Temporal Onset of Hypoxia and Oxidative Stress After Pulmonary Irradiation

Purpose: To investigate the temporal onset of hypoxia following irradiation, and to show how it relates to pulmonary vascular damage, macrophage accumulation, and the production of reactive oxygen species and cytokines. Our previous studies showed that tissue hypoxia in the lung after irradiation contributed to radiation-induced injury.

Methods and Materials: Female Fisher 344 rats were irradiated to the right hemithorax with a single dose of 28 Gy. Serial studies were performed up to 20 weeks following irradiation. Radionuclide lung-perfusion studies were performed to detect changes in pulmonary vasculature. Immunohistochemical studies were conducted to study macrophages, tissue hypoxia (carbonic anhydrase-9 marker), oxidative stress (8-hydroxy-2′-deoxyguanosine), and the expression of profibrogenic (transforming growth factor-β [TGF-β]) and proangiogenic (vascular endothelial growth factor [VEGF]) cytokines.

Results: Significant changes in lung perfusion along with tissue hypoxia were observed 3 days after irradiation. Significant oxidative stress was detected 1 week after radiation, whereas macrophages started to accumulate at 4 weeks. A significant increase in TGF-β expression was seen within 1 day after radiation, and for VEGF at 2 weeks after radiation. Levels of hypoxia, oxidative stress, and both cytokines continued to rise with time after irradiation. The steepest increase correlated with vast macrophage accumulation.

Conclusions: Early changes in lung perfusion, among other factors initiate, the development of hypoxia and chronic oxidative stress after irradiation. Tissue hypoxia is associated with a significant increase in the activation of macrophages and their continuous production of reactive oxygen species, stimulating the production of fibrogenic and angiogenic cytokines, and maintaining the development of chronic radiation-induced lung injury.

Introduction

Radiation-induced pulmonary toxicity is a significant cause of morbidity and mortality in patients treated for tumors in the thoracic region (1, 2). Apparent clinical, radiographic, and histologic changes caused by radiation usually develop within weeks to months after irradiation, and are well-documented (3, 4). However, the mechanisms underlying their pathogenesis are still uncertain. The classic linear “target cell” concept, referring exclusively to the depletion of clonogenic cells by irradiation, is insufficient to explain the pathogenesis of radiation-induced lung injury. In the last decade, it was well-established that a cascade of events on the cellular and molecular levels begins immediately after exposure to radiation, and proceeds during a period of clinically occult pulmonary injury (5, 6). Radiation activates various cellular signaling pathways that lead to the expression and activation of proinflammatory and profibrotic cytokines, vascular injury, and the activation of a coagulation cascade, leading to the development of edema, inflammatory responses, and the initiation of wound-healing processes (7, 8, 9, 10, 11). Multiple factors in this process have been investigated, but the main initiating events and driving forces in the perpetuation of radiation-induced lung injury are unknown, hampering the development of protective strategies and the causal treatment of late radiation sequelae, as well as the search for predictive markers.

Our previous studies showed that hypoxia develops in rodent lung tissue after radiation. and is associated with increased macrophage accumulation and activation, oxidative stress, and profibrogenic and proangiogenic cytokine activity contributing to radiation-induced pulmonary injury (12, 13, 14). In general, tissue hypoxia is considered a major signal in wound-healing and tissue-remodeling processes (15, 16). Another important element in tissue-remodeling processes, and specifically in fibrogenesis, is the balance of reactive oxygen species (ROS) and nitrogen species (RNS) (17). In our in vitro studies, we demonstrated that hypoxia elicits macrophages to produce higher levels of transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and superoxide, leading to enhanced oxidative stress (14). In addition, superoxide contributed to increased macrophage cytokine production, which was diminished by applying an antioxidant superoxide dismutase mimetic (14).

Hence, we hypothesize that hypoxia (and consequently, oxidative stress) is one of the driving forces in initiating and perpetuating radiation-induced lung injury. The goal of this study was to determine the temporal onset and course of hypoxia after irradiation of the right hemithorax of rats, and to determine how hypoxia correlates with vascular damage, macrophage accumulation, ROS, and cytokines, which are known to be involved in processes leading to both immediate and chronic lung-tissue injury.