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

Critical Care Vol12No4 Circulating angiopoietin-1 and angiopoietin-2 in critically ill patients: development and clinical application of two new immunoassays Alexander Lukasz 1 Julian Hellpap 1 Rdiger Horn 0 Jan T Kielstein 1 Sascha David 1 Hermann Haller 1 Philipp Kmpers 1 Corresponding author: Philipp Kmpers 0 Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School , Carl-Neuberg-Strae 1, Hannover 30625 , Germany 1 Department of Nephrology, Hannover Medical School , Carl-Neuberg-Strae 1, 30625 Hannover , Germany Introduction In 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. Methods We 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. - Introduction Endothelial activation denotes a devastating key event in sepsis pathophysiology that is characterized by increased expression of luminal adhesion molecules, leukocyte recruitment, and altered vasomotor tone, resulting in vascular barrier breakdown [1-3]. The endothelial-specific angiopoietinTie ligandreceptor system has recently emerged as a nonredundant regulator of endothelial activation [4-6]. Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are antagonistic ligands that bind to the extracellular domain of the Tie2 receptor, which is almost exclusively expressed by endothelial cells. Binding of Ang-1 to Tie2 promotes vessel integrity, inhibits vascular leakage and suppresses inflammatory gene expression [7,8]. Ang-2 is stored in WeibelPalade bodies and is rapidly secreted and induced upon stimulation, whereas Ang-1 is constitutively expressed by pericytes and vascular Ang-1 = angiopoietin-1; Ang-2 = angiopoietin-2; BSA = bovine serum albumin; EDD = extended daily dialysis; EDTA = ethylenediamine tetraacetic acid; ELISA = enzyme-linked immunosorbent assay; ICU = intensive care unit; IL = interleukin; ILMA = immunoluminometric sandwich assay; IRMA = immunoradiometric sandwich assay; PAB = polyclonal anti-human Ang-1 affinity-purified goat IgG antibody; PBST = phosphate-buffered saline with 0.05% Tween-20; SOFA = Sequential Organ Failure Assessment. smooth muscle cells [5,9,10]. Binding of antagonistic Ang-2 completely disrupts protective Tie2 signaling in the majority of experimental studies [7,11,12]. Ang-2 has also been identified as a Tie2 agonist, however, especially when administered in a supramaximal dose [13,14]. Several pilot studies suggest that measuring circulating Ang1 and Ang-2 in critically ill patients might provide valuable information on vascular barrier properties. A marked imbalance of the angiopoietinTie system in favor of Ang-2 was detected consistently in critically ill patients [15-19]. Elevated Ang-2 concentrations correlate with severity of illness as assessed by the injury severity score [15], the organ failure index [17], and the Acute Physiology and Chronic Health Evaluation (APACHE) II score or Sequential Organ Failure Assessment (SOFA) score [16,18,19]. Circulating Ang-2 predicted outcome in two studies [15,16]. Circulating Ang-2 and the respective Ang-2/Ang-1 ratio therefore constitute potential new biomarkers for endothelial activation in critical illness. Preanalytic performance, detailed assay characteristics, and clinical applicability of Ang-1 and Ang-2 immunoassays have not been reported. The aim of the present study was to develop, characterize and validate immunoassays for the detection of circulating Ang-1 and Ang-2. Materials and methods Angiopoietin-1 immunoradiometric sandwich assay A polyclonal anti-human Ang-1 affinity-purified goat IgG antibody (PAB) and a monoclonal anti-human Ang-1 mouse antibody were obtained from R&D Systems (Minneapolis, MN, USA). Recombinant human Ang-1 (90% purity recombinant, expressed in a murine nonsecreting NSO myeloma cell line) was purchased from Sigma-Aldrich (Munich, Germany). Maxisorp Startubes (Nunc, Roskilde, Denmark) were coated for 2 hours at 4C with 0.5 g/tube monoclonal anti-human Ang-1 mouse antibody in 0.1 M sodium carbonate buffer (pH 9.5), and were then washed twice with phosphate-buffered saline with 0.05% Tween-20 (PBST). Serum samples (100 l) were then diluted 1:1 with assay buffer (30 g/l BSA, 10 g/l bovine IgG, 1% goat serum, 0.1% NaN3, 1 M NaCl, 40 mM sodium phosphate buffer, pH 7.4), were added to the tubes, and were incubated for about 24 hours at 4C. PAB was iodinated with 125Iod (Hartmann, Braunschweig, Germany) using IODO-GEN (Perbio Science, Bonn, Germany). Unbound 125I was separated by desalting on a 10 ml Sephadex G-25 column (Pharmacia, Uppsala, Sweden). The tubes were washed twice with PBST. Two hundred microliters of assay buffer containing 10 ng 125I-iodinated PAB (specific activity approximately 0.74 MBq/g) (tracer) were added to each tube, and were incubated for 4 hours at room temperature. After three washing steps, bound radioactivity was quantified in a gamma counter (LKB Wallac 1261; Perkin-Elmer, Waltham, Massachusetts, USA). In each experiment, a standard curve was generated with various dilutions of Ang-1. The curve was then used to calculate the Ang-1 concentrations in individual samples. Angiopoietin-1 immunoluminometric sandwich assay In the case of immunoluminometric detection, PAB was conjugated with Acridinium C2 NHS Ester (Assay Designs, Ann Arbor, MI, USA). The conjugated PAB was then quantified in a System Luminometer (Nichols Institute Diagnostics, San Juan Capistrano, California, USA). Angiopoietin-2 ELISA Ang-2 was measured using antibodies included in the DuoSet methodology ELISA (R&D Systems). Recombinant human Ang-2 (95% purity, murine nonsecreting NSO derived; R&D Systems) served as the standard. ELISA plates (Nunc Maxisorb, Roskilde, Denmark) were coated overnight at 4C with 2 g/ml monoclonal Ang-2 antibody in 0.1 M sodium carbonate buffer (pH 9.5), and were then washed three times with 300 l PBST. Serum samples (50 l) were then diluted 1:1 with assay buffer 1 (30 g/l BSA, 10 g/l bovine IgG, 1% goat serum, 0.1% NaN3, 1 M NaCl, 40 mM sodium phosphate buffer, pH 7.4), were added to the tubes, and were incubated for 2 hours at (...truncated)