Philipp Kümpers

Circulating miR-210 Predicts Survival in Critically Ill Patients with Acute Kidney Injury

BACKGROUND AND OBJECTIVES MicroRNAs (miRNAs) are small ribonucleotides regulating gene expression. MicroRNAs are present in the blood in a remarkably stable form. We tested whether circulating miRNAs in the plasma of critically ill patients with acute kidney injury (AKI) at the inception of renal replacement therapy are deregulated and may predict survival. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We profiled miRNAs using RNA isolated from the plasma of patients with AKI and healthy controls. The results were validated in 77 patients with acute kidney injury, 30 age-matched healthy controls, and 18 critically ill patients with acute myocardial infarction by quantitative real-time PCR. RESULTS Circulating levels of miR-16 and miR-320 were downregulated in the plasma of kidney injury AKI patients, whereas miR-210 was upregulated compared with healthy controls (all P < 0.0001) and disease controls (miR-210 and miR-16: P < 0.0001; miR-320: P = 0.03). Cox regression (P < 0.05) and Kaplan-Meier curve analysis (P = 0.03) revealed miR-210 as an independent and powerful predictor of 28-day survival. CONCLUSIONS Circulating miRNAs are altered in patients with kidney injury AKI. MiR-210 predicts mortality in this patient cohort and may serve as a novel biomarker AKI reflecting pathophysiological changes on a cellular level.

Excess circulating angiopoietin-2 is a strong predictor of mortality in critically ill medical patients

IntroductionThe endothelial specific angiopoietin (Ang)-Tie2 ligand-receptor system has been identified as a non-redundant mediator of endothelial activation in experimental sepsis. Binding of circulating Ang-1 to the Tie2 receptor protects the vasculature from inflammation and leakage, whereas binding of Ang-2 antagonises Tie2 signalling and disrupts endothelial barrier function. Here, we examine whether circulating Ang-1 and/or Ang-2 independently predict mortality in a cohort of critically ill medical patients.MethodsCirculating vascular endothelial growth factor (VEGF), Ang-1 and Ang-2 were prospectively measured in sera from 29 healthy controls and 43 medical ICU patients by immunoradiometric assay (IRMA) and ELISA, respectively. Survival after 30 days was the primary outcome studied.ResultsMedian serum Ang-2 concentrations were increasingly higher across the following groups: healthy controls, patients without sepsis, patients with sepsis and patients with septic shock. In contrast, Ang-1 and VEGF concentrations were significantly lower in all patient groups compared with healthy controls. Ang-2 correlated with partial pressure of oxygen in arterial blood (PaO2)/fraction of inspired oxygen (FiO2), tissue hypoxia, Sequential Organ Failure Assessment (SOFA) and Physiology and Chronic Health Evaluation II (APACHE II) score. Multivariate Cox regression analyses confirmed a strong independent prognostic impact of high Ang-2 as a novel marker of 30-day survival.ConclusionsA marked imbalance of the Ang-Tie system in favour of Ang-2 is present in critically ill medical patients. Our findings highlight the independent prognostic impact of circulating Ang-2 in critical illness. Ang-2 may be used as a readily available powerful predictor of outcome and may open new perspectives to individualise treatment in the ICU.

Bench-to-bedside review: Angiopoietin signalling in critical illness – a future target?

Multiple organ dysfunction syndrome (MODS) occurs in response to major insults such as sepsis, severe haemorrhage, trauma, major surgery and pancreatitis. The mortality rate is high despite intensive supportive care. The pathophysiological mechanism underlying MODS are not entirely clear, although several have been proposed. Overwhelming inflammation, immunoparesis, occult oxygen debt and other mechanisms have been investigated, and – despite many unanswered questions – therapies targeting these mechanisms have been developed. Unfortunately, only a few interventions, usually those targeting multiple mechanisms at the same time, have appeared to be beneficial. We clearly need to understand better the mechanisms that underlie MODS. The endothelium certainly plays an active role in MODS. It functions at the intersection of several systems, including inflammation, coagulation, haemodynamics, fluid and electrolyte balance, and cell migration. An important regulator of these systems is the angiopoietin/Tie2 signalling system. In this review we describe this signalling system, giving special attention to what is known about it in critically ill patients and its potential as a target for therapy.

Serum neutrophil gelatinase-associated lipocalin at inception of renal replacement therapy predicts survival in critically ill patients with acute kidney injury

IntroductionNeutrophil gelatinase-associated lipocalin (NGAL) is a promising novel biomarker that correlates with the severity and outcome of acute kidney injury (AKI). However, its prognostic utility during the late course of AKI, especially in patients that require renal replacement therapy (RRT) remains unknown. The aim of this study was to evaluate the predictive value of serum NGAL in patients with established AKI at inception of RRT in the intensive care unit (ICU).MethodsSerum NGAL (ELISA methodology) was measured in 109 critically ill patients with AKI at inception of RRT in 7 ICUs of a tertiary care university hospital. The primary outcome studied was 28-day mortality. Secondary outcome measures were ICU length of stay, ventilator-free days, and renal recovery at day 28.ResultsThere was a significant difference in serum NGAL between healthy subjects (median [interquartile range] 39.0 [37.5-42.75] ng/mL), critically ill patients with systemic inflammatory response syndrome (SIRS) (297 [184-490] ng/mL), and critically ill patients with sepsis (708 [365-1301] ng/mL; P < 0.0001), respectively. Multiple linear regression showed that NGAL levels were independently related to the severity of AKI and the extent of systemic inflammation. NGAL levels were higher in non-survivors (430 [303-942] ng/mL) compared to survivors (298 [159-506] ng/mL; P = 0.004). Consistently, Cox proportional hazards regression analysis identified NGAL as a strong independent predictor for 28-day survival (hazard ratio 1.6 (95% confidence interval [CI] 1.15 - 2.23), P = 0.005).ConclusionsThis is the first prospective evaluation of serum NGAL as an outcome-specific biomarker in critically ill patients at initiation of RRT. The results from this study indicate that serum NGAL is as an independent predictor of 28-day mortality in ICU patients with dialysis-dependent AKI.

The synthetic Tie2 agonist peptide vasculotide protects against vascular leakage and reduces mortality in murine abdominal sepsis

IntroductionAngiopoietin-1 (Angpt1), the natural agonist ligand for the endothelial Tie2 receptor, is a non-redundant endothelial survival and vascular stabilization factor that reduces endothelial permeability and inhibits leukocyte-endothelium interactions. Here we evaluate the efficacy of a novel polyethylene glycol (PEG)-clustered Tie2 agonist peptide, vasculotide (VT), to protect against vascular leakage and mortality in a murine model of polymicrobial abdominal sepsis.MethodsPolymicrobial abdominal sepsis in C57BL6 mice was induced by cecal-ligation-and-puncture (CLP). Mice were treated with different dosages of VT or equal volume of phosphate-buffered saline (PBS). Sham-operated animals served as time-matched controls.ResultsSystemic administration of VT induced long-lasting Tie2 activation in vivo. VT protected against sepsis-induced endothelial barrier dysfunction, as evidenced by attenuation of vascular leakage and leukocyte transmigration into the peritoneal cavity. Histological analysis revealed that VT treatment ameliorated leukocyte infiltration in kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression. VT-driven effects were associated with significantly improved organ function and reduced circulating cytokine levels. The endothelial-specific action of VT was supported by additional in vitro studies showing no effect of VT on either cytokine release from isolated peritoneal macrophages, or migratory capacity of isolated neutrophils. Finally, administration of VT pre-CLP (hazard ratio 0.39 [95% confidence interval 0.19-0.81] P < 0.001) and post-CLP reduced mortality in septic mice (HR 0.22 [95% CI 0.06-0.83] P < 0.05).ConclusionsWe provide proof of principle in support of the efficacious use of PEGylated VT, a drug-like Tie2 receptor agonist, to counteract microvascular endothelial barrier dysfunction and reduce mortality in a clinically relevant murine sepsis model. Further studies are needed to pave the road for clinical application of this therapeutic concept.

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.

Rituximab as rescue therapy in anti-neutrophil cytoplasmic antibody-associated vasculitis: a single-centre experience with 15 patients

BACKGROUND B-cell depletion with rituximab, a chimeric anti-CD20 antibody, is a novel treatment for refractory and relapsing ANCA-associated small-vessel vasculitis. Data are limited and most reports describe single patients or small numbers of patients followed prospectively. METHODS We report a single-centre experience with 15 patients who received rituximab for refractory or relapsing ANCA-associated vasculitis. All patients had been treated with corticosteroids and cyclophosphamide and a variety of other second-line immunosuppressive agents. None of the patients had evidence of infection and received four infusions of 375 mg/m(2) of rituximab. Disease activity was assessed in accordance with the Birmingham Vasculitis Activity Score (BVAS). BVAS, C-reactive protein and ANCA titres were recorded at baseline and during follow-up. RESULTS B-cell depletion was achieved in all patients. Partial or complete remission was seen in 14 of 15 patients with a significant decline in BVAS compared to baseline (P < 0.007). One patient with granulomatous ANCA-associated vasculitis did not respond to rituximab. There were no side effects during rituximab infusion. Transient leucopenia was observed in two patients. One patient with bronchial stenosis died of pneumonia 5.5 months after the initiation of rituximab treatment. One initially anti-HBc-positive/HBsAg-negative patient experienced a reactivation of hepatitis B, developed end-stage renal failure and died after refusal of dialysis. CONCLUSIONS We report the largest case series of rituximab use for ANCA-associated vasculitis so far. Our data support that the drug is capable of inducing partial or complete remission in refractory or relapsing patients. Leucopenia and infectious complications remain a matter of concern.

Circulating angiopoietins in idiopathic pulmonary arterial hypertension

AIMS To determine the diagnostic utility of circulating angiopoietin-1 (Ang-1) and its antagonist angiopoietin-2 (Ang-2) as potential biomarkers of disease severity or response to treatment in idiopathic pulmonary arterial hypertension (IPAH). Imbalances in angiogenic factors including vascular endothelial cell growth factor (VEGF) and the angiopoetin-Tie2 receptor system have been implicated in the pathogenesis of IPAH. METHODS AND RESULTS Plasma Ang-1, Ang-2, soluble Tie2 (sTie2), and VEGF were determined by in-house immunoassays in two cohorts of IPAH patients: a retrospective cohort (n = 81) and a prospective cohort (n = 25). Ten patients with normal pulmonary artery pressures and 14 apparently healthy subjects served as controls. Plasma levels of all angiogenic factors were elevated in IPAH patients compared with controls (all P < 0.005). Angiopoietin-2, but not Ang-1, sTie2, and VEGF correlated with cardiac index (r = -0.53, P < 0.001), pulmonary vascular resistance (PVR) (r= 0.60, P < 0.001), and mixed venous oxygen saturation (SvO(2)) (r= -0.63, P < 0.001). In multivariate analysis, elevated Ang-2 was an independent risk factor of mortality (P = 0.004). The patients in the prospective cohort were studied longitudinally at baseline and 3 months after initiation of therapy. Changes in Ang-2 after initiation of therapy correlated with changes in mean right atrial pressure (r = 0.6, P = 0.008), PVR (r = 0.51, P = 0.04), and inversely related to changes in SvO(2) (r = -0.75, P < 0.001). Histological studies showed that the expression of Ang-2 mRNA and protein was up-regulated in plexiform lesions from IPAH lung tissue samples. CONCLUSION Ang-2 may be involved in the pathogenesis of IPAH, and plasma Ang-2 might serve as a promising new biomarker of disease severity and response to treatment in patients with IPAH.

Circulating angiopoietin-1 and angiopoietin-2 in critically ill patients: development and clinical application of two new immunoassays.

IntroductionIn critically ill patients, the massive release of angiopoietin-2 (Ang-2) from endothelial Weibel–Palade bodies interferes with constitutive angiopoietin-1 (Ang-1)/Tie2 signaling in endothelial cells, thus leading to vascular barrier breakdown followed by leukocyte transmigration and capillary leakage. The use of circulating Ang-1 and Ang-2 as novel biomarkers of endothelial integrity has therefore gained much attention. The preclinical characteristics and clinical applicability of angiopoietin immunoassays, however, remain elusive.MethodsWe developed sandwich immunoassays for human Ang-1 (immunoradiometric sandwich assay/immunoluminometric sandwich assay) and Ang-2 (ELISA), assessed preanalytic characteristics, and determined circulating Ang-1 and Ang-2 concentrations in 30 healthy control individuals and in 94 critically ill patients. In addition, Ang-1 and Ang-2 concentrations were measured in 10 patients during a 24-hour time course with respect to interference by intravenous antibiotic treatment and by extended daily dialysis.ResultsThe assays had detection limits of 0.12 ng/ml (Ang-1) and 0.2 ng/ml (Ang-2). Inter-assay and intra-assay imprecision was ≤8.8% and 3.7% for Ang-1 and was ≤4.6% and 5.2% for Ang-2, respectively. Angiopoietins were stable for 24 hours and were resistant to four freeze–thaw cycles. Angiopoietin concentrations were not associated with age, body mass index or renal function in healthy individuals. Ang-1 and Ang-2 concentrations correlated with severity of illness in critically ill patients. Angiopoietin concentrations were not influenced by antibiotic treatment or by extended daily dialysis.ConclusionAng-1 and Ang-2 might serve as a novel class of biomarker in critically ill patients. According to preclinical and clinical validation, circulating Ang-1 and Ang-2 can be reliably assessed by novel immunoassays in the intensive care unit setting.

Effects of a synthetic PEG-ylated Tie-2 agonist peptide on endotoxemic lung injury and mortality

A synthetic 7-mer, HHHRHSF, was recently identified by screening a phage display library for binding to the Tie-2 receptor. A polyethylene-oxide clustered version of this peptide, termed vasculotide (VT), was reported to activate Tie-2 and promote angiogenesis in a mouse model of diabetic ulcer. We hypothesized that VT administration would defend endothelial barrier function against sepsis-associated mediators of permeability, prevent lung vascular leakage arising in endotoxemia, and improve mortality in endotoxemic mice. In confluent human microvascular endothelial cells, VT prevented endotoxin-induced (lipopolysaccharides, LPS O111:B4) gap formation, loss of monolayer resistance, and translocation of labeled albumin. In 8-wk-old male C57Bl6/J mice given a ∼70% lethal dose of endotoxin (15 mg/kg ip), VT prevented lung vascular leakage and reversed the attenuation of lung vascular endothelial cadherin induced by endotoxemia. These protective effects of VT were associated with activation of Tie-2 and its downstream mediator, Akt. Echocardiographic studies showed only a nonsignificant trend toward improved myocardial performance associated with VT. Finally, we evaluated survival in this mouse model. Pretreatment with VT improved survival by 41.4% (n = 15/group, P = 0.02) and post-LPS administration of VT improved survival by 33.3% (n = 15/group, P = 0.051). VT-mediated protection from LPS lethality was lost in Tie-2 heterozygous mice, in agreement with VTs proposed receptor specificity. We conclude that this synthetic Tie-2 agonist, completely unrelated to endogenous Tie-2 ligands, is sufficient to activate the receptor and its downstream pathways in vivo and that the Tie-2 receptor may be an important target for therapeutic evaluation in conditions of pathological vascular leakage.