aureus. The caspase 9 antagonist, which blocks only the intrinsic apoptosis pathway, reduced apoptosis triggered by E. coli and S. aureus supernatant but not by P. falciparum supernatant. Thus, the extrinsic apoptosis pathway may contribute to apoptosis in sepsis and in P. falciparum malaria, while the intrinsic apoptosis pathway may contribute to apoptosis only in sepsis but not in P. falciparum malaria. The different contributions of the extrinsic and intrinsic pathways to apoptosis in sepsis and malaria could be tested further by administering selective inhibitors of intrinsic or extrinsic apoptosis in animal models of sepsis or malaria (Imao et al. 2006). Because activated protein C blocks the extrinsic apoptosis pathway, it has the potential to protect the vascular endothelium from apoptosis not only in sepsis but also in malaria. Activated protein C interacts not only with endothelial cells but also with neutrophils. Both cells possess the EPCR (Sturn et al. 2003). Activated protein C inhibits neutrophil chemotaxis in vitro (Sturn et al. 2003), prevents the activation of neutrophils after ischaemia/reperfusion injury in rats (Hirose et al. 2000) and reduces endotoxin-induced pulmonary leucocyte accumulation in human volunteers (Nick et al. 2004). This is in agreement with our observation that activated protein C protects endothelial cells from apoptosis in the presence of neutrophils and that direct interaction of activated protein C with neutrophils contributes to its protective effect. We found no significant protection in the absence of neutrophils. However, because activated protein C interacts both with neutrophils and with endothelial cells, it cannot be excluded that a smaller protective effect of activated protein C in the absence of neutrophils went undetected because of the small sample size. Using the same model, but a larger sample size, an in vitro study of myocardial infarctions found that activated protein C (but not ascorbic acid or ulinastatin) protected endothelial cells from apoptosis both in the presence and in the absence of neutrophils (Frimmel et al. 2008). Neutrophils may contribute to endothelial apoptosis not only in infections but also in inflammations of other aetiologies as well, including myocardial infarction. The presence of similar pathomechanisms in different inflammatory diseases suggests that anti-apoptotic therapies might offer benefit not only in infections but in other inflammatory states as well. In our experiments, the anti-apoptotic effect of activated protein C was amplified when applied simultaneously with ascorbic acid or ulinastatin. Activated protein C and ascorbic acid both counteract apoptosis by inhibiting the upregulation of NF-kappaB and the pro-apoptotic change in the Bax/BCL2 expression ratio (Haendeler et al. 1996). Activated protein C and ulinastatin both counteract apoptosis by decreasing serum levels of tumour necrosis factor alpha and interleukin 1 (Kakinuma et al. 1997; Okajima 2004; Zhang et al. 2005). However, while activated protein C binds to receptors on its target cells and alters cell functions, ascorbic acid and ulinastatin neutralize neutrophil secretory products (Hemmer et al. 2005). This might explain why the anti-apoptotic effects of the combination of activated protein C either with ascorbic acid or with ulinastatin are greater than the effect of each combination partner alone. A large randomized study has shown that administration of activated protein C improves survival in severe sepsis (Bernard et al. 2001). In life-threatening P. falciparum malaria, several case reports describe marked clinical improvement after administration of activated protein C (Kapadia & Shirwadkar 2006; Kendrick et al. 2006; Nau et al. 2006; Bruneel et al. 2007; Rankin & Austin 2007; Robak et al. 2010). Therefore, the anti-apoptotic effects of activated protein C in both malaria and sepsis support the notion that activated protein C may offer clinical benefit not only in sepsis but also in severe malaria. We are indebted to Dr Rolf D. Walter (Hamburg, Germany) for providing culture supernatants of P. falciparum-parasitized and of non-parasitized erythrocytes. The help of Dr Joachim Rychly (Rostock) and Dr Eckhard Koepcke (Rostock) is greatly appreciated. Financial support was provided by the B. Braun Foundation (Melsungen, Germany), the German Research Foundation, and the Research Support Program, University of Rostock, Faculty of Medicine." @default.
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• W1657909877 title "Activated protein C protects vascular endothelial cells from apoptosis in malaria and in sepsis" @default.
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