Inhibition of platelet-derived growth factor signaling attenuates pulmonary fibrosis

Abstract

Pulmonary fibrosis is the consequence of a variety of diseases with no satisfying treatment option. Therapy-induced fibrosis also limits the efficacy of chemotherapy and radiotherapy in numerous cancers. Here, we studied the potential of platelet-derived growth factor (PDGF) receptor tyrosine kinase inhibitors (RTKIs) to attenuate radiation-induced pulmonary fibrosis. Thoraces of C57BL/6 mice were irradiated (20 Gy), and mice were treated with three distinct PDGF RTKIs (SU9518, SU11657, or Imatinib). Irradiation was found to induce severe lung fibrosis resulting in dramatically reduced mouse survival. Treatment with PDGF RTKIs markedly attenuated the development of pulmonary fibrosis in excellent correlation with clinical, histological, and computed tomography results. Importantly, RTKIs also prolonged the life span of irradiated mice. We found that radiation up-regulated expression of PDGF (A-D) isoforms leading to phosphorylation of PDGF receptor, which was strongly inhibited by RTKIs. Our findings suggest a pivotal role of PDGF signaling in the pathogenesis of pulmonary fibrosis and indicate that inhibition of fibrogenesis, rather than inflammation, is critical to antifibrotic treatment. This study points the way to a potential new approach for treating idiopathic or therapy-related forms of lung fibrosis.

Figure 1.

Radiation-induced paracrine activation of fibroblasts in a coculture system. (A) Fibroblast proliferation induced by exposure to coculture medium (Control) or by prior 10 Gy irradiation of HUVECs or A549 cells in the absence (RT) or presence of SU9518 (SU9518+RT) in the fibroblast medium. Mean ± SD. *, P < 0.05. (B) Phosphorylation status (anti-phosphorylated tyrosine antibody, anti-pY) of PDGFRβ in quiescent fibroblasts, fibroblasts exposed to medium from 10 Gy irradiated endothelial cells (6 and 72 h after radiation, RT), or with additional exposure to PDGF RTKI, SU9518 (RT+SU). Equal loading of lanes was demonstrated with anti-PDGFRβ. (C) Real-time quantitative RT-PCR of PDGF-A, PDGF-B, PDGF-C, and PDGF-D isoforms at 6, 12, 24, 48, and 72 h after 10 Gy irradiation of HLMVECs and A549 cells. Data are means ± SD from at least three independent measurements and show relative expression levels compared with the nonirradiated control cells at each time point.

Figure 2.

Inhibition of PDGF signaling prolongs survival of irradiated mice. (A) Pharmacokinetic analysis of plasma SU9518 (structure shown as inset) concentration after a single dose of 100 mg/kg administered s.c. to C57BL/6 mice. T_max ≤ 2 h; C_max = 706 ng/ml; T_1/2 = 73.6 h. (B) Kaplan-Meier analysis of mouse survival after thoracic irradiation and SU9518 treatment. Death was considered complete (cause specific due to radiation) in all cases except those of planned euthanasia for histological assessment, which were considered as censored. Radiation (20 or 40 Gy) reduced survival (P < 0.001 vs. the control [100 μl carboxymethylcellulose twice weekly]; log-rank). SU9518 had no influence on survival of unirradiated mice (P > 0.95 vs. the control). The difference between prevention (SU9518 started 1 d before irradiation) and intervention (SU9518 started 3 d after irradiation) was significant for the 20 Gy group but not for the 40 Gy group (P = 0.035/20 Gy; P = 0.41/40 Gy). SU9518 prevention in both the 20 and 40 Gy group tended to increase survival versus radiation alone, but was not statistically significant (P = 0.15/20 Gy; P = 0.22/40 Gy). If SU9518 administration was started 3 d after 20 Gy irradiation (intervention, 20Gy+SU9518, 40Gy+SU9518), mouse survival increased markedly versus radiation alone (P = 0.006/20 Gy; P = 0.04/40 Gy). (C) Kaplan-Meier analysis of mouse survival from independent confirmation experiments using two other PDGF RTKIs (Imatinib and SU11657) with administration starting 3 d after thoracic irradiation (20 Gy, intervention). Both Imatinib and SU11657 significantly increased survival of irradiated mice (P < 0.01).

Figure 3.

Constitutive activation of PDGF signaling in mouse lungs. (A) Immunohistochemical analysis demonstrates strong induction of PDGF-B and PDGFR-β expression in lung sections 4 wk after 20 Gy total thoracic irradiation (RT). Phosphorylation of PDGFR-β (p-PDGFR-β) was also enhanced in irradiated (RT) versus control mice. Treatment with SU9518 (RT+SU) significantly reduced phosphorylation of PDGFR-β. Bar, 100 μm. (B) 2, 4, and 20 wk after 20 Gy irradiation (RT) and administration of SU9518 (SU+RT and RT+SU), mice were killed and the lungs were harvested for protein isolation. The amount of phosphorylated PDGFR-β (anti-pY) was determined by immunoblotting. Equal loading of lanes was demonstrated with anti-PDGFRβ. (C) Real-time quantitative RT-PCR of PDGF-A, PDGF-B, PDGF-C, and PDGF-D isoforms detected in RNA from mouse lung after SU9518 treatment, 20 Gy thoracic irradiation (RT), and SU9518 administered after lung radiation (RT+SU9518). In irradiated lung specimen, significant up-regulation of all four PDGF isoforms are shown at 2 and 4 wk after irradiation (*, P < 0.01) as well as for PDGF-A, PDGF-B, and PDGF–C isoforms at 20 wk (§, P < 0.001). The expression of the PDGF-A, PDGF-B, and PDGF-C isoforms is significantly impaired at 20 wk after irradiation in SU9518-treated mice (#, P < 0.02), whereas the PDGF expression at earlier time points is not markedly affected by PDGF RTK. This data underscore that the inhibition of fibrosis is the principal target for PDGF RTKIs. Bars are means ± SD from three independent measurements and show relative expression levels compared with the nonirradiated lung tissue at 2, 4, and 20 wk after radiation.

Figure 4.

High resolution CT as a noninvasive tool for qualitative and quantitative longitudinal monitoring of pulmonary fibrosis progression in mice. Treatment with SU9518 started either 1 d before 20 Gy irradiation (SU9518+RT) or 3 d after irradiation (RT+SU9518). (A) Representative CT scans showing progression of pulmonary fibrosis in mice after 20 Gy whole thorax irradiation (RT) and treatment with SU9518 treatment starting 3 d after RT. Fibrosis (black arrows) is characterized by diffuse bilateral areas of “ground-glass” attenuation and intralobular reticular opacities. (B, C, and D) Quantitative lung density values derived from CT scans. The same 10 randomly chosen mice in each treatment group were examined in a longitudinal study by CT every 2 wk. Five regions of interest were randomly selected in the lungs, and the lung density (in HU) was determined for each region of interest. The time course in B shows that intervention and prevention schedule were both effective in attenuating radiation-induced lung density, and SU9518 intervention (RT+SU9518) was slightly more effective than SU9518 prevention after week 20 (P < 0.05). Bars are mean ± SE. *, P < 0.001 versus the control; #, P < 0.01 versus RT (B and D). (E) Independent confirmatory experiment using Imatinib and SU11657. Inhibitor administration started on day 3 after 20 Gy thoracic irradiation (20 Gy). Both compounds are very effective in attenuating radiation-induced enhancement of lung density. Bars show lung density derived from CT scans (mean ± SE). *, P < 0.01 versus control; #, P < 0.01 versus RT.

Figure 5.

Histological assessment of lung fibrosis. (A) Photomicrographs of hematoxylin and eosin–stained lung tissue sections from control mice, irradiated mice (20 Gy, RT), and mice treated with SU9518 after thoracic irradiation (intervention, RT+SU9518) at 12, 16, 20, and 24 wk after radiation. Bar, 100 μm. (B) Masson's trichrome staining of lung sections 12, 16, 20, and 24 wk after irradiation without (RT) or with administration of SU9518 (RT+SU9518). Examples of focal fibrotic lesions (*), areas of “normal” lung (black arrows), and inflammatory cell infiltration (L, red) are marked. Scale bar, 100 μm. (C) Time course of septal edema development in irradiated mice (RT) with maximum edema at 72 h after radiation (P < 0.05 RT vs. control for each time from 0 h to 4 wk). The preventive or interventive administration of SU9518 (SU9518+RT, RT+SU9518) had significant suppressive effects on edema development, except at 72 h after radiation (P < 0.05 vs. RT). (D) Septal fibrosis develops progressively several weeks after radiation (P < 0.05 RT vs. control for each time point after week 2). Both the SU9518 intervention (RT+SU9518) and prevention (SU9518+RT) treatment significantly reduced the progression of septal fibrosis, as measured by the extent of alveolar wall thickness, at all time points after week 2 (P < 0.05 vs. RT for each time point). However, septal fibrosis was not completely inhibited by the compound (P < 0.05 vs. the control after week 2). (E) Quantitative analysis of leukocyte numbers at various times after irradiation (20 Gy, RT) reveals two peaks. SU9518 before (SU9518+RT) or after (RT+SU9518) irradiation did not significantly attenuate the early leukocyte peak (P > 0.2). Both the preventive (SU9518+RT) and interventional (RT+SU9518) administration of SU9518 significantly inhibited the fibrosis-associated second inflammatory response during weeks 2–26 (P < 0.05 vs. RT for each time point). However, the second fibrosis-associated inflammation was not completely inhibited by SU9518 (P < 0.05 vs. the control after week 2). Bars are mean ± SD.

Figure 6.

Model of fibrosis development integrating our findings. The data suggest that a fibrosis-generating stimulus such as ionizing radiation initiates an inflammatory response consisting of leukocyte infiltration and the release of proinflammatory/profibrotic cytokines such as PDGF. This acute tissue response initiates a constitutive chronic stimulation and activation of the fibroblasts by PDGF, with subsequent proliferation and extracellular matrix deposition. PDGF RTKIs inhibits fibrogenesis induced by the release of PDGF from injured endothelial cells, epithelial cells, and infiltrated macrophages.