Matijs van Meurs

Time course of angiopoietin-2 release during experimental human endotoxemia and sepsis

IntroductionEndothelial activation leading to vascular barrier breakdown denotes a devastating event in sepsis. Angiopoietin (Ang)-2, a circulating antagonistic ligand of the endothelial specific Tie2 receptor, is rapidly released from Weibel-Palade and has been identified as a non-redundant gatekeeper of endothelial activation. We aimed to study: the time course of Ang-2 release during human experimental endotoxemia; the association of Ang-2 with soluble adhesion molecules and inflammatory cytokines; and the early time course of Ang-2 release during sepsis in critically ill patients.MethodsIn 22 healthy volunteers during a 24-hour period after a single intravenous injection of lipopolysaccharide (LPS; 4 ng/kg) the following measurement were taken by immuno luminometric assay (ILMA), ELISA, and bead-based multiplex technology: circulating Ang-1, Ang-2, soluble Tie2 receptor, the inflammatory molecules TNF-alpha, IL-6, IL-8 and C-reactive protein, and the soluble endothelial adhesion molecules inter-cellular adhesion molecule-1 (ICAM-1), E-selectin, and P-selectin. A single oral dose of placebo or the p38 mitogen activated protein (MAP) kinase inhibitor drug, RWJ-67657, was administered 30 minutes before the endotoxin infusion. In addition, the course of circulating Ang-2 was analyzed in 21 septic patients at intensive care unit (ICU) admission and after 24 and 72 hours, respectively.ResultsDuring endotoxemia, circulating Ang-2 levels were significantly elevated, reaching peak levels 4.5 hours after LPS infusion. Ang-2 exhibited a kinetic profile similar to early pro-inflammatory cytokines TNF-alpha, IL-6, and IL-8. Ang-2 levels peaked prior to soluble endothelial-specific adhesion molecules. Finally, Ang-2 correlated with TNF-alpha levels (r = 0.61, P = 0.003), soluble E-selectin levels (r = 0.64, P < 0.002), and the heart rate/mean arterial pressure index (r = 0.75, P < 0.0001). In septic patients, Ang-2 increased in non-survivors only, and was significantly higher compared with survivors at baseline, 24 hours, and 72 hours.ConclusionsLPS is a triggering factor for Ang-2 release in men. Circulating Ang-2 appears in the systemic circulation during experimental human endotoxemia in a distinctive temporal sequence and correlates with TNF-alpha and E-selectin levels. In addition, not only higher baseline Ang-2 concentrations, but also a persistent increase in Ang-2 during the early course identifies septic patients with unfavorable outcome.

Acute administration of recombinant Angiopoietin-1 ameliorates multiple-organ dysfunction syndrome and improves survival in murine sepsis

INTRODUCTION Endothelial activation leading to vascular barrier breakdown plays an essential role in the pathophysiology of multiple-organ dysfunction syndrome (MODS) in sepsis. Increasing evidence suggests that the function of the vessel-protective factor Angiopoietin-1 (Ang-1), a ligand of the endothelial-specific Tie2 receptor, is inhibited by its antagonist Angiopoietin-2 (Ang-2) during sepsis. In order to reverse the effects of the sepsis-induced suppression of Ang-1 and elevation of Ang-2 we aimed to investigate whether an intravenous injection of recombinant human (rh) Ang-1 protects against MODS in murine sepsis. METHODS Polymicrobiological abdominal sepsis was induced by cecal ligation and puncture (CLP). Mice were treated with either 1 μg of intravenous rhAng-1 or control buffer immediately after CLP induction and every 8h thereafter. Sham-operated animals served as time-matched controls. RESULTS Compared to buffer-treated controls, rhAng-1 treated septic mice showed significant improvements in several hematologic and biochemical indicators of MODS. Moreover, rhAng-1 stabilized endothelial barrier function, as evidenced by inhibition of protein leakage from lung capillaries into the alveolar compartment. Histological analysis revealed that rhAng-1 treatment attenuated leukocyte infiltration in lungs and kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression in rhAng-1 treated mice. Finally, the protective effects of rhAng-1 treatment were reflected by an improved survival time in a lethal CLP model. CONCLUSIONS In a clinically relevant murine sepsis model, intravenous rhAng-1 treatment alone is sufficient to significantly improve a variety of sepsis-associated organ dysfunctions and survival time, most likely by preserving endothelial barrier function. Further studies are needed to pave the road for clinical application of this therapy concept.

Early organ-specific endothelial activation during hemorrhagic shock and resuscitation

Multiple organ dysfunction syndrome (MODS) is a complication of hemorrhagic shock (HS) and related to high morbidity and mortality. Interaction of activated neutrophils and endothelial cells is considered to play a prominent role in the pathophysiology of MODS. Insight in the nature and molecular basis of endothelial cell activation during HS can assist in identifying new rational targets for early therapeutic intervention. In this study, we examined the kinetics and organ specificity of endothelial cell activation in a mouse model of HS. Anesthetized male mice were subjected to controlled hemorrhage to a MAP of 30 mmHg. Mice were killed after 15, 30, 60, or 90 min of HS. After 90 min of hemorrhagic shock, a group of mice was resuscitated with 6% hydroxyethyl starch 130/0.4. Untreated mice and sham shock mice that underwent instrumentation and 90 min of anesthesia without shock served as controls. Gene expression levels of inflammatory endothelial cell activation (P-selectin, E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1) and hypoxia-responsive genes (vascular endothelial growth factor and hypoxia-inducible factor 1&agr;) were quantified in kidney, liver, lung, brain, and heart tissue by quantitative reverse-transcription-polymerase chain reaction. Furthermore, we examined a selection of these genes with regard to protein expression and localization using immunohistochemical analysis. Induction of inflammatory genes occurred early during HS and already before resuscitation. Expression of adhesion molecules was significantly induced in all organs, albeit to a different extent depending on the organ. Endothelial genes CD31 and VE-cadherin, which function in endothelial cell homeostasis and integrity, were not affected during the shock phase except for VE-cadherin in the liver, which showed increased mRNA levels. The rapid inflammatory activation was not paralleled by induction of hypoxia-responsive genes. This study demonstrated the occurrence of early and organ-specific endothelial cell activation during hemorrhagic shock, as presented by induced expression of inflammatory genes. This implies that early therapeutic intervention at the microvascular level may be a rational strategy to attenuate MODS.

Shock-induced stress induces loss of microvascular endothelial Tie2 in the kidney which is not associated with reduced glomerular barrier function

Both hemorrhagic shock and endotoxemia induce a pronounced vascular activation in the kidney which coincides with albuminuria and glomerular barrier dysfunction. We hypothesized that changes in Tie2, a vascular restricted receptor tyrosine kinase shown to control microvascular integrity and endothelial inflammation, underlie this loss of glomerular barrier function. In healthy murine and human kidney, Tie2 is heterogeneously expressed in all microvascular beds, although to different extents. In mice subjected to hemorrhagic and septic shock, Tie2 mRNA and protein were rapidly, and temporarily, lost from the renal microvasculature, and normalized within 24 h after initiation of the shock insult. The loss of Tie2 protein could not be attributed to shedding as both in mice and healthy volunteers subjected to endotoxemia, sTie2 levels in the systemic circulation did not change. In an attempt to identify the molecular control of Tie2, we activated glomerular endothelial cell cultures and human kidney slices in vitro with LPS or TNF-alpha, but did not observe a change in Tie2 mRNA levels. In parallel to the loss of Tie2 in vivo, an overt influx of neutrophils in the glomerular compartment, which coincided with proteinuria, was seen. As neutrophil-endothelial cell interactions may play a role in endothelial adaptation to shock, and these effects cannot be mimicked in vitro, we depleted neutrophils before shock induction. While this neutrophil depletion abolished proteinuria, Tie2 was not rescued, implying that Tie2 may not be a major factor controlling maintenance of the glomerular filtration barrier in this model.

Glutamine and antioxidants in critically ill patients.

To the Editor: Heyland et al. (April 18 issue)1 have shown that glutamine supplementation (approximately 65 g daily) of a diet that does not supply adequate energy (910 kcal) or protein (45 g) did not affect clinical outcome and increased mortality. However, the addition of a supplement that constituted 60% of total dietary protein introduced an amino acid imbalance with the potential for toxicity. Other, similar examples, such as the lack of arginine as part of dietary protein2 or the use of essential amino acids only3 in otherwise complete diets, have been shown to produce hyperammonemia in humans. Furthermore, glutamine itself is known to exacerbate defects in ammonia metabolism.4 Low-protein diets that are also low in sulfur-containing amino acids alter methionine metabolism, increasing homocysteine levels, which may affect the long-term health of human infants.5 Thus, manipulation of the amino acid composition of the diet, whether by eliminating one (arginine) or a group (all nonessential amino acids) or providing inadequate amounts of key amino acids (e.g., methionine or cysteine), or by grossly distorting the balance (such that glutamine constitutes more than 50%), may have adverse clinical consequences, despite a seemingly modest difference in the amounts of the various amino acids.

Angiopoietin-1 Treatment Reduces Inflammation but Does Not Prevent Ventilator-Induced Lung Injury

Background Loss of integrity of the epithelial and endothelial barriers is thought to be a prominent feature of ventilator-induced lung injury (VILI). Based on its function in vascular integrity, we hypothesize that the angiopoietin (Ang)-Tie2 system plays a role in the development of VILI. The present study was designed to examine the effects of mechanical ventilation on the Ang-Tie2 system in lung tissue. Moreover, we evaluated whether treatment with Ang-1, a Tie2 receptor agonist, protects against inflammation, vascular leakage and impaired gas exchange induced by mechanical ventilation. Methods Mice were anesthetized, tracheotomized and mechanically ventilated for 5 hours with either an inspiratory pressure of 10 cmH2O (‘low’ tidal volume ∼7.5 ml/kg; LVT) or 18 cmH2O (‘high’ tidal volume ∼15 ml/kg; HVT). At initiation of HVT-ventilation, recombinant human Ang-1 was intravenously administered (1 or 4 µg per animal). Non-ventilated mice served as controls. Results HVT-ventilation influenced the Ang-Tie2 system in lungs of healthy mice since Ang-1, Ang-2 and Tie2 mRNA were decreased. Treatment with Ang-1 increased Akt-phosphorylation indicating Tie2 signaling. Ang-1 treatment reduced infiltration of granulocytes and expression of keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP)-2, monocyte chemotactic protein (MCP)-1 and interleukin (IL)-1β caused by HVT-ventilation. Importantly, Ang-1 treatment did not prevent vascular leakage and impaired gas exchange in HVT-ventilated mice despite inhibition of inflammation, vascular endothelial growth factor (VEGF) and Ang-2 expression. Conclusions Ang-1 treatment downregulates pulmonary inflammation, VEGF and Ang-2 expression but does not protect against vascular leakage and impaired gas exchange induced by HVT-ventilation.

Off-pump CABG surgery reduces systemic inflammation compared with on-pump surgery but does not change systemic endothelial responses: a prospective randomized study.

ABSTRACT Coronary artery bypass graft (CABG) surgery can result in severe postoperative organ failure. During CABG surgery, cardiopulmonary bypass (CPB) with cardiac arrest is often used (on-pump CABG), which often results in a systemic inflammatory response. To reduce this inflammatory response, off-pump CABG was reintroduced, thereby avoiding CPB. There is increasing evidence that the endothelium plays an important role in the pathophysiology of organ failure after CABG surgery. In this study, 60 patients who were scheduled for elective CABG surgery were randomized to have surgery for on-pump or off-pump CABG. Blood was collected at four time points: start, end, 6 h, and 24 h postoperatively. Levels of inflammatory cytokines, soluble adhesion molecules, and angiogenic factors and their receptors were measured in the plasma. No differences were found in preoperative characteristics between the patient groups. The levels of tumor necrosis factor-&agr;, interleukin 10, and myeloperoxidase, but not interleukin 6, were increased to a greater extent in the on-pump CABG compared with off-pump CABG after sternum closure. The soluble endothelial adhesion molecules E-selectin, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 were not elevated in the plasma during and after CABG surgery in both on-pump and off-pump CABG. Angiopoietin 2 was only increased 24 h after surgery in both on-pump and off-pump CABG. Higher levels of sFlt-1 were found after sternum closure in off-pump CABG compared with on-pump CABG. Avoiding CPB and aortic cross clamping in CABG surgery reduces the systemic inflammatory response. On-pump CABG does not lead to an increased release of soluble endothelial adhesion molecules in the circulation compared with off-pump CABG.

The flow dependency of Tie2 expression in endotoxemia

RationaleTie2 is predominantly expressed by endothelial cells and is involved in vascular integrity control during sepsis. Changes in Tie2 expression during sepsis development may contribute to microvascular dysfunction. Understanding the kinetics and molecular basis of these changes may assist in the development of therapeutic intervention to counteract microvascular dysfunction.ObjectiveTo investigate the molecular mechanisms underlying the changes in Tie2 expression upon lipopolysaccharide (LPS) challenge.Methods and resultsStudies were performed in LPS and pro-inflammatory cytokine challenged mice as well as in mice subjected to hemorrhagic shock, primary endothelial cells were used for in vitro experiments in static and flow conditions. Eight hours after LPS challenge, Tie2 mRNA loss was observed in all major organs, while loss of Tie2 protein was predominantly observed in lungs and kidneys, in the capillaries. A similar loss could be induced by secondary cytokines TNF-α and IL-1β. Ang2 protein administration did not affect Tie2 protein expression nor was Tie2 protein rescued in LPS-challenged Ang2-deficient mice, excluding a major role for Ang2 in Tie2 down regulation. In vitro, endothelial loss of Tie2 was observed upon lowering of shear stress, not upon LPS and TNF-α stimulation, suggesting that inflammation related haemodynamic changes play a major role in loss of Tie2 in vivo, as also hemorrhagic shock induced Tie2 mRNA loss. In vitro, this loss was partially counteracted by pre-incubation with a pharmacologically NF-кB inhibitor (BAY11-7082), an effect further substantiated in vivo by pre-treatment of mice with the NF-кB inhibitor prior to the inflammatory challenge.ConclusionsMicrovascular bed specific loss of Tie2 mRNA and protein in vivo upon LPS, TNFα, IL-1β challenge, as well as in response to hemorrhagic shock, is likely an indirect effect caused by a change in endothelial shear stress. This loss of Tie2 mRNA, but not Tie2 protein, induced by TNFα exposure was shown to be controlled by NF-кB signaling. Drugs aiming at restoring vascular integrity in sepsis could focus on preventing the Tie2 loss.

Pleiotropic effects of angiopoietin-2 deficiency do not protect mice against endotoxin-induced acute kidney injury

BACKGROUND In sepsis and various other inflammatory conditions, elevated circulating levels of angiopoietin-2 (Ang2) are detected, but the precise functional role of Ang2 in these conditions is not well understood. Here, we investigated the contribution of Ang2 to the inflammatory response and renal function impairment in a mouse model of endotoxaemia. METHODS Ang2-deficient mice and wild-type littermates were challenged with lipopolysaccharide [LPS; 1500 EU/g, intraperitoneal (i.p.)]. In additional experiments, wild-type C57Bl/6 mice were depleted of circulating neutrophils by antibody treatment (NIMPR14) prior to LPS challenge to study the role of neutrophils in regulating LPS-induced cytokine release. After 8 or 24 h of LPS challenge, the mice were sacrificed and organs were harvested. Quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were performed for endothelial adhesion molecules (P-selectin, E-selectin, VCAM-1 and ICAM-1) and plasma cytokines (TNF-α, IL-6, KC, MIP-2), respectively. To assess renal function, blood urea nitrogen levels in plasma and albumin-to-creatinine ratio in urine were measured. RESULTS Upon LPS challenge, expression levels of various endothelial adhesion molecules in Ang2-deficient mice were reduced in an organ-specific manner. In contrast, in these mice, plasma levels of TNF-α and IL-6 were significantly increased compared with their wild-type littermates, possibly due to decreased neutrophil glomerular influx. Importantly, the absence of Ang2 did not protect the mice from acute kidney injury (AKI) upon LPS challenge. CONCLUSIONS The absence of Ang2 release upon LPS challenge induces pleotropic effects with regard to endothelial activation and systemic inflammation, but does not protect mice from LPS-induced AKI.

ADIPONECTIN DIMINISHES ORGAN-SPECIFIC MICROVASCULAR ENDOTHELIAL CELL ACTIVATION ASSOCIATED WITH SEPSIS

Experimental sepsis was induced in male C57BL/6j, adiponectin-deficient mice (ADPNKO), and wild-type littermates by i.p. injection of 16 mg/kg lipopolysaccharide or cecal ligation and puncture. Blood and tissue samples were harvested 24 h after model induction. Circulating adiponectin is reduced in mice with endotoxemic challenge and after cecal ligation and puncture compared with healthy control mice. Quantitative reverse transcriptase–polymerase chain reaction for adiponectin reveals a pattern of response that is both model- and organ-specific. When challenged with sepsis, adiponectin deficiency results in increased expression of endothelial adhesion and coagulation molecules in the lung, liver, and kidney as quantified by reverse transcriptase–polymerase chain reaction, increased macrophage and neutrophil infiltration by immunohistochemistry, and vascular leakage in the liver and kidney. Adiponectin-deficient mice have reduced survival following cecal ligation and puncture and increased blood levels of interleukin 6, soluble vascular endothelial growth factor receptor 1, and soluble endothelial adhesion molecules E-selectin and intercellular adhesion molecule 1. Finally, ADPNKO promoted end-organ injury in the liver and kidney, whereas the lungs were not affected. These data suggest a protective role of adiponectin in diminishing microvascular organ-specific endothelial cell activation during sepsis. ABBREVIATIONS LPS—lipopolysaccharide CLP—cecal ligation and puncture sVEGFR1—soluble vascular endothelial growth factor receptor 1 IL-6 – interleukin 6 ICAM-1—intercellular adhesion molecule 1 VCAM-1—vascular cell adhesion molecule 1