Pulmonary Irradiation Fibrosis Is Preceded By Increased Endothelial Cell Gene Expression

Irradiation of the C57BL/6NHsd mouse lung results in pulmonary fibrosis. An acute phase (days 1 to 28) following irradiation recognized by an inflammatory response is followed by a latent phase (day 28 to day 125) where lung appears normal. Fibrosis occurs from day 125 till death characterized by bone marrow stromal cell migration to and proliferation in lung and increased collagen deposition. To investigate the role of endothelial cells, C57BL/6NHsd female mice were irradiated to 20 Gy to the pulmonary cavity shielding the rest of the body. Lung was excised at serial times, endothelial cells separated and tested for expression of endothelial genes vWF, VEGF, FGF1, CCL13 and CTGF; inflammatory genes IL-6; IGFbp7 and genes associated with fibrosis including MnSOD, Nrf2, NfkB, TLR4 and TGF-B. Endothelial cells were separated by using antibodies to CD45, or PECAM and sorting using flow cytometry. RNA was extracted using Triazol and gene expression determined using Real Time Polymerase Chain Reaction (RT-PCR). In irradiated total lung there was significant increase in endothelial cell markers at all times, compared to nonirradiated lung. Transcripts for vWF increased by 474.1 ± 388.5% (p = 0.017) by 2 days after irradiation, 520 ± 55.4% (p < 0.0001) at day 50, and 484.8 ± 26.8% (p < 0.0001) by day 200. VEGF and CTGF also remained elevated. In contrast, total lung MnSOD gene expression increased by 140.6 ± 46.1% at day 2 (p = 0.0185), decreased at day 50 to 17.9 ± 11.2% (p = 0.2808), and increased to 31.5 ± 11.4% by day 200 (p = 0.023). However, purified endothelial cells compared to alveolar cells, showed increased expression of not only the endothelial cell genes but also MnSOD, Nrf2, NfkB, and TGF-B. MnSOD expression in endothelial compared to alveolar cells at day 28 after irradiation was higher at 244.5 ± 7.8 vs 80.5 ± 4.9 (p = 0.0016), at day 150 expression was 140.1 ± 25.0 vs 5.4 ± 1.2 (p < 0.0001) and day 200 expression was 368.5 ± 6.4 vs 51.8 ± 10.3 (p = 0.0007). Lung endothelial cells display critical biomarkers of irradiation injury leading to fibrosis.