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Heparin-binding protein is important for vascular leak in sepsis Bentzer, Peter; Fisher, Jane; Kong, HyeJin J; Mörgelin, Mattias; Boyd, John H; Walley, Keith R; Russell, James A; Linder, Adam Oct 4, 2016

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RESEARCH Open AccessHeparin-binding protein is important forvascular leak in sepsisPeter Bentzer1,2,4, Jane Fisher3,4, HyeJin Julia Kong4, Mattias Mörgelin3, John H. Boyd4, Keith R. Walley4,James A. Russell4 and Adam Linder3,4** Correspondence:adam.linder@med.lu.se3Department of Infectious Diseases,University of Lund and SkåneUniversity Hospital, Getingevägen,Lund SE-221 85, Sweden4Centre for Heart Lung Innovation,Division of Critical Care Medicine,St. Paul’s Hospital, University ofBritish Columbia, Vancouver, BC,CanadaFull list of author information isavailable at the end of the articleAbstractBackground: Elevated plasma levels of heparin-binding protein (HBP) are associatedwith risk of organ dysfunction and mortality in sepsis, but little is known aboutcausality and mechanisms of action of HBP. The objective of the present study wasto test the hypothesis that HBP is a key mediator of the increased endothelialpermeability observed in sepsis and to test potential treatments that inhibit HBP-induced increases in permeability.Methods: Association between HBP at admission with clinical signs of increasedpermeability was investigated in 341 patients with septic shock. Mechanisms ofaction and potential treatment strategies were investigated in cultured humanendothelial cells and in mice.Results: Following adjustment for comorbidities and Acute Physiology and ChronicHealth Evaluation (APACHE) II, plasma HBP concentrations were weakly associatedwith fluid overload during the first 4 days of septic shock and the degree ofhypoxemia (PaO2/FiO2) as measures of increased systemic and lung permeability,respectively. In mice, intravenous injection of recombinant human HBP induced alung injury similar to that observed after lipopolysaccharide injection. HBP increasedpermeability of vascular endothelial cell monolayers in vitro, and enzymatic removalof luminal cell surface glycosaminoglycans (GAGs) using heparinase III andchondroitinase ABC abolished this effect. Similarly, unfractionated heparins and lowmolecular weight heparins counteracted permeability increased by HBP in vitro.Intracellular, selective inhibition of protein kinase C (PKC) and Rho-kinase pathwaysreversed HBP-mediated permeability effects.Conclusions: HBP is a potential mediator of sepsis-induced acute lung injurythrough enhanced endothelial permeability. HBP increases permeability through aninteraction with luminal GAGs and activation of the PKC and Rho-kinase pathways.Heparins are potential inhibitors of HBP-induced increases in permeability.Keywords: Septic shock, Heparin-binding protein (HBP), Vascular leak, Acuterespiratory distress syndrome, PermeabilityBackgroundA key feature in the pathophysiology of adult respiratory distress syndrome (ARDS)and septic shock is increased microvascular permeability; however, the current under-standing of the underlying mechanisms is limited [1]. Furthermore, no therapy directlytargeting increased microvascular permeability is currently available in sepsis.Intensive Care MedicineExperimental© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 InternationalLicense (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, andindicate if changes were made.Bentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 DOI 10.1186/s40635-016-0104-3Heparin-binding protein (HBP), also known as azurocidin or CAP37, is stored insecretory vesicles and azurophilic granules of neutrophils and is released early uponneutrophil adhesion and during neutrophil extravasation. Bacterial products induce re-lease of HBP leading to increased vascular leakage by acting on endothelial cellsthrough largely unknown mechanisms [2]. It has been shown that HBP binds to cellsurface proteoglycans, but the importance of this binding for permeability increases hasnot been investigated [3, 4]. Moreover, the importance of HBP on the pathophysiologyand outcomes of sepsis is unclear. Support for the importance of the permeability-increasing effect of HBP may be inferred from the observation that elevated plasmalevels of HBP are associated with shock in septic patients [5–7]. Furthermore, whilepatients with severe ARDS have been shown to have higher HBP levels than those withless severe ARDS [8], it is unclear if increased levels of HBP merely reflect injury sever-ity or if there is a causal relationship between HBP and ARDS.Based on these considerations, the first objective of the present study was to test thehypothesis that elevated levels of HBP are associated with severity of fluid overload andARDS—as indirect markers of increased permeability in a cohort of patients with septicshock. Based on findings compatible with a role for HBP in vascular leakage in septicshock, our second objective was to use in vitro and in vivo models to investigate thecellular mechanisms involved in the permeability-increasing effect of HBP and, finally,to explore the potential for heparins to mitigate HBP-induced increased permeability.MethodsHuman studiesPatient populationPlasma concentrations of HBP were analyzed in a subgroup of patients included in theVasopressin and Septic Shock Trial (VASST) cohort of septic shock patients [9]. VASSTwas a multicenter randomized double-blind controlled trial in which adult patients withseptic shock requiring vasopressor support (at least 5 μg/min of noradrenaline) for atleast 6 h despite adequate fluid resuscitation were eligible for inclusion (n = 778). Pa-tients were randomized to receive either masked vasopressin or noradrenaline to reacha target mean arterial pressure of 65 to 75 mmHg until shock had resolved. Infusion ofthe study drug was started 12 ± 9 h after meeting inclusion criteria and plasma that wascollected at baseline (within 2 h of start of infusion of the study drug) was available for341 patients. The research ethics boards of all 27 participating centers approved theVASST (Current Controlled Trials number, ISRCTN94845869). Written informedconsent was obtained from patients, next of kin, or surrogate decision-makers as ap-propriate. Plasma concentration of HBP was measured in duplicate blinded to clinicaloutcomes using a commercial HBP ELISA (Axis-Shield Diagnostics Ltd.). Plasmaconcentration of IL-6 was measured in duplicate using a Luminex multiplex bead assay(Luminex, Austin, TX).Outcome measuresThe primary outcome was the relationship between HBP concentration in plasma andpercent fluid overload. Percent fluid overload is a marker of increased vascular leakageand was calculated at 6, 12, 24, and 48 h after inclusion using the following formula:(fluid intake − fluid output)/(body weight (kg))*100 [10, 11]. The secondary outcomeBentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 2 of 16was the association between plasma HBP concentration at baseline and lung permeabil-ity. Because radiologic data and ventilator settings were unavailable, PaO2/FiO2 wasused as a surrogate marker of increased lung permeability and ARDS. In addition, weexamined the association between plasma HBP concentration at baseline and severityof shock measured as norepinephrine dose and plasma lactate concentration during thefirst 5 days after admission.In vitro studiesHuman endothelial cell modelImmortalized human umbilical vein endothelial cells (EA.hy926, American TypeCulture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10 % fetal bovine serum (FBS) (both from Life Technologies). Forall permeability measurements, cells were cultured to confluence (3 days) on ThinCerttransparent inserts with a 3-μm pore size (Greiner Bio-One). Experiments with thesecells were carried out in serum-free DMEM. Human recombinant HBP was used for allin vitro experiments (R&D Systems) and in vivo experiments (Novoprotein).Permeability assaysTrans-endothelial electrical resistance (TEER) reflects paracellular small molecule perme-ability, and a decrease in TEER reflects an increased permeability. TEER was measuredusing an EVOM system with STX2 electrodes (World Precision Instruments) as describedpreviously [12]. Confluence of the monolayer was confirmed by microscopic inspectionand by measuring a resistance across the monolayer of at least 50 Ω more than that of awell with no cells. To validate that changes in TEER reflected physiologically importantpermeability changes, macromolecule permeability was determined by measuring diffu-sion of streptavidin-conjugated horseradish peroxidase (HRP) (MW ≈ 100 kDa) in someof the experiments as described in detail in Additional file 1.Glycosaminoglycan digestionCells cultured on permeable inserts were treated with 15 mU/mL of Heparinase III(New England Biolabs) or 2.5 mU/mL Chondroitinase ABC (R&D Systems) for 1 h at37 °C [13]. Cells were then stimulated with HBP and TEER, and HRP passage was mea-sured after 1.5 h.Evaluation of HBP signaling pathwaysFor inhibition of signaling pathways, cells were pretreated for 1 h at 37 °C with Y-27632 (Tocris) at a concentration of 1 μM [14] or Calphostin C (Tocris) at a dose of50 nM [15] to inhibit Rho-kinase and protein kinase C (PKC), respectively.Effects of heparin compounds on HBP-induced increased permeabilityThe putative inhibitors of HBP were unfractionated heparin (UFH) (Leo) and three lowmolecular weight heparins (LMWHs): dalteparin (Pfizer), enoxaparin (Sanofi-Aventis),and tinzaparin (Leo). Varying doses of UFH were tested to determine the minimallyeffective dose. Therapeutic plasma levels of heparin in humans are 0.3–0.7 U/mL, anda concentration range of 0.01–100 U/mL was examined [16]. The LMWHs were dosedto obtain the clinical therapeutic level of 1 anti-Xa IE/mL [17]. HBP (10 μg/mL) wasmixed with the various heparins, incubated at 37 °C for 20 min, and cells were subse-quently stimulated with this mixture.Bentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 3 of 16In vivo studiesLund University Ethical Committee for Animal Research approved the experimentalprotocol. Adult male C57BL/6 mice (Taconic) weighing 26 ± 2 g were used. Mice weretreated in accordance with the National Institutes of Health for the Care and Use forLaboratory Animals. After induction of anesthesia and preparation, as described indetail in Additional file 1, animals received one of three treatments: (1) HBP 100 μgintravenously followed by an infusion of 2.5 μg/g/h for 1 h (n = 3), (2) UFH at a dose of0.4 U/g followed by HBP 100 μg intravenously followed by infusion of HBP as de-scribed above (n = 3). This dose has previously been shown to double activated partialthromboplastin time in mice and is therefore in the therapeutic range [18]. (3) Control:bolus dose of 100 μL of vehicle (10 mM phosphate-buffered saline (PBS)) followed byinfusion of vehicle at 1.25 μL/g/h (n = 3).At 1 h after start of treatment, animals were killed by exsanguination and the lungswere collected for electron microscopy and histologic analysis as described inAdditional file 1. An investigator blinded to the treatment status of the animals per-formed preparation and analysis of electron microscopic and histologic images as de-scribed in detail previously [19]. Briefly, histologic analysis was done by scoring threelung sections from each animal for alveolar thickness, capillary congestion, and cellular-ity using a score from 0 to 3 with 3 being the highest injury score. An overall score wascalculated by averaging the three indices of injury. The shed blood was collected formeasurement of plasma concentrations of HBP as described above. Electron micros-copy and histology preparations were compared to those obtained from mice sacrificedat 4 h after intraperitoneal injection of lipopolysaccharide (LPS) from Escherichia coli0111:B4 (Sigma-Aldrich) in a dose of 0.25 mg [20] after preparation as described above.Statistical analysisComparisons between groups were made using the non-parametric Mann-Whitney test,Student’s t test, one-way ANOVA, and two-way repeated measures ANOVA as appropriate.Spearman’s non-parametric correlation coefficient (rho) was used to assess correlations be-tween HBP levels and percent fluid overload and PaO2/FiO2. Two-tailed P values of lessthan 0.05 were considered to be significant. Adjusted analyses were done by a logistic re-gression model for presence of severe ARDS (PaO2/FiO2 ≤100 mmHg [≤13.3 kPa]) andadjusting for age, gender, Acute Physiology and Chronic Health Evaluation (APACHE) II,comorbidities, and physiological parameters and laboratory variables that differed signifi-cantly between the patients with PaO2/FiO2 ≤100 or >100 mmHg, respectively (Table 1).Data are expressed as mean ± standard deviation unless stated otherwise. Data were ana-lyzed using GraphPad Prism (version 6.0, GraphPad Software, Inc.) and SPSS (version 19.0).ResultsPlasma HBP is associated with fluid overloadMedian plasma concentration of HBP at baseline for the whole cohort was 25 ng/mL(range, 0–361, interquartile range (IQR) 8–71). For comparison, median HBP levels arereported to be 6 (range, 2–9 ng/mL) in healthy controls using a similar methodology[21]. We tested the hypothesis that increased vascular leakage, as reflected by percentfluid overload, was correlated with HBP concentration. Increased plasma HBP was veryweakly correlated with percent fluid overload at 6 h (rho 0.13, P = 0.01, Fig. 1a).Bentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 4 of 16HBP is associated with severity of hypoxemiaIncreased plasma concentration of HBP correlated weakly with the lowest PaO2/FiO2 atany time in the first 5 days after admission, as an indicator of severity of ARDS (rho−0.25, P < 0.001, Fig. 1b). Patients with severe hypoxemia as defined by a PaO2/FiO2≤100 mmHg at any time in the first 5 days after admission were hemodynamically morecompromised at baseline and had higher APACHE scores than patients with PaO2/FiO2 >100 mmHg (Table 1). Plasma HBP concentration was 47 (IQR 23–123) and 22(IQR 7–62) in the groups with PaO2/FiO2 ≤100 mmHg and PaO2/FiO2 >100 mmHg,respectively (P < 0.01). Logistic regression adjusting for ethnicity, gender, age, APACHEII, site of infection, chronic heart failure, COPD, chronic steroid treatment, chronic dia-lysis and chronic hepatic failure, lactate concentration, norepinephrine dose and IL-6concentrations showed that plasma HBP concentrations remained associated with pres-ence of severe hypoxemia as defined above (P = 0.003). Plasma HBP was also positivelycorrelated with severity of shock as indicated by dosage of noradrenaline and plasmalactate concentrations at days 1–4 (see Additional file 2: Figure S1 for day 1 data).Table 1 Patient characteristics at baselineAll patients(n = 341)PaO2/FiO2 >100(n = 283)PaO2/FiO2 ≤100(n = 53)P valueMale, n (%) 201 (59) 163 (57) 37 (70) 0.13Age, years (median (IQR)) 63 (50.6–72.4) 63 (50.6–72.2) 64 (49.8–73.6) 0.93Caucasian, n (%) 307 (90) 261 (92) 42 (79) <0.01APACHE II (median (IQR)) 26 (21–32) 26 (21–32) 29 (24–35) <0.01Comorbidities, n (%)Chronic heart failure 26 (8) 21 (7.4) 5 (9.4) 0.82COPD 58 (17) 53 (19) 5 (9.4) 0.15Chronic steroids 72 (21) 60 (21) 11 (21) 1.0Chronic dialysis 30 (9) 23 (8.1) 6 (11) 0.62Chronic hepatic failure 37 (11) 29 (10) 8 (15) 0.43Infection site, n (%)Lung 147 (43) 112 (40) 34 (64) <0.01Abdomen 89 (26) 80 (28) 8 (15) 0.07Other 105 (31) 91 (32) 11 (21) 0.14Physiological and laboratory variables at baseline, median (IQR)MAP (mmHg) 56 (50–62) 56 (50–62) 55 (48–61) 0.22Lactate (mmol/L) 1.7 (0.9–3.4) 1.6 (0.8–3.2) 2.5 (1.3–5.0) <0.01Norepinephrine (μg/min) 13 (8–25) 12 (8–22) 23 (11–38) <0.01WBC (109 cells/L) 14 (8–21) 14 (8–21) 11 (7–19) 0.18Platelets 172 (90–259) 174 (90–268) 153 (108–238) 0.25Temperature (°C) 38.6 (37.7–39.3) 38.5 (37.7–39.3) 38.7 (37.8–39.2) 0.49PaO2/FiO2 192 (142–260) 205.3 (162–271) 99 (82.5–110.8) <0.01IL-6 (pM) 4.3 (1.5–37) 5.6 (1.5–24) 18.9 (2.1–422) <0.01Outcomes other than mortality, median (IQR)DAF ventilator support 9 (0–21) 13 (1–22) 0 (0–2) <0.01DAF renal replacement therapy 27 (7–28) 28 (12–28) 4 (1–16) <0.01Groups were compared using the Student’s t test, or Mann-Whitney test, or chi-squared test as appropriateDAF days alive and free, WBC white blood cell count, PaO2 arterial partial pressure of oxygen, FiO2 fraction ofinspired oxygenBentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 5 of 16HBP increases in vitro permeabilityHaving established that elevated HBP is associated with physiological findings in keep-ing with enhanced vascular permeability in septic patients, we went on to further inves-tigate HBP’s mechanisms of action in human endothelial cells. To confirm previousresults demonstrating a permeability-increasing effect of HBP on endothelial cellmonolayers [2], effects of HBP upon the permeability of endothelial cell monolayerswere measured 1.5 h following HBP stimulation. Cell monolayers stimulated with HBPhad lower TEER and higher HRP passage than controls indicating that HBP inducedincreased human endothelial cell permeability (Fig. 2a, b). Cell monolayers stimulatedwith HBP reached a minimum TEER by 30 min to 1 h following stimulation (Fig. 2a).Similarly, monolayers stimulated with HBP had a higher HRP passage at the earliestFig. 1 Elevated plasma HBP levels are associated with markers of increased vascular leakage. a Scatterplot ofplasma HBP levels at baseline and percent fluid overload at 6 h after admission. Dotted lines mark median valuefor HBP and percent fluid overload, respectively. b Scatterplot of plasma HBP levels at baseline and lowestPaO2/FiO2 during the first 5 days after admission. Dotted lines mark median value for HBP and PaO2/FiO2 fluidoverload, respectively. Spearman’s non-parametric correlation coefficient (rho) is given in the figuresBentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 6 of 16time points measured at 1 h, after which HRP diffusion reached equilibrium in all con-ditions (Fig. 2b).HBP acts by binding endothelial proteoglycansIn order test our hypothesis that binding of HBP to endothelial proteoglycans is re-quired for the increased permeability, Heparinase III or Chondroitinase ABC were usedto selectively cleave either heparan sulfate or chondroitin sulfate/dermatan sulfate, re-spectively, from the endothelial surface [3, 22–24]. Pre-treatment with Heparinase IIIand Chondroitinase ABC did not affect basal permeability but attenuated HBP-inducedincrease in permeability suggesting a role for proteoglycans as receptors in HBP-induced vascular endothelial permeability increase (Fig. 3).HBP increases permeability through PKC and the Rho-kinase pathwaysBecause activation of the PKC pathway may increase endothelial permeability [25] andbecause HBP is shown to stimulate PKC activity [26], we tested the hypothesis thatFig. 2 HBP increases the permeability of human endothelial cell monolayers. a EA.hy926 cells were grown toconfluence on permeable supports and stimulated with HBP. TEER across the filter was monitored over time.The overall difference was determined by two-way ANOVA (treatment effect P = 0.001, time effect P < 0.001).b HRP was added to the top chamber, and HRP passage was monitored over time. Values are normalized tothe TEER of empty inserts. The overall difference was determined by two-way repeated measures ANOVA(treatment effect P = 0.009, time effect P < 0.001). In both experiments, Sidak’s multiple comparison post hoctest was used to compare HBP treatment and control at each time point. Error bars are standard error of themean, n = 3. Some error bars are not visible due to scale. *P < 0.05, **P < 0.01, ***P < 0.001Bentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 7 of 16HBP increases permeability via the PKC pathway. HBP-induced permeability increasecould be blocked by the unselective PKC inhibitor Calphostin C. Activation of PKC isreported to increase permeability of endothelial cells via the Rho-kinase pathway [27].The Rho-kinase inhibitor Y-27 632 also attenuated HBP-induced increases in perme-ability (Fig. 4).HBP alone induced acute lung injury in vivoWhile HBP is associated with findings suggestive of increased vascular permeability inhuman septic shock, this association does not prove causality because many othermediators contribute to human septic ARDS. Therefore, we used a murine model totest the hypothesis that HBP causes acute lung injury (ALI) in vivo. HBP administrationinduced histologic features characteristic of ALI including increased cellularity andoverall lung injury score compared to vehicle-treated control animals (Table 2 andFig. 5). Electron microscopy showed protein deposits and almost complete disappear-ance of alveoli. HBP induced histological and electron microscopic changes very similarFig. 3 Effects of enzymatic removal of heparan sulfate or chondroitin sulfate by Heparinase III and ChondroitinaseABC on HBP-induced permeability increases. EA.hy926 cells were grown to confluence on permeable supportsand treated with heparinase III (Hep. III) or Chondroitinase ABC (Chondro. ABC) for 1 h and then stimulated withHBP. a TEER was measured after 1.5 h after HBP stimulation. b HRP was also added to the top chamber, and HRPpassage was measured 2 h after HBP stimulation. TEER values are normalized to the TEER of empty inserts.Error bars are standard error of the mean, n = 3 for each condition. In both experiments, one-way ANOVAwith Dunnett’s test for multiple comparisons was used to compare each group to the condition with HBPand no enzyme treatment (far right). *P < 0.05, **P < 0.01, ***P < 0.001Bentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 8 of 16to those observed after LPS administration (Fig. 5). Plasma concentration of HBP at theend of the experiment was 400 ± 157 ng/mL in HBP-treated animals and 1.1 ± 1.5 ng/mLin control animals.The effect of HBP is inhibited by heparinsBased on the observation that heparins are known to bind to HBP [28], we hypothesizedthat heparins could prevent the permeability-increasing effects of HBP. Heparin pre-vented HBP-induced increased permeability: there was dose-dependent inhibition of al-tered TEER and HRP permeability following co-stimulation of human endothelial cellswith HBP and unfractionated heparin (UFH) (Fig. 6). Maximum inhibition of permeabilityincreases by HBP was seen at concentrations of UFH in the range of 0.1–1 U/mL. Lowmolecular weight heparins in the therapeutic range also inhibited the TEER-increasing ef-fect of HBP (Fig. 7). In another set of experiments, UFH was added to cells after 1 h ofstimulation with HBP. TEER increased to 89 ± 1.0 % of baseline 1 h after UFH was addedwhereas HBP-treated cells to which no UFH was added remained at 77 ± 4.0 % of baseline(P < 0.01, Fig. 8). Treatment with UFH prior to administration of HBP in vivo appeared toprevent both histological and electron microscopic appearance, but no significant changein histologic score could be detected (Fig. 5 and Table 2).Fig. 4 Effect of inhibition of signaling pathways in HBP-induced permeability increases. EA.hy926 cells weregrown to confluence on permeable supports and treated with Y-27 632 (Rho-kinase inhibitor) and CalphostinC (PKC inhibitor) for 1 h and then stimulated with HBP. TEER across the filter was measured 1.5 h after HBPstimulation and is normalized to empty inserts. Error bars are standard error of the mean, n = 3 for eachcondition. One-way ANOVA with Dunnett’s test for multiple comparisons was used to compare each groupto the condition with HBP and no inhibitor (far right). *P < 0.05, **P < 0.01, ***P < 0.001Table 2 Lung histologic injury scoreAlveolar thickness Capillary congestion Cellularity Overall scoreVehicle (n = 3) 0.33 ± 0.88 0.5 ± 0.05 0.67 ± 0.29 0.5 ± 0.33HBP (n = 3) 1.89 ± 0.84 1.78 ± 0.69 2.06 ± 0.59* 1.91 ± 0.7*HBP + UFH (n = 3) 0.67 ± 1.15 2.67 ± 0.58 0.33 ± 0.58# 1.22 ± 0.69LPS (n = 3) 2.5 ± 0.71* 2.25 ± 0.35* 2.00 ± 0.0* 2.25 ± 0.35*One-way ANOVA. The Sidak method was used to correct for multiple comparisonsUFH unfractionated heparin, LPS lipopolysaccharide*P < 0.05 compared to vehicle, #P < 0.05 compared to HBPBentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 9 of 16DiscussionOur results show that increased plasma HBP concentration in human septic shock isassociated with increased vascular leak as reflected indirectly by percent fluid overloadand the severity of hypoxemia. In addition, HBP increased permeability in vitro. Fur-thermore, administration of HBP in a murine model rapidly induces a lung injury simi-lar to that observed after LPS administration. The presence of heparan sulfate andchondroitin sulfate moieties on the endothelial cell surface is required for HBP-inducedincreased permeability as shown by enzymatic degradation of these compounds, whichFig. 5 HBP-induced signs of acute lung injury in mice in vivo. Mice were injected with intravenous heparin-binding protein (HBP) and/or unfractionated heparin (UFH) followed by continuous infusion for 1 h. Controlsreceived vehicle (10 mM phosphate-buffered saline). The lungs were stained with hematoxylin and eosin (left)or analyzed by scanning electron microscopy (right). Hematoxylin and eosin-stained sections were scored foralveolar thickness, capillary congestion, and cellularity (see Table 2). Images from sections with a median overallscore are shown. Histologic and electron microscopic images of mice treated with intraperitoneal lipopolysaccharide(LPS) from Escherichia coli 0111:B4 in a dose of 0.25 mg for 4 h are presented in lower panelsBentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 10 of 16completely abolished the permeability-increasing effect of HBP. Downstream signalingoccurs via the protein kinase C and Rho-kinase pathways as shown by experimentsusing selective inhibitors. The permeability increase by HBP in human endothelial cellswas inhibited by pre-treatment and post-treatment with UFH and low molecular weightheparins.To the best of our knowledge, this is the first report showing that increased plasmaHBP levels are associated with the presence or development of severe hypoxemia in acohort of patients suffering from septic shock. These results are in line with a studyshowing that increased plasma HBP at admission in trauma patients is correlated withdevelopment of ARDS [29]. The results also align with the association between hypox-emia and plasma levels of HBP in a small cohort of patients with influenza infection[30]. However, our results are in contrast with a recent study in which there was nocorrelation between HBP and development of hypoxemia in patients with severe sepsis[31]. Given the small number of septic patients in that study (n = 83), it could be hy-pothesized that difference in results could be related to a possible false negative due tolower power in the study by Tydén et al. compared to the higher sample size (n = 341)and statistical power of our study. The finding that HBP concentrations at baseline inVASST are correlated with indirect markers of increased systemic vascular leakageFig. 6 Unfractionated heparin blocked HBP-induced permeability increases. EA.hy926 cells were grown toconfluence on permeable supports and stimulated with 10 μg/mL HBP, pre-incubated with the indicateddose of heparin. a TEER was measured 1.5 h after stimulation and is normalized to empty inserts. b HRPwas also added to the top chamber, and HRP passage was measured 2 h after stimulation. Error bars are standarderror of the mean, n= 3 for each condition. One-way ANOVA with Dunnett’s test for multiple comparisons wasused to compare each group to the condition with HBP and no heparin (far left).*P< 0.05, **P< 0.01, ***P< 0.001Bentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 11 of 16during the first days of septic shock corroborates previous results suggesting that in-creased plasma HBP levels at hospital admission predict shock [5–7]. Taken together,these results indicate that plasma levels of HBP may be used to identify patients at riskfor development of ARDS and increased systemic vascular leakage and indicate thatHBP may be a clinically important mediator of permeability increase in septic shock.The finding that the associations between HBP and markers suggestive of increased per-meability were weak could reflect the complex pathophysiology of sepsis with multiple re-dundant pathways leading to increased permeability [1]. Moreover, indirect markers ofincreased permeability are likely to be influenced by factors, which are unrelated tochanges in permeability. The timing of the plasma sampling may also falsely underesti-mate the association between HBP and markers of permeability, as discussed below.Fig. 7 Low molecular weight heparins blocked HBP-induced permeability increases. EA.hy926 cells weregrown to confluence on permeable supports and stimulated with 10 μg/mL HBP, pre-incubated with theindicated inhibitor. TEER was measured after 1.5 h after stimulation and is normalized to empty inserts. Errorbars are standard error of the mean, n = 3 for each condition. One-way ANOVA with Dunnett’s test formultiple comparisons was used to compare each group to the condition with HBP and no inhibitor (far left).UFH unfractionated heparin. *P < 0.05, **P < 0.01Fig. 8 Unfractionated heparin reversed HBP-induced permeability increases. EA.hy926 cells were grown toconfluence on permeable supports and stimulated with 10 μg/mL HBP. At 1 h following stimulation, HBP-containing media were removed and replaced with fresh media (gray line, treatment 1), or heparin wasadded to a final concentration of 3 U/mL (dashed line, treatment 2), or no change was made (black line,treatment 3). TEER across the filter was monitored over time and is normalized to empty inserts. The overalldifference was determined by two-way repeated measures ANOVA (treatment effect P = 0.002, time effectP < 0.001). Sidak’s multiple comparisons post hoc test was used to compare treatments 1 and 2 to treatment 3at each time point. Error bars are standard error of the mean, n = 3 for each intervention. UFH unfractionatedheparin. **P < 0.01, ***P < 0.00Bentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 12 of 16Topical application of HBP has previously been show to induce rapid leakage ofFITC-labeled dextran in the hamster cheek pouch preparation [2]. However, to date,the effects of intravenous HBP on a whole animal model have not been investigated.Our finding that HBP rapidly induced histological changes consistent with acute lunginjury supports the hypothesis that HBP induces increased capillary leak in the lung invivo and supports a causal relationship between HBP and ARDS.HBP binds to proteoglycans on endothelial cells, but the functional importance ofthis binding for increases in permeability has not been investigated before [3, 4]. Pro-teoglycans are membrane-bound molecules with a protein core to which polysaccha-rides containing heparan sulfate (HS) and chondroitin sulfate (CS) chains are bound.The syndecans [1–4] are composed of a transmembrane protein core to which HSchains and sometimes CS chains are attached. The glypicans [1–6], with the exceptionof glypican-5, carry only HS side chains [32, 33]. To date, the expression of all synde-cans and glypican-1 and glypican-4 has been described on endothelial cells andsyndecan-4 is the predominant syndecan in cultured human endothelial cells [34, 35].Our result showing that cleavage of both HS and of CS can inhibit the permeability-increasing effect of HBP indicates that syndecans act as receptors for HBP [36]. Proteo-glycans are known to act both as primary receptors and as co-receptors that facilitatebinding of agonists to other receptors, and thus, the involvement of other receptors forHBP remains a possibility [32, 34].Our screening of potential intracellular signaling pathways identified PKC as one of themost likely pathways mediating the permeability-increasing effect of HBP on endothelialcells. The result that inhibition of PKC inhibits the permeability-increasing effect of HBPaligns with previous results showing that HBP increases intracellular calcium and activatesPKCα in endothelial cells [2, 26]. Interestingly, activation of syndecan-4 has been sug-gested to influence stress fiber formation in fibroblasts through a calcium-independentPKCα activation mechanism indicating that increased intracellular calcium may not be aprerequisite for increases in permeability [37, 38]. At present, it is unclear if Rho-kinaseactivation is downstream of PKC activation or if it represents a parallel pathway.One small high-quality RCT has investigated the effect of heparin treatment on mor-tality in sepsis and could not demonstrate a benefit of heparin treatment on mortalityor severity of ARDS [39]. However, several retrospective analyses and meta-analyseshave indicated that heparin treatment may reduce mortality in septic shock [40–42]while one retrospective study could not demonstrate an effect of heparins on ARDS[43]. Given the association between HBP and clinical signs consistent with vascular leakand the permeability-increasing effect in vitro, HBP represents a potential target fortherapeutic intervention in sepsis. Our result that both UFH and low molecular weightheparins inhibited permeability increases induced by HBP suggests a cogent rationalefor further studies of heparin(s) to prevent sepsis-induced ARDS and vascular leak. Fur-thermore, our results raise the possibility that HBP levels in plasma may be used toidentify a subset of patients with septic shock that may benefit from treatment withheparin in future trials. It should be noted that a recent meta-analysis suggested thatsafety aspects of heparin in sepsis are underreported, and we conclude that risk ofbleeding is a potential concern for the application of heparin in this setting [41].We acknowledge that this study has several limitations. Firstly, baseline blood sam-ples in the VASST cohort were collected within 2 h of start of treatment with the studyBentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 13 of 16drug, which occurred about 12 ± 9 h after meeting inclusion criteria [9]. Given thatplasma HBP levels change rapidly [6], we cannot exclude that the variability in the tim-ing of blood sampling could have influenced our results and potentially underestimatedthe association between HBP and our indirect clinical markers of increased permeabil-ity. Secondly, the high cost of recombinant HBP limited the number and length of invivo experiments that could be performed and prevented us from a more detailedevaluation of physiological effects by HBP in vivo.ConclusionsTaken together, our clinical and experimental data suggest a causal relationship be-tween HBP, increased permeability, and ARDS in human sepsis. Unfractionated heparinand low molecular weight heparins are potential drugs to prevent excessive HBP-induced increases in vascular leak in sepsis.Additional filesAdditional file 1: Online data supplement. (DOCX 104 kb)Additional file 2: Figure S1. Elevated plasma HBP levels are associated with increased plasma lactate andmaximum dose of norepinephrine at day 1 after admission. (A) Scatterplot of plasma HBP levels and maximumdose of norepinephrine on day 1. (B) Scatterplot of plasma HBP levels and plasma lactate concentration at day 1.Dotted lines mark median value for HBP and norepinephrine dose or plasma lactate concentration, respectively.Spearman’s non-parametric correlation coefficient (rho) is given in the figures. (JPEG 886 kb)AbbreviationsALI: Acute lung injury; APACHE: Acute Physiology and Chronic Health Evaluation; ARDS: Adult respiratory distresssyndrome; CS: Chondroitin sulfate; DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal bovine serum;GAGs: Glycosaminoglycans; HBP: Heparin-binding protein; HRP: Horseradish peroxidase; HS: Heparan sulfate;LMWH: Low molecular weight heparin; LPS: Lipopolysaccharide; PBS: Phosphate-buffered saline; PKC: Protein kinase C;TEER: Trans-endothelial electrical resistance; UFH: Unfractionated heparin; VASST: Vasopressin and Septic Shock TrialFundingThis study received funding from the Anna and Edwin Berger Foundation (PB), Region Skåne (PB), Swedish Research CouncilPost-Doc grants (AL), Swedish Medical Association (Läkaresällskapet) (AL), Canadian Institutes of Health Research (CIHR) IMPACTStrategic Training Post-Doctoral Fellowship (AL), Groschinky Foundation (AL), and Österlunds Foundation (AL).Authors’ contributionsAll authors participated in the conception and design of the study and in the critical revision of the manuscript forimportant intellectual content. PB, JF, JB, KW, JR, and AL participated in the data acquisition. PB, JF, and AL performedthe data analysis. PB, JF, MM, JB, JR, and AL interpreted the data. PB, JF, JB, KW, JR, and AL produced the draft of themanuscript. All authors had full access to all of the data (including statistical reports and tables) in the study and cantake responsibility for the integrity of the data and the accuracy of the data analysis. 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Crit Care Med 43:511–51842. Zarychanski R, Doucette S, Fergusson D, Roberts D, Houston DS, Sharma S, Gulati H, Kumar A (2008) Earlyintravenous unfractionated heparin and mortality in septic shock. Crit Care Med 36:2973–297943. Hofstra J, Vlaar A, Prins D, Koh G, Levi M, Schultz M, Binnekade J, Juffermans N (2012) Early intravenousunfractionated heparin and outcome in acute lung injury and acute respiratory distress syndrome—aretrospective propensity matched cohort study. BMC Pulm Med 12:43Submit your manuscript to a journal and benefi t from:7 Convenient online submission7 Rigorous peer review7 Immediate publication on acceptance7 Open access: articles freely available online7 High visibility within the fi eld7 Retaining the copyright to your article    Submit your next manuscript at 7 springeropen.comBentzer et al. Intensive Care Medicine Experimental  (2016) 4:33 Page 16 of 16


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