Pulmonary Endothelial Cell Irradiation Damage Signaling Initiates Late Fibrosis

Abstract 4682

Thoracic irradiation of C57BL/6NHsd mice produces an acute lung inflammatory period (0 to 30 days after irradiation), followed by a stable latent period (30 to 100 days), followed by a delayed fibrosis reaction (100 –150 days). Fibrosis involves migration of bone marrow derived mesenchymal stem cells (fibroblast progenitors) into the lung. The molecular mechanisms which initiate migration and homing of fibroblast progenitors are unknown. Increased pulmonary TGF-β expression has been correlated with the onset of fibrosis. To identify the cells and genetic pathways involved, C57BL/6NHsd female mice were irradiated with 20 Gy to the thoracic cavity and sacrificed at serial time points including days 0, 2, 14, 28, 50, 75, 100, 110, 125, 150, and 200, lungs were removed and RNA extracted from either whole lung or purified cell populations. Real Time Polymerase Chain Reaction (RT-PCR) was used to quantitate gene expression.

In whole lung tissue, when compared to un-irradiated lungs, a significant elevation in expression of multiple inflammation and chemotaxis associated genes (including TGF-β, TNF-α,and MnSOD) and stress response genes (including NFkB, AP-1, SP-1,and Nrf2) was detected both during the acute inflammatory phase (p= 0.0112, 0.0037, 0.0245 and p= 0.0001, < 0.0001, =0.0010, =0.0181 respectively) and late-onset fibrosis phase (p= 0.0079, 0.0459, 0.0001, and p < 0.0001, = 0.0006, =0.0128, < 0.0001 respectively), but were either not elevated or significantly decreased during the latent period ( p< 0.0001, = 0.3785, 0.8123, and p=.0243, < 0.0001, < 0.0001, < 0.0001 respectively). In contrast, increased expression of endothelial cell associated genes including: vWF, VEGF, CTGF, FGF1 and CCL13 was detected during the latent period, as well as during the acute and chronic reaction phases. Purified lung endothelial, but not epithelial cells, showed stable vascular gene elevations of vWF, VEGF, CTGF, FGF1 and CCL13 during the entire time course including day 7 (p < 0.0001, < 0.0001, =0.0035, =0.0009, =0.0056), day 75 (p=.00263, 0.0104, 0.0284, 0.0038, and 0.0007), and day 150 (p < 0.0001, < 0.0001, =0.0005, =0.0039, =0.0014 respectively). The data supports a significant role of vascular gene expression in the activation and recruitment of cells involved in radiation induced late pulmonary fibrosis.

Further analysis of lung endothelial cells may elucidate the mechanisms involved in the late effects of thoracic irradiation, and identify new targets for drug development to prevent irradiation pulmonary fibrosis.