Functional characterization of sEpoR.

<p><b>3a.</b> Western blot showing increasedphospho-Stat-5 in the presence of increasing erythropoietin (25 to 5000 mU/ml) in BaF3/EpoR cell lysates. <b>3b</b>. Representative western blot showing total phospho-Stat-5 and Stat-5 in the presence of erythropoietin 5000 mU/ml and varying concentrations of recombinant sEpoR-Fc (50 -5000 ng/ml). Phospho-Stat-5 decreases with increasing recombinant sEpoR. <b>3c</b>. Quantification of sEpoR-Fc inhibition of phospho-Stat 5 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009246#pone-0009246-g003" target="_blank">Figure 3b</a>). Ratios of phospho/total Stat-5 (mean ± SD, n = 3) represented as a percentage of control (Epo alone). *represents p<0.05. <b>3d</b>. Serum from patients with high sEpoR blocks erythropoietin mediated Stat-5 phosphorylation. Shown is the ratio of phospho-Stat-5 to Stat-5 as measured by densitometry. Serum starved BaF3/EpoR cells were exposed to vehicle (negative control), erythropoietin at 50 mU/ml (positive control) and erythropoietin plus 10% serum with Low sEpoR (≤62.5 pg/ml) or serum with high sEpoR (≥4000 pg/ml) for 10 minutes. Cells were lysed in RIPA buffer and 10 ug protein/lane was run on a 4-12% denaturing gel. Gels were transferred and blotted with anti-Stat-5 and anti-phospho-Stat 5. <b>3e</b>. Quantification of western data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009246#pone-0009246-g003" target="_blank">Figure 3d</a>). Ratios of phospho/total Stat-5 (mean ± SD, n = 5 individual patient samples each for low sEpoR and high sEpoR) represented as a percentage of control (Epo alone). *represents p<0.05.</p

sEpoR is regulated by proinflammatory cytokines.

<p><b>4a.</b> IL-6, TNF-α and PMA increase sEpoR in the supernatant of K562 cells. K562 were plated in serum free media and exposed for 48 h to vehicle, PMA, IL-6 and TNF-α. At the end of the incubation cells were pelleted and the supernatant subjected to ELISA for sEpoR. sEpoR measurements were corrected for total protein concentration. * represents p value of <0.05 when compared to the control group. <b>4b.</b> Mean IL-6 levels in subjects with low (n = 32) and high sEpoR (n = 32) are shown. * represents p value of <0.05 when compared to low sEpoR group.</p

sEpoR characterization in uremic serum.

<p><b>1a</b>. Soluble EpoR is detectable in serum from dialysis patients by western blot. Human serum was subjected to immunoprecipitation with goat anti-human erythropoietin receptor antibody (R&D Systems, AF-322-PB) followed by western blotting with mouse monoclonal anti-human erythropoietin receptor (R&D Systems, MAB307). Both antibodies recognize the extracellular domain of the receptor. Lanes 1–6 are serum from 6 representative dialysis patients, lane 7 is blank and lane 8 is recombinant sEpoR (Sigma Aldrich E0643, Saint Louis MI). Shown in the serum samples is a band of expected molecular weight of approximately 27 kDa. The control sEpoR with Fc tag is consistent with the manufacturers reported molecular weight of 32 kDa. <b>1b</b>. Soluble EpoR is also detected using the same dialysis patient serum samples by performing immunoprecipitation in reverse order. In this experiment immunoprecipitation was done with mouse monoclonal anti-human erythropoietin receptor (R&D Systems, MAB307) followed by western blotting with goat anti-human erythropoietin receptor (R&D Systems, AF-322-PB). Lanes 1 to 3 are serum from 3 dialysis patients, and lane 4 is recombinant sEpoR-Fc (Sigma, 307) as positive control.</p

Serum from People with Sepsis Disrupts Endothelial Architecture and This Effect Resolves with Clinical Improvement, Correlates with Measured Ang-2, and Is Reversed by Ang-1

<div><p>Ten percent FBS or 10% serum from one of two patients with sepsis was incubated with EC monolayers to assess effects on endothelial architecture. High Ang-2 serum (Patient CE4, Ang-2 = 89 ng/ml) induced thick actin stress fibers and intercellular gap formation (D–F), whereas low Ang-2 serum (CF1, Ang-2 = 8.9 ng/ml) did not (G–I). The gap-promoting effect of Patient CE4′s serum was reversed with addition of 100 ng/ml recombinant human Ang-1 (J–L) and was indistinguishable from control cells that exhibit thin actin fibers and no intercellular gaps (A–C).</p> <p>Serum was then taken from one patient (Patient CG), drawn on hospital day 2 (Patient CG2, Ang-2 = 78 ng/ml) and hospital day 16 (Patient CG12, Ang-2 = 6.3 ng/ml), and was added at 10% to HMVEC monolayers. Again, high-Ang-2 serum (CG2) induced gap formation and thick actin stress fibers (M–O), effects not seen in the serum of the same patient at discharge (CG12) (P–R) and effects that were reversed with the addition of 100 ng/ml Ang-1 (S–U). Arrows indicate intercellular gaps.</p></div

Ang-2 Alone Disrupts Endothelial Architecture at Physiologic Concentrations

<div><p>(A) Control (vehicle) or recombinant human Ang-2 (100 ng/ml) was added to HMVEC monolayers. These cells were then fixed and stained for F-actin and VE-cadherin. Shown are healthy control cells (panels a–c) versus Ang-2 treated cells (panels d–f), which exhibit thick actin stress fibers and disrupted junctions, leaving intercellular gaps (arrows).</p> <p>(B) HMVECs were grown to confluence on Transwell membranes coated with fibronectin. Monolayers were treated with vehicle or Ang-2 (400 ng/ml in luminal chamber) plus FITC-albumin. P_a was calculated after 8 h as described in the Methods section. P_a values are expressed as percentage of control cells.*<i>p</i> < 0.01.</p></div

Serum Ang-2 at Study Enrollment

<p>Ang-2 was measured in serum specimens obtained prospectively from patients meeting criteria for sepsis (<i>n</i> = 22) and from randomly selected hospitalized patients (<i>n</i> = 29), used as a control, with a variety of illnesses ranging from infectious (e.g., pyelonephritis, aseptic meningitis, pneumonia) to cardiovascular (e.g., angina, syncope) and neurologic (e.g., stroke) diseases. Patients with sepsis were further subdivided into those with severe sepsis—defined by the presence of shock or multi-organ dysfunction (<i>n</i> = 17)—and those without severe sepsis (mild sepsis, <i>n</i> = 5). Individuals who were controls (marked Controls) and individuals with sepsis without shock or multi-organ dysfunction (marked Mild Sepsis) had low serum Ang-2 at enrollment (3.5 ± 0.6 ng/ml and 4.8 ± 1.5 ng/ml, respectively). Patients hospitalized with severe sepsis (marked Severe Sepsis) had significantly higher serum Ang-2 at enrollment (23.2 ± 9.1 ng/ml, <i>p</i> = 0.0071) compared with control patients. During the course of the hospitalization, only the severe sepsis group had higher peak Ang-2 (32.4 ± 8.7 ng/ml), whereas those patients in the control group and those patients with mild sepsis maintained stable Ang-2 < 10 ng/ml (unpublished data).</p

Peak Circulating Ang-2 Correlates with Impaired Pulmonary Gas Exchange

<div><p>(A) Impaired oxygenation of blood, as assessed by the nadir PaO₂/FiO₂ ratio, correlates with significant differences in circulating Ang-2, *<i>p</i> = 0.0195.</p> <p>(B) Circulating Ang-2 does not correlate with survival to discharge. Among the five patients who did not survive, medical care was withdrawn from three patients in accordance with family wishes; the remaining two died despite full measures.</p> <p>(C) APACHE II is a commonly used scoring system to rate overall severity of critical illness. Ang-2 does not differ significantly among individuals with high (more severe illness) or low (less severe illness) APACHE II scores.</p> <p>(D) History of congestive heart failure (defined by clinical documentation in medical record of measured ejection fraction = < 40%) does not correlate with significant differences in circulating Ang-2.</p></div

Temporal Trends of Circulating Ang-2 in Three Illustrative Hospitalized Patients

<p>Patient CH (—▪—), a 74-y-old woman, was admitted to the medical intensive care unit with severe sepsis. She was treated with broad-spectrum antibiotics, initially required three vasoactive agents to manage shock, and was mechanically ventilated. Patient CH's nadir PaO₂/FiO₂ = 240 occurred on hospital day 2, correlating with her peak circulating Ang-2. Enterococcus was grown from her urine. She progressively convalesced and was extubated prior to discharge. Patient AP (—▴—), a 92-y-old woman, was admitted to the general medicine service from a nursing home for increased confusion over her baseline dementia. She had no evidence of sepsis, shock, or respiratory compromise—PaO₂/FiO₂ > 300. She was treated for a foot wound infection with two antibiotics and was discharged in stable condition back to the nursing home. Patient AG (—○—), a 77-y-old man, was first admitted to the general medicine service with hypotension following excessive fluid removal at hemodialysis—there was no evidence of infection, systemic inflammatory response, or respiratory compromise with PaO₂/FiO₂ > 300 (hospital days 1–3). However, 3 mo later (graphed as hospital days 6–8 for purposes of illustration), the same patient (—○—) was re-admitted to the intensive care unit following emergent right leg amputation for gangrene complicated by shock and inability to extubate. Nadir PaO₂/FiO₂ = 144 occurred on the same day as peak Ang-2 (depicted as hospital day 8), when he died despite full care.</p

Systemic Administration of Ang-2 Promotes Pulmonary Hyperpermeability and Water Accumulation

<div><p>(A) After injection of vehicle or Ang-2 (10 μg, intraperitoneal), mice were injected in the retro-orbital sinus with Evans blue (2%, 50 μl); after sacrifice, intravascular Evans blue was washed out with PBS and vascular leakage was evaluated by measuring extravasated Evans blue. The amount of Evans blue in organ homogenates was spectrophotometrically quantified. Evans blue content significantly increased in the lung and liver of Ang-2–treated mice, indicating leakage out of the vasculature and impregnation within the tissue, *<i>p</i> < 0.01.</p> <p>(B) Representative photographs of lungs were taken after washout of intravascular Evans blue with PBS for 10 min. The lung from a control (vehicle intraperitoneal) mouse (left) appears blanched in contrast to the purple-tinted, congested lung from an Ang-2-treated mouse (right).</p> <p>(C) The lung W/D weight ratio was determined as described in the Methods section. Ang-2 treatment for 16 h increased lung W/D weight ratio, consistent with congestion due to water accumulation, *<i>p</i> < 0.01.</p></div

Systemic Ang-2 Administration Provokes Rapid and Progressive Pulmonary Congestion

<div><p>Ang-2 was administered intraperitoneally (10 μg), and lung sections were assessed for histologic changes. Control lung is shown at 100× in (A). Note the thin alveolar septa, particularly in the inset (400×).</p> <p>(B) 3 h after Ang-2, there is noticeable expansion of alveolar septa with increase in cellularity, reduction in air space, and some leakage of cells into the alveolar space.</p> <p>(C) These changes are more advanced after 2 d of systemic Ang-2 administration (total dose 20 μg).</p></div