Increased local expression of coagulation factor X contributes to the fibrotic response in human and murine lung injury
Chris J. Scotton, … , Oliver Eickelberg, Rachel C. Chambers
Chris J. Scotton, … , Oliver Eickelberg, Rachel C. Chambers
Published August 3, 2009
Citation Information: J Clin Invest. 2009;119(9):2550-2563. https://doi.org/10.1172/JCI33288.
View: Text | PDF
Research Article Pulmonology

Increased local expression of coagulation factor X contributes to the fibrotic response in human and murine lung injury

Abstract

Uncontrolled activation of the coagulation cascade contributes to the pathophysiology of several conditions, including acute and chronic lung diseases. Coagulation zymogens are considered to be largely derived from the circulation and locally activated in response to tissue injury and microvascular leak. Here we report that expression of coagulation factor X (FX) is locally increased in human and murine fibrotic lung tissue, with marked immunostaining associated with bronchial and alveolar epithelia. FXa was a potent inducer of the myofibroblast differentiation program in cultured primary human adult lung fibroblasts via TGF-β activation that was mediated by proteinase-activated receptor–1 (PAR1) and integrin αvβ5. PAR1, αvβ5, and α-SMA colocalized to fibrotic foci in lung biopsy specimens from individuals with idiopathic pulmonary fibrosis. Moreover, we demonstrated a causal link between FXa and fibrosis development by showing that a direct FXa inhibitor attenuated bleomycin-induced pulmonary fibrosis in mice. These data support what we believe to be a novel pathogenetic mechanism by which FXa, a central proteinase of the coagulation cascade, is locally expressed and drives the fibrotic response to lung injury. These findings herald a shift in our understanding of the origins of excessive procoagulant activity and place PAR1 central to the cross-talk between local procoagulant signaling and tissue remodeling.

Authors

Chris J. Scotton, Malvina A. Krupiczojc, Melanie Königshoff, Paul F. Mercer, Y.C. Gary Lee, Naftali Kaminski, John Morser, Joseph M. Post, Toby M. Maher, Andrew G. Nicholson, James D. Moffatt, Geoffrey J. Laurent, Claudia K. Derian, Oliver Eickelberg, Rachel C. Chambers

×

Figure 4

FXa-induced α-SMA protein expression is dependent on TGF-β activity.

Options: View larger image (or click on image) Download as PowerPoint
FXa-induced α-SMA protein expression is dependent on TGF-β activity. (A)...
(A) Effect of TFLLR-NH2 on TGF-β activation. pHALFs were grown in coculture with tMLECs (see Methods). Activation was blocked by the pan-specific TGF-β–neutralizing antibody 1D11. (B and C) Time course of SMAD2 phosphorylation by FXa. (B) Representative immunoblot for pSMAD2 and total SMAD2/3 loading control. (C) Densitometric analysis. (D and E) Effect of ALK5 inhibition with SB431542 (D) and SD-208 (E) on FXa-induced α-SMA protein expression, analyzed by Western blotting. Cell cultures were preincubated with inhibitors for 30 minutes prior to 36 hours FXa exposure. Densitometric analyses are shown. (F and G) Effect of αvβ5-neutralizing antibodies on FXa-induced α-SMA protein expression. Cell cultures were preincubated with 10 μg/ml anti-αvβ5 or isotype control antibody for 1 hour prior to 36 hours FXa exposure. (F) Representative immunoblots for α-SMA and ERK2 loading control. (G) Densitometric analysis (mean ± SEM). (H) Effect of Y-27632 on FXa-induced α-SMA protein expression. Cell cultures were preincubated with Y-27632 for 1 hour prior to 36 hours FXa exposure; densitometric analysis is shown (mean ± SEM). (I and J) Effect of αvβ5-neutralizing antibodies on FXa-induced SMAD2 phosphorylation. Cell cultures were preincubated with 10 μg/ml anti-αvβ5 or isotype control antibody for 1 hour prior to 10 hours FXa exposure. (I) Representative immunoblots. (J) Densitometric analysis, representative of 2 separate experiments (mean ± SEM). Unless otherwise indicated, data (mean ± SD) are representative of 3 separate experiments, with 3 replicates per group. *P < 0.05, **P < 0.001, ANOVA.