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. 2016 Dec;4(1):33.
doi: 10.1186/s40635-016-0104-3. Epub 2016 Oct 4.

Heparin-binding protein is important for vascular leak in sepsis

Peter Bentzer  1   2   3 , Jane Fisher  4   3 , HyeJin Julia Kong  3 , Mattias Mörgelin  4 , John H Boyd  3 , Keith R Walley  3 , James A Russell  3 , Adam Linder  5   6
Affiliations

Heparin-binding protein is important for vascular leak in sepsis

Peter Bentzer et al. Intensive Care Med Exp. 2016 Dec.

Erratum in

Abstract

Background: Elevated plasma levels of heparin-binding protein (HBP) are associated with risk of organ dysfunction and mortality in sepsis, but little is known about causality and mechanisms of action of HBP. The objective of the present study was to test the hypothesis that HBP is a key mediator of the increased endothelial permeability 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 increased permeability was investigated in 341 patients with septic shock. Mechanisms of action and potential treatment strategies were investigated in cultured human endothelial cells and in mice.

Results: Following adjustment for comorbidities and Acute Physiology and Chronic Health Evaluation (APACHE) II, plasma HBP concentrations were weakly associated with fluid overload during the first 4 days of septic shock and the degree of hypoxemia (PaO2/FiO2) as measures of increased systemic and lung permeability, respectively. In mice, intravenous injection of recombinant human HBP induced a lung injury similar to that observed after lipopolysaccharide injection. HBP increased permeability of vascular endothelial cell monolayers in vitro, and enzymatic removal of luminal cell surface glycosaminoglycans (GAGs) using heparinase III and chondroitinase ABC abolished this effect. Similarly, unfractionated heparins and low molecular weight heparins counteracted permeability increased by HBP in vitro. Intracellular, selective inhibition of protein kinase C (PKC) and Rho-kinase pathways reversed HBP-mediated permeability effects.

Conclusions: HBP is a potential mediator of sepsis-induced acute lung injury through enhanced endothelial permeability. HBP increases permeability through an interaction with luminal GAGs and activation of the PKC and Rho-kinase pathways. Heparins are potential inhibitors of HBP-induced increases in permeability.

Keywords: Acute respiratory distress syndrome; Heparin-binding protein (HBP); Permeability; Septic shock; Vascular leak.

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Figures

Fig. 1
Fig. 1
Elevated plasma HBP levels are associated with markers of increased vascular leakage. a Scatterplot of plasma HBP levels at baseline and percent fluid overload at 6 h after admission. Dotted lines mark median value for HBP and percent fluid overload, respectively. b Scatterplot of plasma HBP levels at baseline and lowest PaO2/FiO2 during the first 5 days after admission. Dotted lines mark median value for HBP and PaO2/FiO2 fluid overload, respectively. Spearman’s non-parametric correlation coefficient (rho) is given in the figures
Fig. 2
Fig. 2
HBP increases the permeability of human endothelial cell monolayers. a EA.hy926 cells were grown to confluence 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 to the 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 hoc test was used to compare HBP treatment and control at each time point. Error bars are standard error of the mean, n = 3. Some error bars are not visible due to scale. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
Effects of enzymatic removal of heparan sulfate or chondroitin sulfate by Heparinase III and Chondroitinase ABC on HBP-induced permeability increases. EA.hy926 cells were grown to confluence on permeable supports and treated with heparinase III (Hep. III) or Chondroitinase ABC (Chondro. ABC) for 1 h and then stimulated with HBP. a TEER was measured after 1.5 h after HBP stimulation. b HRP was also added to the top chamber, and HRP passage 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 ANOVA with Dunnett’s test for multiple comparisons was used to compare each group to the condition with HBP and no enzyme treatment (far right). *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4
Effect of inhibition of signaling pathways in HBP-induced permeability increases. EA.hy926 cells were grown to confluence on permeable supports and treated with Y-27 632 (Rho-kinase inhibitor) and Calphostin C (PKC inhibitor) for 1 h and then stimulated with HBP. TEER across the filter was measured 1.5 h after HBP stimulation and is normalized to empty inserts. Error bars are standard error of the mean, n = 3 for each condition. One-way ANOVA with Dunnett’s test for multiple comparisons was used to compare each group to the condition with HBP and no inhibitor (far right). *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 5
Fig. 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. Controls received 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 for alveolar thickness, capillary congestion, and cellularity (see Table 2). Images from sections with a median overall score 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 panels
Fig. 6
Fig. 6
Unfractionated heparin blocked HBP-induced permeability increases. EA.hy926 cells were grown to confluence on permeable supports and stimulated with 10 μg/mL HBP, pre-incubated with the indicated dose of heparin. a TEER was measured 1.5 h after stimulation and is normalized to empty inserts. b HRP was also added to the top chamber, and HRP passage was measured 2 h after stimulation. Error bars are standard error of the mean, n = 3 for each condition. One-way ANOVA with Dunnett’s test for multiple comparisons was used to compare each group to the condition with HBP and no heparin (far left).*P < 0.05, **P < 0.01, ***P < 0.001
Fig. 7
Fig. 7
Low molecular weight heparins blocked HBP-induced permeability increases. EA.hy926 cells were grown to confluence on permeable supports and stimulated with 10 μg/mL HBP, pre-incubated with the indicated inhibitor. TEER was measured after 1.5 h after stimulation and is normalized to empty inserts. Error bars are standard error of the mean, n = 3 for each condition. One-way ANOVA with Dunnett’s test for multiple comparisons was used to compare each group to the condition with HBP and no inhibitor (far left). UFH unfractionated heparin. *P < 0.05, **P < 0.01
Fig. 8
Fig. 8
Unfractionated heparin reversed HBP-induced permeability increases. EA.hy926 cells were grown to confluence 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 was added 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 overall difference was determined by two-way repeated measures ANOVA (treatment effect P = 0.002, time effect P < 0.001). Sidak’s multiple comparisons post hoc test was used to compare treatments 1 and 2 to treatment 3 at each time point. Error bars are standard error of the mean, n = 3 for each intervention. UFH unfractionated heparin. **P < 0.01, ***P < 0.00
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