Clemens Feistritzer

Protective Signaling by Activated Protein C Is Mechanistically Linked to Protein C Activation on Endothelial Cells

Activated protein C (APC) has endothelial barrier protective effects that require binding to endothelial protein C receptor (EPCR) and cleavage of protease activated receptor-1 (PAR1) and that may play a role in the anti-inflammatory action of APC. In this study we investigated whether protein C (PC) activation by thrombin on the endothelial cell surface may be linked to efficient protective signaling. To minimize direct thrombin effects on endothelial permeability we used the anticoagulant double mutant thrombin W215A/E217A (WE). Activation of PC by WE on the endothelial cell surface generated APC with high barrier protective activity. Comparable barrier protective effects by exogenous APC required a 4-fold higher concentration of APC. To demonstrate conclusively that protective effects in the presence of WE are mediated by APC generation and not direct signaling by WE, we used a PC variant with a substitution of the active site serine with alanine (PC S360A). Barrier protective effects of a low concentration of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. WE induced protective signaling only in the presence of wild type PC but not PC S360A and PAR1 cleavage was required for these protective effects. These data demonstrate that the endogenous PC activation pathway on the endothelial cell surface is mechanistically linked to PAR1-dependent autocrine barrier protective signaling by the generated APC. WE may have powerful protective effects in systemic inflammation through signaling by the endogenously generated APC.

Effect of Molecular Weight and Substitution on Tissue Uptake of Hydroxyethyl Starch A Meta-Analysis of Clinical Studies

BackgroundIntravenously infused hydroxyethyl starch (HES) can be found in urine, plasma and tissues. HES remaining in plasma and tissues is thought to increase the risk of clinical complications. HES solutions of lower molecular weight and substitution have been developed to increase urinary excretion and reduce plasma persistence. However, their effect on tissue uptake of HES has not been investigated in human subjects.ObjectiveOur objective was to test the hypothesis that lower molecular weight and substitution decrease tissue uptake of HES.Data sourcesComputer searches were performed of MEDLINE; EMBASE; the Cochrane Library; meeting abstract databases in surgery, anaesthesiology and intensive care; ClinicalTrials.gov; and Google. Supplementary sources were reference lists and electronic tables of journal contents. No time period or language restrictions were imposed.Study SelectionClinical studies were eligible for inclusion in the meta-analysis, if data were reported both for cumulative urinary excretion of HES over 24 hours after infusion and for plasma HES concentration at 24 hours.Data ExtractionData were extracted on 24-hour urinary excretion of HES, 24-hour HES plasma concentration, plasma volume, HES molecular weight and substitution, study design, type and demographics of subjects, indication for fluid infusion, and HES infusion regimen. Tissue uptake of HES was computed as the difference between the infused dose and the sum of urinary excretion and residual plasma HES at 24 hours.Data SynthesisTwenty-five clinical studies totalling 287 subjects were included. Tissue uptake of low-molecular-weight HES (≤200 kD) was 42.3% (95% confidence interval [CI] 39.6, 45.0) compared with 24.6% (CI 17.8, 31.4) for high-molecular-weight HES (p<0.001). Similarly, tissue uptake of lower-substitution HES (≤0.5) was 42.4% (CI 39.5, 45.3) versus 26.6% (CI 19.6, 33.6) for higher-substitution HES (p<0.001). Among the three most often investigated single HES solutions, tissue uptake of 130/0.4 (42.6%; CI 35.0, 50.2) and HES 200/0.5 (43.3%; CI 39.4, 47.2) closely coincided, whereas uptake of HES 450/0.7 (22.2%; CI 14.8, 29.6) was lower (p = 0.001 and p<0.001, respectively).ConclusionsThis meta-analysis did not support the hypothesis that lower molecular weight and substitution decrease tissue uptake of HES. Further clinical studies of HES tissue uptake are needed.

Lipocalin-2 ameliorates granulocyte functionality

Attraction of neutrophils to sites of infection or tissue injury is an essential prerequisite for an efficient innate immune response. Herein, we provide novel evidence that the antimicrobial protein, neutrophil gelatinase associated lipocalin (24p3 or lipocalin‐2, Lcn2) is a central regulator of this process. Lcn2 is produced by several cell types but high amounts are released by neutrophils. Using human and murine neutrophils, we found that the addition of recombinant Lcn2 significantly stimulated their migration, which was independent of IL‐8/keratinocyte chemokine formation. Mechanistically, this could be traced back to Lcn2‐mediated changes of Erk1/2 signaling. Accordingly, the i.p. injection of Lcn2 into C57BL/6 mice stimulated the mobilization of neutrophils while we found a significantly reduced neutrophil chemotactic activity of cells obtained from Lcn2 KO mice. This observation transmitted to a reduced accumulation of neutrophils in intra‐dermal lesions infected with Salmonella typhimurium in Lcn2 KO mice as compared to WT mice. This was not only due to a reduced chemotaxis but also to an impaired cellular adhesion of neutrophils in the absence of Lcn2. We herein describe a novel role of Lcn2 as an important paracrine chemoattractant and an indispensable factor for neutrophil function in inflammation.

Protection of vascular barrier integrity by activated protein C in murine models depends on protease-activated receptor-1

Protease activated receptor-1 (PAR1) mediates barrier protective signalling of activated protein C (APC) in human endothelial cells in vitro and may contribute to APCs beneficial effects in patients with severe sepsis. Mouse models are of key importance for translational research but species differences may limit conclusions for the human system. We analysed whether mouse APC can cleave, activate and induce signalling through murine PAR1 and tested in newly established mouse models if long-term infusion of APC prevents from vascular leakage. Cell surface immunoassays demonstrated efficient cleavage of endogenous murine endothelial PAR1 by either murine or human APC. Pharmacological concentrations of APC of either species had powerful barrier protective effects on cultured murine endothelial cells that required PAR1 cleavage. Vascular endothelial growth factor-mediated hyperpermeability in the skin was reduced by either endogenously generated as well as directly infused recombinant mouse APC in wild-type mice. However APC did not significantly alter the vascular barrier function in PAR1-deficient mice. In endotoxin-challenged mice, infused APC significantly prevented from pulmonary fluid accumulation in the wild-type mice but not in mice lacking PAR1. Our results directly show that murine APC cleaves and signals through PAR1 in mouse endothelial cells. APC reduces vascular permeability in mouse models and PAR1 plays a major role in mediating these effects. Our data in vitro and in vivo support the paradigm that PAR1 contributes to protective effects of APC on vascular barrier integrity in sepsis.

The immune modulator FTY720 targets sphingosine–kinase-dependent migration of human monocytes in response to amyloid beta-protein and its precursor

Accumulation of inflammatory mononuclear phagocytes in Alzheimers senile plaques, a hallmark of the innate immune response to β‐amyloid fibrils, can initiate and propagate neurodegeneration characteristic of Alzheimers disease. Phagocytes migrate toward amyloid β‐ protein involving formyl peptide receptor like‐1‐dependent signaling. Using human peripheral blood monocytes in Boyden chamber micropore filter assays, we show that the amyloid β‐ protein‐ and amyloid β‐precursor protein‐induced migration was abrogated by dimethylsphingosine, a sphingosine kinase inhibitor. Amyloid β‐protein stimulated in monocytes the gene expression for sphingosine‐1‐phosphate receptors 2 and 5, but not 1, 3, and 4. FTY720 that acts as a sphingosine‐1‐phosphate receptor agonist after endogenous phosphorylation by sphingosine kinase, as well as various neuropeptides that are known to be monocyte chemoattractants, dose‐dependently inhibited amyloid β‐protein‐induced migration. These data demonstrate that the migratory effects of β‐amyloid in human monocytes involve spingosine‐1‐ phosphate signaling. Whereas endogenous neuropeptides may arrest and activate monocytes at sites of high β‐amyloid concentrations, interference with the amyloid β‐protein‐dependent sphingosine‐1‐phosphate pathway in monocytes by FTY720, a novel immunomodulatory drug, suggests that FTY720 may be efficacious in β‐amyloid‐related inflammatory diseases.

Effects of anticoagulant strategies on activation of inflammation and coagulation

Acute inflammatory events, such as those that occur in sepsis, lead to dysregulation of the coagulation cascade. The hemostatic imbalance in sepsis, characterized by the excessive activation of procoagulant pathways and the impairment of anticoagulant activity, leads to disseminated intravascular coagulation and results in microvascular thrombosis, tissue hypoperfusion and, ultimately, multiple organ failure and death. Furthermore, natural anti-inflammatory mechanisms of the endogenous anticoagulants are diminished by the impaired coagulation. Supportive strategies aiming at inhibiting activation of coagulation and inflammation by treatment with exogenous anticoagulants have been found to be beneficial in experimental and initial clinical studies. This review summarizes the available experimental and clinical data regarding the interaction between coagulation and inflammation, focusing on the two anticoagulants which are in clinical use, antithrombin and activated protein C. Identification of the different biological mechanisms of the two endogenous anticoagulants might help to determine target patient populations as well as to develop new anticoagulant analogs that differ in there respective effects in coagulation and inflammation.

Angiopoietin Affects Neutrophil Migration

Objective: After an ischemic event vascular growth factors are involved in regulating leukocyte infiltration in inflammatory processes. This study focused on effects of 2 other angiogenic growth factors, angiopoietin‐1 and angiopoietin‐2, on human neutrophils and on the involvement of the angiopoietin receptor Tie‐2.

Treatment of intractable pruritus in drug induced cholestasis with albumin dialysis: a report of two cases.

Cholestatic liver injury can be caused by a variety of drugs and is difficult to treat. We report two patients, a 22 year old male and a 55 year old female, with drug induced cholestasis caused by anabolic-androgenic steroids (silabolin and nandrolone) and by fluoxetine, respectively. Both patients presented with massive jaundice and severe pruritus resulting in sleep deprivation and suicide ideation. Laboratory examination revealed signs of cholestasis. Medical treatment was ineffective; therefore, extra-corporeal albumin dialysis using the molecular adsorbent recirculating system (MARS) was started. Three treatments with a mean duration of 16 hours were performed in each patient. The procedure was well tolerated by the patients and resulted in a sustained relief of pruritus as well as in a decline of plasma bilirubin and serum 3&agr;-hydroxy bile acid levels. The mean plasma bilirubin concentration decreased from 25.27 mg/dl to 10.7 mg/dl; the mean serum 3&agr;-hydroxy bile acid concentration decreased from 299 μmol/L to 88 μmol/L. After 2 months, the pruritus had nearly vanished in both patients, and there was a further decline of bilirubin levels after discharge from hospital. In conclusion, extracorporeal albumin dialysis appears to be a therapeutic option in severe drug induced cholestasis refractory to medical treatment.

Divergent modulation of Chlamydia pneumoniae infection cycle in human monocytic and endothelial cells by iron, tryptophan availability and interferon gamma

Chlamydia pneumoniae is an obligatory intracellular bacterium causing chronic inflammatory diseases in humans. We studied the role of the nutritive factors, iron and tryptophan, towards the course of infection and immune response pathways in C. pneumoniae infected endothelial cells and monocytes. Human endothelial (EA.hy923) and monocytic cells (THP-1) were infected with C. pneumoniae, supplemented with iron or 1-methyltryptophan (1-MT), an inhibitor of the tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO), and subsequently stimulated with IFN-gamma or left untreated. The number of infected cells, the morphology and quantity of C. pneumoniae inclusion bodies, IDO activity and innate immune effector pathways were analysed. While neither iron challenge, IDO inhibition or IFN-gamma treatment had a significant effect on C. pneumoniae morphology or numbers within THP-1 monocytic cells, iron supplementation to EA.hy926 cells resulted in promotion of C. pneumoniae proliferation and differentiation while IFN-gamma had an inhibitory effect. Furthermore, the number of infected endothelial cells was significantly decreased upon 1-MT treatment. C. pneumoniae infection induced a pro-inflammatory immune response as evidenced by increased IDO activity, neopterin formation or TNF-alpha production in THP-1 but not in endothelial cells. These pathways were superinduced upon IFN-gamma treatment and partly modulated by iron supplementation. Our results demonstrate that the infectious cycle of C. pneumoniae behaves differently between monocytic and endothelial cells. While the intracellular pathogen remains in a persistent form within monocytes, it can differentiate and proliferate within endothelial cells indicating that endothelial cells are a preferred environment for Chlamydia. Nutritive factors such as iron have subtle effects on C. pneumoniae biology in endothelial, but not monocytic cells. Our results contribute to a better understanding of C. pneumoniae infection and its role in chronic inflammatory diseases such as atherosclerosis.

Cd40-ligand-dependent induction of Cox-2 gene expression in endothelial cells by activated platelets: inhibitory effects of atorvastatin

Increasing evidence shows the importance of platelet–endothelial cell interactions in the progression of atherosclerosis. Platelets contribute to coronary events both as major components of thrombi and as a triggering factor in inflammation that leads to plaque vulnerability. Recent data suggest that statins, besides their lipid-lowering properties, exert pleiotropic effects that may be beneficial in atherosclerosis. Whether activated platelets influence cyclooxygenase-2 (COX-2) expression in human umbilical vein endothelial cells (HUVEC), the effect of atorvastatin, and possible mechanisms were investigated. COX-2 gene expression in HUVEC was studied using real-time polymerase chain reaction. CD40 ligand surface expression of platelets was tested by fluorescence-activated cell sorting analyses. Activated platelets significantly up-regulated COX-2 gene expression in HUVEC. Co-incubation of platelets with atorvastatin was shown to reverse this up-regulation via reduction of CD40 ligand surface expression on platelets. Data suggest that atorvastatin influences CD40–CD40-ligand-dependent platelet–endothelial interaction and that this influence affects platelet-induced COX-2 expression in HUVEC.