Radiation therapy causes loss of dermal lymphatic vessels and interferes with lymphatic function by TGF-beta1-mediated tissue fibrosis

Abstract

Although radiation therapy is a major risk factor for the development of lymphedema following lymphadenectomy, the mechanisms responsible for this effect remain unknown. The purpose of this study was therefore to determine the effects of radiation on lymphatic endothelial cells (LECs) and lymphatic function. The tails of wild-type or acid sphingomyelinase (ASM)-deficient mice were treated with 0, 15, or 30 Gy of radiation and then analyzed for LEC apoptosis and lymphatic function at various time points. To analyze the effects of radiation fibrosis on lymphatic function, we determined the effects of transforming growth factor (TGF)-beta1 blockade after radiation in vivo. Finally, we determined the effects of radiation and exogenous TGF-beta1 on LECs in vitro. Radiation caused mild edema that resolved after 12-24 wk. Interestingly, despite resolution of tail edema, irradiated animals displayed persistent lymphatic dysfunction. Radiation caused loss of capillary lymphatics and was associated with a dose-dependent increase in LEC apoptosis. ASM-/- mice had significantly less LEC apoptosis; however, this finding did not translate to improved lymphatic function at later time points. Short-term blockade of TGF-beta1 function after radiation markedly decreased tissue fibrosis and significantly improved lymphatic function but did not alter LEC apoptosis. Radiation therapy decreases lymphatic reserve by causing depletion of lymphatic vessels and LECs as well as promoting soft tissue fibrosis. Short-term inhibition of TGF-beta1 activity following radiation improves lymphatic function and is associated with decreased soft tissue fibrosis. ASM deficiency confers LEC protection from radiation-induced apoptosis but does not prevent lymphatic dysfunction.

Fig. 1.

Radiation therapy causes severe, long-term lymphatic dysfunction. A: tail volume changes from baseline after irradiation with 0, 15, or 30 Gy. Although there was a significant, dose-dependent increase in tail volume 4 wk after radiation therapy (P < 0.008 and 0.002, respectively), these differences resolved in long-term follow-up (12, 24 wk). B: analysis of lymphatic function with lymphoscintigraphy 4, 12, and 24 wk after radiation with 0, 15, or 30 Gy. Decay-adjusted maximal uptake of ⁹⁹Tc-labeled sulfur colloid in the lymph nodes at the base of the tail after injection at the tip of the tail is presented. Note significant, dose-dependent impairment in lymph node uptake at the 4 wk time point (*P < 0.001 for both doses compared with nonirradiated control, #P < 0.05 for 15 Gy compared with 30 Gy at 4 wk after radiation therapy). Also note sustained and progressive lymphatic dysfunction in animals treated with 15 or 30 Gy and analyzed after 12 and 24 wk (*P < 0.001 for both doses). C: representative heat maps of ⁹⁹Tc radioisotope uptake in a nonirradiated animal (top) and an animal 24 wk after exposure to 30 Gy (middle). Red color indicates areas of most intense radioactivity, and yellow, green, and blue represent progressively lower activities. Arrowheads indicate the site of radiolabeled colloid near the tip of the tail, and arrows indicate uptake in the lymph node basin at the base of the tail. Note far greater uptake in the lymph nodes of the nonirradiated mouse at the conclusion of the study, indicating more efficient lymphatic transport. A picture of a mouse tail is presented for orientation at bottom.

Fig. 2.

Radiation therapy decreases number of dermal capillary lymphatic vessels and lymphatic endothelial cells (LECs). A: analysis of capillary lymphatic vessels in the skin after irradiation with 0, 15, or 30 Gy. Tissues were stained with podoplanin antibody, and lymphatic vessel counts were performed in 3–5 high-power field (HPF) sections in a minimum of 3 animals per group by 2 blinded reviewers. Vessel counts are presented as means ± SD. Note significant and sustained decrease in the number of podoplanin-positive luminal structures in irradiated tissue sections compared with nonirradiated control sections (*P < 0.05 compared with nonirradiated control). B: representative ×200 micrographs of tissue sections from a nonirradiated animal (0 Gy; top) and an animal 24 wk after a 30-Gy dose of radiation (30 Gy; bottom) stained immunohistochemically for podoplanin (arrow demonstrates positively stained lymphatic). C: analysis of the number of LECs/HPF using immunofluorescent staining for lymphatic vessel endothelial receptor-1 (LYVE-1). Cell counts were performed in 3–5 HPFs/animal in a minimum of 3 animals per time point/group by blinded reviewers and are presented as means ± SD. Again, note significant decrease in LEC counts in irradiated animals beginning 4 wk after irradiation (*P < 0.05 for all doses and time points compared with baseline; P < 0.001 at 24 wk) D: representative ×100 micrographs of sections stained for LYVE-1 (red) and DAPI nuclear counterstain (blue) in animal exposed to 0 Gy (top) or 30 Gy (bottom). Note grossly observed decreased number of LECs (red cells).

Fig. 3.

Radiation is associated with LEC apoptosis and promotes cellular senescence in vitro. A: identification of apoptotic LECs by colocalization of LYVE-1 and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL). Individual LECs were identified by LYVE-1 staining and counted in 3–5 HPFs in a minimum of 3 animals per time point/group by blinded reviewers. Percentage of apoptotic LECs was calculated by similarly counting the number of LYVE-1+, TUNEL+ cells and expressing this number as a function of total LEC number. Data are presented as means ± SD. Increased LEC apoptosis was noted as early as 4 h after radiation and peaked at the 10 h time point in a dose-dependent manner (*P < 0.01). B: representative immunofluorescent micrographs (×200) of colocalization of the LEC marker (red) with TUNEL (green) and DAPI nuclear stain (blue) 10 h after irradiation with 0, 15, or 30 Gy. Note the dose-dependent increase in the presence of TUNEL-positive nuclei. C: flow cytometric TUNEL analysis of isolated LECs irradiated in vitro demonstrated that LECs in this setting are relatively radioresistant, requiring a 30-Gy dose of radiation to induce significant apoptosis (*P < 0.0001). Data are presented as means ± SD of triplicate experiments. D: this finding was confirmed by immunofluorescence for annexin V and caspase-3. In the presented representative images annexin V is shown by red immunofluorescence, caspase-3 with green immunofluorescence, and DAPI nuclear counterstain in blue. Note the greater number of cells staining for both annexin V and caspase-3 in the 30-Gy-treated cells (bottom). E: radiation therapy induces LEC senescence 4 days after treatment, at doses that are much lower than those required to induce apoptosis. Cells were radiated with 0, 4, 8, or 12 Gy of ionizing radiation, and senescence was analyzed with β-galactosidase staining at pH 6. Positively stained (blue) cells were identified with light microscopy and quantified in 3–5 HPFs/treatment group. All experiments were performed in triplicate, and data are presented as means ± SD. Note a significant, dose-dependent increase in LEC senescence (*P < 0.001 for all compared with nonirradiated control). F: representative photomicrographs (×100) of β-galactosidase staining of nonirradiated (0 Gy; top) and irradiated (30 Gy; bottom) LECs.

Fig. 4.

Protection of LECs from radiation-induced apoptosis does not prevent induction of lymphatic dysfunction. A: identification of apoptotic LECs by colocalization of LYVE-1 (red) and TUNEL (green) in acid sphingomyelinase (ASM)−/− and wild-type (WT) mice (means ± SD). Note significant decrease in LEC apoptosis 10 h after radiation in ASM−/− mice (*P < 0.034) B: representative immunofluorescent micrographs (×200) of colocalization of the LEC marker (red) with TUNEL (green) and DAPI nuclear stain (blue) 10 h after irradiation with 15 Gy. C: lymphoscintigraphy of ASM −/− and WT mice 4 wk after 15 Gy of irradiation. Data represent mean maximal decay-adjusted uptake of a minimum of 3 animals per time point/group (not significant). D: scar index of tissue sections from ASM−/− and WT animals that received 15 Gy of irradiation 4 wk after radiation (not significant). E: representative ×200 micrographs of tissue sections from ASM−/− (top) or WT (bottom) mice 4 wk after radiation with 15 Gy. No differences are observed.

Fig. 5.

Radiation causes soft tissue fibrosis and increased transforming growth factor (TGF)-β1 activity. A: calculation of the scar index using polarized light microscopy of Sirius red-stained sections. Mean scar index was calculated from analysis of 3–5 sections/animal/group, and data are presented as means ± SD. Note significant and progressive increase in scar index after radiation with 15 or 30 Gy (*P < 0.04 for all compared with baseline except for the 15 Gy dose at 12 wk, which approached but did not achieve significance, P < 0.08). B: representative photomicrographs (×200) of nonirradiated (0 Gy; top) and irradiated (30 Gy; bottom) sections 24 wk after radiation. Note increased fibrosis in irradiated section as demonstrated by more red and orange birefringence. In contrast, note yellow-green birefringence of nonirradiated skin. C: radiation therapy increases the number of phosphorylated Smad3 (pSmad3)-positive cells in tissue sections beginning as early as 4 h after radiation and persisting as long as 24 wk later (*P < 0.03 for all compared with baseline). Sections were stained with pSmad3 antibodies, and the number of positively stained cells was assessed in 3–5 HPFs per section in a minimum of 3 animals per time point/group (mean ± SD). D: representative ×200 micrographs of pSmad3 immunohistochemical staining in nonirradiated (0 Gy; top) and irradiated (30 Gy; bottom) sections 4 h after radiation. Note that nonirradiated sections demonstrate virtually no positive dermal cells, while large numbers of positively stained cells are noted in the irradiated tissue section.

Fig. 6.

Treatment with LY-364947, a small-molecule inhibitor of TGF-β1 receptor (TGF-BR)I, effectively blocks TGF-β1 signaling. A and B: Western blot (A) and quantification (B) for TGF-β-induced (TGFBi) in vehicle- and LY-364947-treated animals 1 wk after irradiation with 15 Gy. Each lane represents protein isolated from an individual animal. Note marked reduction (nearly 5-fold decrease) in TGFBi protein expression in LY-364947-treated animals, indicating inhibition of TGF-β1 signaling (*P < 0.02). C: calculation of the number of pSmad3-stained cells in 3–5 sections/animal in a minimum of 3 animals per group 4 and 12 wk after irradiation with 15 Gy (mean ± SD). Note significant and persistent decrease in the number of pSmad3-positive cells in animals treated with LY-364947 for 1 wk (*P < 0.03). D: representative ×200 micrographs of tissue sections from animals treated with vehicle (top) or LY-364947 (bottom) for 1 wk after irradiation with 15 Gy. Tissue sections were harvested 12 wk after radiation therapy. Note marked decrease in the number of pSmad3-stained cells in the LY-364947-treated animals.

Fig. 7.

Short-term inhibition of TGF-β1 signaling reduces radiation-induced soft tissue fibrosis and lymphatic dysfunction. A: scar index of tissue sections from animals that received 15 Gy of irradiation and were treated with vehicle or LY-364947 for 1 wk after irradiation (mean ± SD scar index). Tissues were harvested 4 or 12 wk after radiation. Note marked decrease in scar index in LY-364947-treated animals compared with control animals at both time points evaluated (*P < 0.002, #P < 0.02). B: representative ×200 micrographs of tissue sections from animals treated with vehicle (top) or LY-364947 (bottom) 12 wk after radiation with 15 Gy. Note decreased scarring as represented by decreased red-orange and increased yellow-green birefringence in the LY-364947-treated section. C: tail volume measurements in animals treated with vehicle or LY-364947 4 or 12 wk after irradiation with 15 Gy (mean ± SD). Treatment with LY-364947 resulted in mild but nonsignificant reduction in tail edema after irradiation. D: lymphoscintigraphy of animals treated with vehicle or LY-364947 4 or 12 wk after 15 Gy of irradiation. Data represent mean maximal decay-adjusted uptake of a minimum of 3 animals per time point/group. Note statistically significant increased lymph node uptake in LY-364947-treated animals, particularly at the 4 wk time point (*P < 0.0001). Statistically significant improved lymphatic function was also noted at the 12 wk time point; however, this difference was less marked than the 4 wk analysis (#P < 0.03). E: representative heat maps of ⁹⁹Tc radioisotope uptake in mice irradiated with 15 Gy and treated with either vehicle (left) or LY-364947 (right) at 4 and 12 wk after irradiation. Red color indicates areas of most intense radioactivity, and yellow, green, and blue represent progressively lower activities. Arrowheads indicate the site of radiolabeled colloid near the tip of the tail, and arrows indicate uptake in the lymph node basin at the base of the tail. Note far greater uptake in the lymph nodes of animals treated with LY-364947, indicating more efficient lymphatic transport. F: quantification of the number of LECs in tissue sections from animals treated with vehicle or LY-364947 4 or 12 wk after 15 Gy of irradiation (mean ± SD). No significant differences were noted. G: identification of apoptotic LECs by colocalization of LYVE-1 and TUNEL (mean ± SD; not significant). H: representative immunofluorescent micrographs (×200) of colocalization of the LEC marker (red) with TUNEL (green) and DAPI nuclear stain (blue) 10 h after irradiation with 15 Gy. I: flow cytometric TUNEL analysis of isolated LECs demonstrating no induction of apoptosis in response to treatment with recombinant human (rh)TGF-β1 (10 ng/ml). Data are presented as means ± SD of triplicate experiments.

Fig. 7.

Short-term inhibition of TGF-β1 signaling reduces radiation-induced soft tissue fibrosis and lymphatic dysfunction. A: scar index of tissue sections from animals that received 15 Gy of irradiation and were treated with vehicle or LY-364947 for 1 wk after irradiation (mean ± SD scar index). Tissues were harvested 4 or 12 wk after radiation. Note marked decrease in scar index in LY-364947-treated animals compared with control animals at both time points evaluated (*P < 0.002, #P < 0.02). B: representative ×200 micrographs of tissue sections from animals treated with vehicle (top) or LY-364947 (bottom) 12 wk after radiation with 15 Gy. Note decreased scarring as represented by decreased red-orange and increased yellow-green birefringence in the LY-364947-treated section. C: tail volume measurements in animals treated with vehicle or LY-364947 4 or 12 wk after irradiation with 15 Gy (mean ± SD). Treatment with LY-364947 resulted in mild but nonsignificant reduction in tail edema after irradiation. D: lymphoscintigraphy of animals treated with vehicle or LY-364947 4 or 12 wk after 15 Gy of irradiation. Data represent mean maximal decay-adjusted uptake of a minimum of 3 animals per time point/group. Note statistically significant increased lymph node uptake in LY-364947-treated animals, particularly at the 4 wk time point (*P < 0.0001). Statistically significant improved lymphatic function was also noted at the 12 wk time point; however, this difference was less marked than the 4 wk analysis (#P < 0.03). E: representative heat maps of ⁹⁹Tc radioisotope uptake in mice irradiated with 15 Gy and treated with either vehicle (left) or LY-364947 (right) at 4 and 12 wk after irradiation. Red color indicates areas of most intense radioactivity, and yellow, green, and blue represent progressively lower activities. Arrowheads indicate the site of radiolabeled colloid near the tip of the tail, and arrows indicate uptake in the lymph node basin at the base of the tail. Note far greater uptake in the lymph nodes of animals treated with LY-364947, indicating more efficient lymphatic transport. F: quantification of the number of LECs in tissue sections from animals treated with vehicle or LY-364947 4 or 12 wk after 15 Gy of irradiation (mean ± SD). No significant differences were noted. G: identification of apoptotic LECs by colocalization of LYVE-1 and TUNEL (mean ± SD; not significant). H: representative immunofluorescent micrographs (×200) of colocalization of the LEC marker (red) with TUNEL (green) and DAPI nuclear stain (blue) 10 h after irradiation with 15 Gy. I: flow cytometric TUNEL analysis of isolated LECs demonstrating no induction of apoptosis in response to treatment with recombinant human (rh)TGF-β1 (10 ng/ml). Data are presented as means ± SD of triplicate experiments.

Fig. 8.

Radiation results in coexpression of lymphatic and fibroblast markers in capillary lymphatics, and this effect is inhibited by TGF-β1 blockade. A: representative ×100 (left) and ×250 (right) overlay images of tissue sections stained with podoplanin (brown) and α-smooth muscle actin (α-SMA; fluorescent green) obtained from animals radiated with 0 Gy (top), radiated with 15 Gy and treated with vehicle (middle), or radiated with 15 Gy and treated with LY-364947 (bottom). Note no colocalization of podoplanin and α-SMA in nonirradiated or irradiated/LY-364947-treated tissues. In contrast, note lymphatic vessel stained for both podoplanin and α-SMA in sections obtained from an animal treated with 15 Gy of irradiation and vehicle control. B: Western blot analysis of LECs grown in vitro and stimulated with 10 ng/ml rhTGF-β1 4, 24, and 72 h after initiation of stimulation. Fibroblast and endothelial-mesenchymal transition markers collagen I, N-cadherin, and fibroblast activation protein (FAP) are upregulated by stimulation. Conversely, LEC marker LYVE-1 and endothelial cell marker E-cadherin are greatly downregulated.