Endothelial cell-specific anticoagulation reduces inflammation in a mouse model of acute lung injury

Abstract

Tissue factor (TF)-dependent coagulation contributes to lung inflammation and the pathogenesis of acute lung injury (ALI). In this study, we explored the roles of targeted endothelial anticoagulation in ALI using two strains of transgenic mice expressing either a membrane-tethered human tissue factor pathway inhibitor (hTFPI) or hirudin fusion protein on CD31⁺ cells, including vascular endothelial cells (ECs). ALI was induced by intratracheal injection of LPS, and after 24 h the expression of TF and protease-activated receptors (PARs) on EC in lungs were assessed, alongside the extent of inflammation and injury. The expression of TF and PARs on the EC in lungs was upregulated after ALI. In the two strains of transgenic mice, expression of either of hTFPI or hirudin by EC was associated with significant reduction of inflammation, as assessed by the extent of leukocyte infiltration or the levels of proinflammatory cytokines, and promoted survival after LPS-induced ALI. The beneficial outcomes were associated with inhibition of the expression of chemokine CCL2 in lung tissues. The protection observed in the CD31-TFPI-transgenic strain was abolished by injection of an anti-hTFPI antibody, but not by prior engraftment of the transgenic strains with WT bone marrow, confirming that the changes observed were a specific transgenic expression of anticoagulants by EC. These results demonstrate that the inflammation in ALI is TF and thrombin dependent, and that expression of anticoagulants by EC significantly inhibits the development of ALI via repression of leukocyte infiltration, most likely via inhibition of chemokine gradients. These data enhance our understanding of the pathology of ALI and suggest a novel therapeutic strategy for treatment.

Keywords: CCL2; acute lung injury; anticoagulants; endothelial cells; hirudin; human tissue factor pathway inhibitor (hTFPI); inflammation; lipopolysaccharide.

Fig. 1

Intratracheal LPS induces lung inflammation (a-b, d-f), coagulation activation (c) and an increased expression of TF (g-i), PAR1 (j–l), and PAR2 (m-o) on CD144⁺ (the indicative molecule of ECs) cells in lung during ALI. Compared with NS group. n = 5–10 for each group. ^**P < 0.01; ^***P < 0.001; ns no significance. Data are presented as mean ± SEM of three separate experiments. CON control, LPS lipopolysaccharide, ALI acute lung injury, WBC white blood cell, Neut neutrophil, BALF bronchoalveolar lavage fluid, TF tissue factor, PAR protease-activated receptor, ECs endothelial cells, WT wild type

Fig. 2

CD31-TFPI-Tg and CD31-Hir-Tg mice protect against LPS-induced ALI. Total cell numbers (a), neutrophil numbers (b), and total protein (l) in BALF, lung edema (m) and alveolar-capillary leak (n) after intratracheal instillation of LPS for 24 h and the percentage of survival (o). With quantitative real-time PCR, the gene expression levels of KC (c), MIP-2 (e), IL-6 (g), TNF-α (i), IL-1β (j), and IL-10 (k) were detected. With Quantikine ELISA analysis, the protein expression levels of KC (d), MIP-2 (f), and IL-6 (h) were measured. Representative light photomicrographs (100 × ) of H&E staining of lung tissue (p) and histology score (q). Scale bars = 200 μm. Under the heading of “10⁴ cells/mL”, a value of 1 designates 10 000 cells/mL. n = 6–11 for each group. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ns no significance. Data are presented as mean ± SEM of three separate experiments. CON control, LPS lipopolysaccharide, ALI acute lung injury, BALF bronchoalveolar lavage fluid, EB Evans blue, WT wild type

Fig. 3

The anti-hTFPI-neutralizing antibody abolishes the protective effect of the hTFPI fusion protein expressed by CD31-TFPI-Tg mice. Total cell numbers (a), neutrophil numbers (b), and total protein (l) in BALF after intratracheal instillation of LPS for 24 h. With quantitative real-time PCR, the gene expression levels of KC (c), MIP-2 (e), IL-6 (g), TNF-α (i), IL-1β (j), and IL-10 (k) were detected. With Quantikine ELISA analysis, the protein expression levels of KC (d), MIP-2 (f), and IL-6 (h) were measured. Representative light photomicrographs (100 × ) of H&E staining of lung tissue (m) and histology score (n). Scale bars = 200 μm. Under the heading of “10⁴ cells/mL”, a value of 1 designates 10 000 cells/mL. n = 6–10 for each group. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ns no significance. Data are presented as mean ± SEM of three separate experiments. CON control, LPS lipopolysaccharide, ALI acute lung injury, BALF bronchoalveolar lavage fluid, WT wild type

Fig. 4

CD31-TFPI-Tg and CD31-Hir-Tg mice, engrafted with bone marrow from WT mice (WT/CD31-TFPI-Tg and WT/CD31-Hir-Tg mice respectively) are still protected against LPS-induced ALI. Total cell numbers (a), neutrophil numbers (b), and total protein (l) in BALF, and lung edema (m) after intratracheal instillation of LPS for 24 h. With quantitative real-time PCR, the gene expression levels of KC (c), MIP-2 (e), IL-6 (g), TNF-α (i), IL-1β (j), and IL-10 (k) were detected. With Quantikine ELISA analysis, the protein expression levels of KC (d), MIP-2 (f), and IL-6 (h) were measured. Representative light photomicrographs (100 × ) of H&E staining of lung tissue (o) and histology score (n). Scale bars = 200 μm. Under the heading of “10⁴ cells/mL”, a value of 1 designates 10 000 cells/mL. n = 6–10 for each group. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ns no significance. Data are presented as mean ± SEM of three separate experiments. CON control, LPS lipopolysaccharide, ALI acute lung injury, BALF bronchoalveolar lavage fluid, WT wild type

Fig. 5

The expression level of CCL2 decreases in lung tissues after LPS-induced ALI in CD31-TFPI-Tg and CD31-Hir-Tg mice, either those engrafted with WT BM or those non-engrafted. Quantitative real-time PCR and ELISA analysis respectively detected the expression level of CCL2 in lung tissues in ALI model of CD31-TFPI-Tg and CD31-Hir-Tg mice (a, b), CD31-TFPI-Tg mice receiving anti-hTFPI-neutralizing antibody (c, d), and WT/CD31-TFPI-Tg and WT/CD31-Hir-Tg mice (e, f). n = 6–10 for each group. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ns no significance. Data are presented as mean ± SEM of three separate experiments. CCL2 chemokine (C-C motif) ligand 2, CON control, LPS lipopolysaccharide, ALI acute lung injury, WT wild type

Fig. 6

Rationale for the investigation of the CD31-TFPI-Tg or CD31-Hir-Tg mice. Tissue factor (TF) and downstream coagulation proteases can exert direct influences on the inflammatory cascade. Protease-activated receptor 1 (PAR1) has a cornerstone role in transforming coagulation protease activity into inflammatory signals and thrombin is the main ligand to PAR1. A predominant mechanism by which the fusion proteins, human tissue factor pathway inhibitor (hTFPI) or hirudin, inhibit inflammation is to inhibit the development of local chemokine gradients, particularly CCL2, which in vivo is dependent on thrombin signaling through endothelial cell (EC)-expressed PAR1. The fact that both strains showed the same protective response implies that the predominant effect of the hTFPI fusion protein is to inhibit thrombin generation