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. 2012 May;64(5):1653-64.
doi: 10.1002/art.33482.

Inhibition of focal adhesion kinase prevents experimental lung fibrosis and myofibroblast formation

David Lagares  1 , Oscar Busnadiego, Rosa Ana García-Fernández, Mohit Kapoor, Shangxi Liu, David E Carter, David Abraham, Xu Shi-Wen, Patricia Carreira, Benjamin A Fontaine, Barry S Shea, Andrew M Tager, Andrew Leask, Santiago Lamas, Fernando Rodríguez-Pascual
Affiliations

Inhibition of focal adhesion kinase prevents experimental lung fibrosis and myofibroblast formation

David Lagares et al. Arthritis Rheum. 2012 May.

Abstract

Objective: Enhanced adhesive signaling, including activation of focal adhesion kinase (FAK), is a hallmark of fibroblasts from lung fibrosis patients, and FAK has therefore been hypothesized to be a key mediator of this disease. This study was undertaken to characterize the contribution of FAK to the development of pulmonary fibrosis both in vivo and in vitro.

Methods: FAK expression and activity were analyzed in lung tissue samples from lung fibrosis patients by immunohistochemistry. Mice orally treated with the FAK inhibitor PF-562,271, or with small interfering RNA (siRNA)-mediated silencing of FAK were exposed to intratracheally instilled bleomycin to induce lung fibrosis, and lungs were harvested for histologic and biochemical analysis. Using endothelin 1 (ET-1) as a stimulus, cell adhesion and contraction, as well as profibrotic gene expression, were studied in fibroblasts isolated from wild-type and FAK-deficient mouse embryos. ET-1-mediated FAK activation and gene expression were studied in primary mouse lung fibroblasts, as well as in wild-type and β1 integrin-deficient mouse fibroblasts.

Results: FAK expression and activity were up-regulated in fibroblast foci and remodeled vessels from lung fibrosis patients. Pharmacologic or siRNA-mediated targeting of FAK resulted in marked abrogation of bleomycin-induced lung fibrosis in mice. Loss of FAK impaired the acquisition of a profibrotic phenotype in response to ET-1. Profibrotic gene expression leading to myofibroblast differentiation required cell adhesion, and was driven by JNK activation through β1 integrin/FAK signaling.

Conclusion: These results implicate FAK as a central mediator of fibrogenesis, and highlight this kinase as a potential therapeutic target in fibrotic diseases.

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Figures

Figure 1
Figure 1. FAK expression and activity are upregulated in fibrotic foci in lungs from pulmonary fibrosis (PF) patients
Lung tissue sections from control donors (A) and patients (B–C) were stained for Masson’s trichrome and for FAK, p-FAK and α-SMA. Sections from control donors showed the normal architecture of the lung tissue with restricted expression of α-SMA in bronchial and vascular smooth muscle cells (SMC) (A, left and middle). In contrast, Masson’s trichrome micrographs from patients revealed focal areas of collagen accumulation in the interstitium of major airways, blood vessels and alveolar septa (B, left). These fibrotic foci (FF) often contained an important number of α-SMA-positive myofibroblasts (MF) (B, middle). In healthy donors, overall FAK expression was low, except in bronchial epithelial cells (BEC) (A, right). A marked increase in FAK staining was observed in PF sections, localized mainly to BEC, but also to macrophages (MAC) (B, right), and α-SMA-positive MF within fibrotic foci (FF) (B, middle and left). p-FAK was detected in MF present in the fibrotic foci in PF tissue sections (C, middle). A control section without primary antibody is also shown (C, right). Microphotographs are representative of 8 different patients. Insets show selected regions at higher magnification.
Figure 2
Figure 2. In vivo FAK inhibition with PF-562,271 attenuates bleomycin-induced lung injury
Mice receiving saline or bleomycin were systemically treated with PF-562,271 or vehicle. A) Representative photographs of whole lungs (left) and measurements of lung weight (right) on day 28 after treatment, n=8, *p <0.05. Black/white bars are explained in panel C. B) Lung fibrosis assessed by hematoxylin and eosin staining (upper and middle rows), and Masson’s trichrome blue staining (lower row). Micrographs are representative of at least eight independent experiments. C) Semi-quantitative analysis of lung injury using the Ashcroft score, n =8, *p <0.05. D) Acid-soluble collagen content in lung homogenates, n =8, *p <0.05.
Figure 3
Figure 3. Down-regulation of FAK by siRNA prevents bleomycin-induced lung fibrosis
FAK or control non-targeting siRNA were administered in a single dose to mice by intratracheal instillation together with bleomycin or saline. A) Representative photographs of whole lungs (left) and measurements of lung weight (right) on day 21 after treatment, n =8, *p <0.05. Black/white bars are explained in panel C. B) Lung fibrosis assessed by hematoxylin and eosin staining (upper row), Masson’s trichrome blue staining (middle row) and immunohistochemical detection of α-SMA-positive myofibroblasts (lower row). Micrographs are representative of at least eight independent experiments. C) Semi-quantitative analysis of lung injury using the Ashcroft score, n =8, *p <0.05 (left) and acid-soluble collagen content in lung homogenates, n =8, *p <0.05 (right). D) Detection of p-FAK, FAK, α-SMA and β-actin in lung lysates of mice receiving bleomycin or saline under non-targeting or FAK siRNA treatment. Values above the blots refer to the ratio of the signal p-FAK/FAK or α-SMA/β-actin after densitometric analysis, n=3.
Figure 4
Figure 4. Vascular leak and leukocyte recruitment and activation induced by bleomycin injury is unaltered in siRNA FAK-treated mice
BALF samples were obtained from bleomycin and saline-treated mice and analyzed by cytospin and cytofluorometry, as well as for total protein content. BALF total numbers of leukocytes, macrophages, neutrophils, lymphocytes, and CD4+, CD8+ and CD3+ T cells (only bleomycin-treated mice), n=4. Assessment of lung vascular permeability by BALF total protein determination, n=4. Data presented are from one of two independent experiments with similar results (n.d. not detected).
Figure 5
Figure 5. Loss of FAK in fibroblasts impairs the ability of ET-1 to induce the profibrotic phenotype
Experiments were done in fibroblasts from wild type and FAK-deficient mice as indicated. A) Time- and dose-dependent effect of ET-1 on p-FAK and FAK, n=3. B) Cell adhesion (left) and contraction (right) under basal conditions or after ET-1, n=4, *p <0.05. C) Analysis of profibrotic gene expression. Time course induction by ET-1 of collagen type I and α-SMA mRNA expression, n=3, *p <0.05 (left). Effect of ET-1 on α-SMA protein (middle, n=3). Induction by ET-1 of α-SMA, collagen type I and CTGF mRNA expression in FAK −/− fibroblasts transduced by adenoviral infection with FAK, or with GFP as a control, n=3, *p <0.05 (right). The dotted line represents the normalized value without ET-1. D) Immunolocalization of vinculin (green) and phalloidin staining of actin filaments (red) (scale bar: 50 µm, n=3). The arrows indicate the formation of vinculin-containing focal adhesions. E) Effect of siRNA-mediated knockdown of FAK expression on ET-1-induced α-SMA and collagen type I mRNA, *p <0.05 comparing ET-1-induced versus basal, #p<0.05 comparing FAK-specific versus non-targeting siRNA, (left, n=3); and α-SMA protein expression (right, n=3). Values above the blots refer to the ratio of the signals p-FAK/FAK or α-SMA/β-actin after densitometric analysis.
Figure 6
Figure 6. Induction of FAK phosphorylation by ET-1 is dependent on cell adhesion and integrin β1 signaling
Analysis of FAK phosphorylation (A) and α-SMA protein expression (B) in fibroblasts seeded on standard tissue culture plates (Plastic), culture plates coated with 10 µg/ml poly-lysine (Poly-Lys), and cells left in suspension (Suspended). Detection of p-FAK (C) and α-SMA protein expression (D) in fibroblasts from wild type (ITGβ1 +/+) and integrin β1-deficient (ITGβ1 −/−) mice seeded on standard culture plates. Blots and histograms depicting the ratio p-FAK/FAK show the result of a representative experiment. Values above the blots refer to the ratio of the signal p-FAK/FAK or α-SMA/β-actin after densitometric analysis, n=3.
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