Radiation-induced impairment in lung lymphatic vasculature

Abstract

Background: The lymphatic vasculature has been shown to play important roles in lung injury and repair, particularly in lung fibrosis. The effects of ionizing radiation on lung lymphatic vasculature have not been previously reported.

Methods and results: C57Bl/6 mice were immobilized in a lead shield exposing only the thoracic cavity, and were irradiated with a single dose of 14 Gy. Animals were sacrificed and lungs collected at different time points (1, 4, 8, and 16 weeks) following radiation. To identify lymphatic vessels in lung tissue sections, we used antibodies that are specific for lymphatic vessel endothelial receptor 1 (LYVE-1), a marker of lymphatic endothelial cells (LEC). To evaluate LEC cell death and oxidative damage, lung tissue sections were stained for LYVE-1 and with TUNEL staining, or 8-oxo-dG respectively. Images were imported into ImageJ v1.36b and analyzed. Compared to a non-irradiated control group, we observed a durable and progressive decrease in the density, perimeter, and area of lymphatic vessels over the study period. The decline in the density of lymphatic vessels was observed in both subpleural and interstitial lymphatics. Histopathologically discernible pulmonary fibrosis was not apparent until 16 weeks after irradiation. Furthermore, there was significantly increased LEC apoptosis and oxidative damage at one week post-irradiation that persisted at 16 weeks.

Conclusions: There is impairment of lymphatic vasculature after a single dose of ionizing radiation that precedes architectural distortion and fibrosis, suggesting important roles for the lymphatic circulation in the pathogenesis of the radiation-induced lung injury.

Figures

FIG. 1.

Decrease in lymphatic vessels after a single dose of radiation. One week after a single dose (14 Gy) of radiation, radiated lungs (B) show a marked decrease in immunoreactivity with anti-LYVE-1 antibodies (brown areas indicated by black arrows) compared to control nonradiated lungs (A). At 16 weeks post-radiation, the decrease in immunoreactivity with anti-LYVE-1 antibodies persisted as seen in lung tissue sections from nonradiated (C) and radiated animals (D). Areas of fibrosis (D, enclosed in blue dotted lines) observed 16 weeks after radiation were completely devoid of lymphatic vessels. (Scale bar: 100 μm). Images of the entire lung section were obtained. ImageJ software was used to measure the density, area, and perimeter of lymphatic vessels in tissue sections from radiated and control animals at the indicated time points (n=3 per group per time point). A statistically significant decrease in lymphatic vessel density (E), perimeter (F), and area (G) was observed; *p<0.05; **p<0.01; ***p<0.001, compared to time-matched controls by Student t test. A color version of this figure is available in the online article at www.liebertpub.com/lrb.

FIG. 2.

Morphometric analysis of lymphatic vessels in the subpleural and interstitial compartments. Schematic representation of subpleural and interstitial lymphatics (A). Lung tissue sections showing immunoreactivity with anti-LYVE-1 antibodies, with subpleural and interstitial lymphatic vessels highlighted in (B). (Scale bar: 100 μm). Significant changes in subpleural lymphatic density (C), perimeter (D), and area (E) were observed at the indicated time points following radiation exposure. Changes in interstitial lymphatic density (F), perimeter (G), and area (H) were observed as early as one week post-radiation (n=3 mice per group per time point); *p<0.05; **p<0.01; ***p<0.001, compared to time-matched controls by Student t test. A color version of this figure is available in the online article at www.liebertpub.com/lrb.

FIG. 3.

Age-related changes in lymphatic vessels. 9-week-old mice were allowed to age. Lung tissues were collected at indicated time points, and lymphatic vessels were identified with anti-LYVE-1 antibodies. Images of the entire lung section were obtained. ImageJ software was used to measure the density (A, D, and G), perimeter (B, E, and H), and area (C, F, and I) of total (A–C), subpleural (D–F), and interstitial (G–I) lymphatic vessels in lung tissue sections at the indicated time points (n=3 per group per time point). Results showed that between 10 weeks and 25 weeks of age, there was no significant change in total, subpleural, or interstitial lymphatic vessel density (A, D, and G). Throughout the study period, there was no significant change in subpleural lymphatic vessel perimeter (E) or area (F). A statistically significant age-related increase in the perimeter (B and H) and area ratio (C and I) of total and interstitial lymphatic vessels was observed; *p<0.05; **p<0.01, by 1-way ANOVA. (These data are different representations of the same data from control animals in Figs. 1 and 2. These graphs are meant to illustrate the maturation effects on lung lymphatic vessels).

FIG. 4.

Transient increase in blood capillaries after one dose of radiation. Lung tissue sections were immunostained with anti-VEGFR-2 antibodies (black arrows) to visualize blood capillaries. One week after a single dose of radiation, VEGFR-2 positive blood capillaries were increased (B) compared to control nonradiated lungs (A). At 16 weeks there were no significant differences in VEFGR-2 positive (black arrows) blood capillary density in nonradiated (C) compared to radiated lungs (D). Clusters of irregularly shaped capillaries were observed along the periphery of fibrotic lesions but were virtually absent in the fibrotic areas (D). (Scale bar: 25 μm). (E) Images of the entire lung section were obtained. ImageJ software was used to calculate the density of VEGFR-2 positive blood capillaries in tissue sections from radiated and control animals at the indicated time points (n=3 per group per time point); *p<0.05, compared to time-matched controls by Student t test. A color version of this figure is available in the online article at www.liebertpub.com/lrb.

FIG. 5.

Increased lymphatic endothelial cell death after single dose radiation. Representative images of lymphatic vessels (LYVE-1, green), and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL, red) staining of lung tissue from control and radiated lungs at one (A) and 16 weeks (B) after a single dose of radiation (14 Gy). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). (Scale bar: 100 μm). (C) Apoptotic cell index calculated as the number of TUNEL positive/LYVE-1 positive lymphatic endothelial cells (indicated by white arrows in the merged images in A and B) per field divided by the perimeter of lymphatic vessels in the same field. There was a significant increase in lymphatic endothelial cell apoptosis at one and 16 weeks in radiated versus control lungs (n=3 mice per group per time point); **p<0.01, compared to time-matched controls by Student t test. A color version of this figure is available in the online article at www.liebertpub.com/lrb.

FIG. 6.

Increased lung lymphatic vessel oxidative damage after single dose of radiation. Representative images of lymphatic vessels (LYVE-1, green) and 8-oxo-dG (8-oxo-dG, red) staining of lung tissue sections from control and radiated lungs at one (A) and 16 (B) weeks after a single dose radiation (14 Gy). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). (Scale bar: 100 μm). (C) The number of 8-oxo-dG positive/LYVE-1 positive lymphatic endothelial cells per field (white arrows in the merged images in A and B) was divided by the perimeter of lymphatic vessels in the same field. There was a significant increase in oxidative stress at one and 16 weeks post radiation (n=3 per group per time point); *p<0.05; ***p<0.001, compared to time-matched controls by Student t test. A color version of this figure is available in the online article at www.liebertpub.com/lrb.

FIG. 7.

Immunoreactive VEGF-C and VEGF-D in control and radiated lungs. Representative images of VEGF-C (A and B) and VEGF-D (C and D) immunostaining visualized with diaminobenzidine (DAB) at 16 weeks post-irradiation. VEGF-C immunoreactivity was observed in epithelial cells (red arrow) and alveolar macrophages (black arrow) in both control (A) and radiated lungs (B). VEGF-D immunoreactivity was observed in epithelial cells (red arrowhead) and alveolar macrophages (recognized by their morphology and presence in the alveolar space; black arrowhead) in both control (C) and radiated lungs (D). Fibrotic areas in the radiated mouse lung (red asterisk) exhibited immunoreactivity for VEGF-C (B) and relatively weak immunoreactivity for VEGF-D (D), whereas VEGF-D was expressed within interstitial cells in control lungs (identified by their localization at the basal membrane side of alveolar epithelial cells, blue arrowhead, C). No staining was observed when primary antibodies were replaced with normal IgG (E and F). (Scale bar: 100 μm). A color version of this figure is available in the online article at www.liebertpub.com/lrb.