Temporal expression of hypoxia-regulated genes is associated with early changes in redox status in irradiated lung

Abstract

The development of normal lung tissue toxicity after radiation exposure results from multiple changes in cell signaling and communication initiated at the time of the ionizing event. The onset of gross pulmonary injury is preceded by tissue hypoxia and chronic oxidative stress. We have previously shown that development of debilitating lung injury can be mitigated or prevented by administration of AEOL10150, a potent catalytic antioxidant, 24h after radiation. This suggests that hypoxia-mediated signaling pathways may play a role in late radiation injury, but the exact mechanism remains unclear. The purpose of this study was to evaluate changes in the temporal expression of hypoxia-associated genes in irradiated mouse lung and determine whether AEOL10150 alters expression of these genes. A focused oligo array was used to establish a hypoxia-associated gene expression signature for lung tissue from sham-irradiated or irradiated mice treated with or without AEOL10150. Results were further verified by RT-PCR. Forty-four genes associated with metabolism, cell growth, apoptosis, inflammation, oxidative stress, and extracellular matrix synthesis were upregulated after radiation. Elevated expression of 31 of these genes was attenuated in animals treated with AEOL10150, suggesting that expression of a number of hypoxia-associated genes is regulated by early development of oxidative stress after radiation. Genes identified herein could provide insight into the role of hypoxic signaling in radiation lung injury, suggesting novel therapeutic targets, as well as clues to the mechanism by which AEOL10150 confers pulmonary radioprotection.

Figure 1. The temporal progression of tissue hypoxia in lung after radiation

Tissue hypoxia is first noticeable around three days (CAIX, Pimonidazole) post-radiation and progressively increases throughout the follow-up period (6 months). At six weeks, the first histopathologic lesions are seen (H&E). These are generally focal in nature and are characterized by thickening of the alveoli wall and increased inflammatory cell infiltrate. By six months, tissue damage had considerably worsened and a greater number of focal lesions were observed. Error bars represent 100 µm.

Figure 2. Temporal expression of HIF mRNA

The amplified PCR fragments were visualized on 1.5% agarose gel containing 0.5 µg/ml ethidium bromide. The GAPDH and HIF genes were amplified in the same reaction. The top band shows a 1 kb GAPDH fragment and the bottom band shows the HIF gene fragment.

Figure 3. Western blot analysis illustrates the dynamic changes in stabilization of HIF-1α (A) and HIF-2α (B) proteins changes over time following irradiation, while HIF-3α (C) remains constant

Western blot bands were visualized and normalized based on α-tubulin expression. The relative intensities were measured and averaged within groups and irradiated animals were compared to time matched controls. 1d: 1 day, 3d: 3 days, 1wk: I week, 3wk: 3 weeks; 6wk: 6 weeks, 6m: 6 months, C: control, R: radiation.

Figure 3. Western blot analysis illustrates the dynamic changes in stabilization of HIF-1α (A) and HIF-2α (B) proteins changes over time following irradiation, while HIF-3α (C) remains constant

Western blot bands were visualized and normalized based on α-tubulin expression. The relative intensities were measured and averaged within groups and irradiated animals were compared to time matched controls. 1d: 1 day, 3d: 3 days, 1wk: I week, 3wk: 3 weeks; 6wk: 6 weeks, 6m: 6 months, C: control, R: radiation.

Figure 3. Western blot analysis illustrates the dynamic changes in stabilization of HIF-1α (A) and HIF-2α (B) proteins changes over time following irradiation, while HIF-3α (C) remains constant

Western blot bands were visualized and normalized based on α-tubulin expression. The relative intensities were measured and averaged within groups and irradiated animals were compared to time matched controls. 1d: 1 day, 3d: 3 days, 1wk: I week, 3wk: 3 weeks; 6wk: 6 weeks, 6m: 6 months, C: control, R: radiation.

Figure 4. Representative Oligo GEArray hybridization images

The control for all of these time points shows a similar pattern. Arrows indicate gene spots with significant increases.

Figure 5. Expression of genes after AEOL10150 treatment

The amplified PCR fragments were visualized on 1.5% agarose gel containing 0.5 µg/ml ethidium bromide. The GAPDH and target gene were amplified in the same reaction. The top band shows a 1 kb GAPDH fragment and the bottom band shows the targeted gene fragment.