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

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.

Effects of nebulized antithrombin and heparin on inflammatory and coagulation alterations in an acute lung injury model in rats

“…In our ALI model, although only AT alone was able to decrease lung weight and edema, nebulized AT alone as well as combined with heparin reduced protein concentration and cellular infiltration into the alveolar space. These results indicate a decrease of lung permeability that could be explained by the partial reestablishment of the alveolar capillary barrier being consistent with previous studies that showed the capacity of anticoagulants to reduce lung permeability . Also, when we determined the histological damage analyzing the hallmarks of ALI, both treated groups with anticoagulants slightly improved the lesion although it was nonsignificant.…”

“…Treatment with AT and AT combined with heparin reduced AM expression of proinflammatory (iNOS) and antiinflammatory (Arg‐I) mediators and decreased the expression of mediators of recruitment (CXCL1 and CCL2), indicating an interaction between both anticoagulants and macrophages. In a previous study, we found that nebulized heparin was able to decrease the effectors of the NF‐kB pathway through AM . In this study, AT and AT combined with heparin also reduced the expression of proinflammatory cytokines (iNOS, CXCL1, and CCL2) regulated by the NF‐kB pathway in AM.…”

“…Herein we focused on the activities of AT and whether they are increased by the combination of heparin. We did not perform a group with heparin alone because in a previous study our group analyzed the effects of nebulized heparin in a 24‐hour model of LPS‐induced ALI and found that heparin reduced pulmonary coagulation and inflammation without producing systemic bleeding . Our results suggest that the local administration of AT and AT combined with heparin by nebulization is safe and effective because the activities of both treatments were focused on the pulmonary compartment and no systemic alterations were observed.…”

“…AT (Anbinex Grífols S.A.) and heparin (unfractionated heparin, Hospira Products Farmac) were used for this experiment. Both anticoagulants have been previously used in rat models with positive results, indicating that there is an interaction between them and the coagulation cascade of the animals . For the nebulization of AT and heparin the AeronebPro nebulizer system (Aerogen Limited) was used.…”

“…Both anticoagulants have been previously used in rat models with positive results, indicating that there is an interaction between them and the coagulation cascade of the animals. 21,28 For the nebulization of AT and heparin the Numbers were randomly assigned to each animal and the samples;…”