Nebulized Heparin Attenuates Pulmonary Coagulopathy and Inflammation through Alveolar Macrophages in a Rat Model of Acute Lung Injury

Laura Chimenti, Marta Camprubí-Rimblas, Raquel Guillamat-Prats, Maria Nieves Gomez, Jessica Tijero, Lluis Blanch, Antonio Artigas, Laura Chimenti, Marta Camprubí-Rimblas, Raquel Guillamat-Prats, Maria Nieves Gomez, Jessica Tijero, Lluis Blanch, Antonio Artigas

Abstract

Objective Alveolar macrophages play a key role in the development and resolution of acute respiratory distress syndrome (ARDS), modulating the inflammatory response and the coagulation cascade in lungs. Anti-coagulants may be helpful in the treatment of ARDS. This study investigated the effects of nebulized heparin on the role of alveolar macrophages in limiting lung coagulation and inflammatory response in an animal model of acute lung injury (ALI). Methods Rats were randomized to four experimental groups. In three groups, ALI was induced by intratracheal instillation of lipopolysaccharide (LPS) and heparin was nebulized at constant oxygen flow: the LPS/Hep group received nebulized heparin 4 and 8 hours after injury; the Hep/LPS/Hep group received nebulized heparin 30 minutes before and 4 and 8 hours after LPS-induced injury; the LPS/Sal group received nebulized saline 4 and 8 hours after injury. The control group received only saline. Animals were exsanguinated 24 hours after LPS instillation. Lung tissue, bronchoalveolar lavage fluid (BALF) and alveolar macrophages isolated from BALF were analysed. Results LPS increased protein concentration, oedema and neutrophils in BALF as well as procoagulant and proinflammatory mediators in lung tissue and alveolar macrophages. In lung tissue, nebulized heparin attenuated ALI through decreasing procoagulant (tissue factor, thrombin-anti-thrombin complexes, fibrin degradation products) and proinflammatory (interleukin 6, tumour necrosis factor alpha) pathways. In alveolar macrophages, nebulized heparin reduced expression of procoagulant genes and the effectors of transforming growth factor beta (Smad 2, Smad 3) and nuclear factor kappa B (p-selectin, CCL-2). Pre-treatment resulted in more pronounced attenuation. Conclusion Nebulized heparin reduced pulmonary coagulopathy and inflammation without producing systemic bleeding, partly by modulating alveolar macrophages.

Conflict of interest statement

Conflict of Interest: The authors have declared that no conflict of interest exists.The work was performed in Institut d'Investigació i Innovació Parc Taulí (I3PT), Sabadell, Spain.

Schattauer GmbH Stuttgart.

Figures

Fig.1
Fig.1
Experimental design.(a) Treatment group and (b) pre-treatment group.
Fig. 2
Fig. 2
Bronchoalveolar lavage analysis and wet/dry weight ratio. Absolute (a) neutrophil (PMN) and (b) alveolar macrophage (AM) cell counts in the bronchoalveolar lavage fluid of rats 24 hours after the induction of the injury. (c) Wet/dry weight ratio and (d) protein concentration in the bronchoalveolar lavage fluid. Data are presented as mean ± SEM. ANOVA followed by the post hoc Fisher's PLSD test were used. *p < 0.05; **p < 0.001; ***p < 0.0001.
Fig. 3
Fig. 3
Lung tissue analysis. (a–d) Representative images of haematoxylin and eosin staining lung tissue sections and (e) histological score in animals 24 hours after induction of the injury. Original magnification ×200. Data are presented as mean ± SEM. ANOVA followed by the post hoc Fisher's PLSD test were used. *p < 0.05; **p < 0.001; ***p < 0.0001.
Fig. 4
Fig. 4
Coagulant effectors and cytokines. (a) Tissue factor (TF), (b) thrombin–anti-thrombin complexes (TATc), (c) fibrin degradation products (FDPs), (c) plasminogen activator inhibitor type-1 activity (PAI-1), (e) IL-6, (f) TNF-α, (g) GRO-κC and (h) IL-10 concentrations were measured in lung homogenate of animals 24 hours after induction of the injury. Data are presented as mean ± SEM. ANOVA followed by the post hoc Fisher's PLSD test were used. *p < 0.05; **p < 0.001; ***p < 0.0001.
Fig. 5
Fig. 5
Activation of alveolar macrophages.Gene expression of proinflammatory (M1): (a) TNF-α, (b) iNOS and alternative (M2), (c) IL-10, (d) arginase-1 mediators in alveolar macrophages isolated from BALF of animals 24 hours after induction of the injury. Data are presented as mean ± SEM. ANOVA followed by the post hoc Fisher's PLSD test were used. *p < 0.05; **p < 0.001; ***p < 0.0001.
Fig. 6
Fig. 6
Inflammatory and coagulation pathways of alveolar macrophages. Gene expression of TGF-β pathway: (a) TGF-β, (b) Smad 2 and (c) Smad 3, NF-κ≡ pathway, (d) IRAK-1, (e) p-selectin and (f) CCL-2 and coagulation pathway, (g) TF, (h) PAI-1 and (i) plasminogen in alveolar macrophages isolated from BALF of animals 24 hours after induction of the injury. Data are presented as mean ± SEM. ANOVA followed by the post hoc Fisher's PLSD test were used. *p < 0.05; **p < 0.001; ***p < 0.0001.

References

    1. Bellani G, Laffey J G, Pham T et al.Epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries. JAMA. 2016;315(08):788–800.
    1. Rubenfeld G D, Caldwell E, Peabody E et al.Incidence and outcomes of acute lung injury. N Engl J Med. 2005;353(16):1685–1693.
    1. Ranieri V M, Rubenfeld G D, Thompson B T et al.Acute respiratory distress syndrome: the Berlin Definition. JAMA. 2012;307(23):2526–2533.
    1. Johnson E R, Matthay M A. Acute lung injury: epidemiology, pathogenesis, and treatment. J Aerosol Med Pulm Drug Deliv. 2010;23(04):243–252.
    1. Johnston L K, Rims C R, Gill S E, McGuire J K, Manicone A M. Pulmonary macrophage subpopulations in the induction and resolution of acute lung injury. Am J Respir Cell Mol Biol. 2012;47(04):417–426.
    1. Aggarwal N R, King L S, D'Alessio F R. Diverse macrophage populations mediate acute lung inflammation and resolution. Am J Physiol Lung Cell Mol Physiol. 2014;306(08):L709–L725.
    1. Herold S, Mayer K, Lohmeyer J. Acute lung injury: how macrophages orchestrate resolution of inflammation and tissue repair. Front Immunol. 2011;2:65.
    1. Bastarache J A, Wang L, Geiser T et al.The alveolar epithelium can initiate the extrinsic coagulation cascade through expression of tissue factor. Thorax. 2007;62(07):608–616.
    1. Gonzales J N, Kim K M, Zemskova M A et al.Low anticoagulant heparin blocks thrombin-induced endothelial permeability in a PAR-dependent manner. Vascul Pharmacol. 2014;62(02):63–71.
    1. Ware L B, Bastarache J A, Wang L. Coagulation and fibrinolysis in human acute lung injury--new therapeutic targets? Keio J Med. 2005;54(03):142–149.
    1. Günther A, Mosavi P, Heinemann Set al.Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia. Comparison with the acute respiratory distress syndrome Am J Respir Crit Care Med 2000161(2, Pt 1):454–462.
    1. Angus D C. Drotrecogin alfa (activated)...a sad final fizzle to a roller-coaster party. Crit Care. 2012;16(01):107.
    1. Warren B L, Eid A, Singer P et al.Caring for the critically ill patient. High-dose antithrombin III in severe sepsis: a randomized controlled trial. JAMA. 2001;286(15):1869–1878.
    1. Abraham E, Reinhart K, Opal S et al.Efficacy and safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sepsis: a randomized controlled trial. JAMA. 2003;290(02):238–247.
    1. Eisele B, Lamy M, Thijs L G et al.Antithrombin III in patients with severe sepsis. A randomized, placebo-controlled, double-blind multicenter trial plus a meta-analysis on all randomized, placebo-controlled, double-blind trials with antithrombin III in severe sepsis. Intensive Care Med. 1998;24(07):663–672.
    1. Laterre P F, Opal S M, Abraham E et al.A clinical evaluation committee assessment of recombinant human tissue factor pathway inhibitor (tifacogin) in patients with severe community-acquired pneumonia. Crit Care. 2009;13(02):R36.
    1. Li Y, Sun J F, Cui X et al.The effect of heparin administration in animal models of sepsis: a prospective study in Escherichia coli-challenged mice and a systematic review and metaregression analysis of published studies. Crit Care Med. 2011;39(05):1104–1112.
    1. MacLaren R, Stringer K A. Emerging role of anticoagulants and fibrinolytics in the treatment of acute respiratory distress syndrome. Pharmacotherapy. 2007;27(06):860–873.
    1. Tuinman P R, Dixon B, Levi M, Juffermans N P, Schultz M J. Nebulized anticoagulants for acute lung injury - a systematic review of preclinical and clinical investigations. Crit Care. 2012;16(02):R70.
    1. Hofstra J J, Vlaar A P, Cornet A D et al.Nebulized anticoagulants limit pulmonary coagulopathy, but not inflammation, in a model of experimental lung injury. J Aerosol Med Pulm Drug Deliv. 2010;23(02):105–111.
    1. Rehberg S, Yamamoto Y, Sousse L E et al.Advantages and pitfalls of combining intravenous antithrombin with nebulized heparin and tissue plasminogen activator in acute respiratory distress syndrome. J Trauma Acute Care Surg. 2014;76(01):126–133.
    1. Dixon B, Santamaria J D, Campbell D J. A phase 1 trial of nebulised heparin in acute lung injury. Crit Care. 2008;12(03):R64.
    1. Dixon B, Schultz M J, Hofstra J J, Campbell D J, Santamaria J D. Nebulized heparin reduces levels of pulmonary coagulation activation in acute lung injury. Crit Care. 2010;14(05):445.
    1. Elsharnouby N M, Eid H E, Abou Elezz N F, Aboelatta Y A. Heparin/N-acetylcysteine: an adjuvant in the management of burn inhalation injury: a study of different doses. J Crit Care. 2014;29(01):1820–1.82E6.
    1. Mu E, Ding R, An X, Li X, Chen S, Ma X. Heparin attenuates lipopolysaccharide-induced acute lung injury by inhibiting nitric oxide synthase and TGF-β/Smad signaling pathway. Thromb Res. 2012;129(04):479–485.
    1. Anastase-Ravion S, Carreno M P, Blondin C et al.Heparin-like polymers modulate proinflammatory cytokine production by lipopolysaccharide-stimulated human monocytes. J Biomed Mater Res. 2002;60(03):375–383.
    1. Hochart H, Jenkins P V, Smith O P, White B. Low-molecular weight and unfractionated heparins induce a downregulation of inflammation: decreased levels of proinflammatory cytokines and nuclear factor-kappaB in LPS-stimulated human monocytes. Br J Haematol. 2006;133(01):62–67.
    1. Camprubí-Rimblas M, Guillamat-Prats R, Lebouvier T et al.Role of heparin in pulmonary cell populations in an in-vitro model of acute lung injury. Respir Res. 2017;18(01):89.
    1. Puig F, Herrero R, Guillamat-Prats R et al.A new experimental model of acid- and endotoxin-induced acute lung injury in rats. Am J Physiol Lung Cell Mol Physiol. 2016;311(02):L229–L237.
    1. Hofstra J J, Cornet A D, de Rooy B F et al.Nebulized antithrombin limits bacterial outgrowth and lung injury in Streptococcus pneumoniae pneumonia in rats. Crit Care. 2009;13(05):R145.
    1. Wang M, He J, Mei B, Ma X, Huo Z. Therapeutic effects and anti-inflammatory mechanisms of heparin on acute lung injury in rabbits. Acad Emerg Med. 2008;15(07):656–663.
    1. Dosquet C, Weill D, Wautier J L. Cytokines and thrombosis. J Cardiovasc Pharmacol. 1995;25 02:S13–S19.
    1. Levi M, Schultz M. The inflammation-coagulation axis as an important intermediate pathway in acute lung injury. Crit Care. 2008;12(02):144.
    1. Lindmark E, Tenno T, Siegbahn A. Role of platelet P-selectin and CD40 ligand in the induction of monocytic tissue factor expression. Arterioscler Thromb Vasc Biol. 2000;20(10):2322–2328.

Source: PubMed

Sponsor e collaboratori

Condizioni mediche

Interventi farmacologici

3
Sottoscrivi