Kathryn Hudak

Mesenchymal stem cells inhibit cutaneous radiation‐induced fibrosis by suppressing chronic inflammation

Exposure to ionizing radiation (IR) can result in the development of cutaneous fibrosis, for which few therapeutic options exist. We tested the hypothesis that bone marrow‐derived mesenchymal stem cells (BMSC) would favorably alter the progression of IR‐induced fibrosis. We found that a systemic infusion of BMSC from syngeneic or allogeneic donors reduced skin contracture, thickening, and collagen deposition in a murine model. Transcriptional profiling with a fibrosis‐targeted assay demonstrated increased expression of interleukin‐10 (IL‐10) and decreased expression of IL‐1β in the irradiated skin of mice 14 days after receiving BMSC. Similarly, immunoassay studies demonstrated durable alteration of these and several additional inflammatory mediators. Immunohistochemical studies revealed a reduction in infiltration of proinflammatory classically activated CD80+ macrophages and increased numbers of anti‐inflammatory regulatory CD163+ macrophages in irradiated skin of BMSC‐treated mice. In vitro coculture experiments confirmed that BMSC induce expression of IL‐10 by activated macrophages, suggesting polarization toward a regulatory phenotype. Furthermore, we demonstrated that tumor necrosis factor‐receptor 2 (TNF‐R2) mediates IL‐10 production and transition toward a regulatory phenotype during coculture with BMSC. Taken together, these data demonstrate that systemic infusion of BMSC can durably alter the progression of radiation‐induced fibrosis by altering macrophage phenotype and suppressing local inflammation in a TNF‐R2‐dependent fashion. Stem Cells 2013;31:2231–2241

Quercetin inhibits radiation-induced skin fibrosis.

Radiation induced fibrosis of the skin is a late toxicity that may result in loss of function due to reduced range of motion and pain. The current study sought to determine if oral delivery of quercetin mitigates radiation-induced cutaneous injury. Female C3H/HeN mice were fed control chow or quercetin-formulated chow (1% by weight). The right hind leg was exposed to 35 Gy of X rays and the mice were followed serially to assess acute toxicity and hind leg extension. Tissue samples were collected for assessment of soluble collagen and tissue cytokines. Human and murine fibroblasts were subjected to clonogenic assays to determine the effects of quercetin on radiation response. Contractility of fibroblasts was assessed with a collagen contraction assay in the presence or absence of quercetin and transforming growth factor-β (TGF-β). Western blotting of proteins involved in fibroblast contractility and TGF-β signaling were performed. Quercetin treatment significantly reduced hind limb contracture, collagen accumulation and expression of TGF-β in irradiated skin. Quercetin had no effect on the radioresponse of fibroblasts or murine tumors, but was capable of reducing the contractility of fibroblasts in response to TGF-β, an effect that correlated with partial stabilization of phosphorylated cofilin. Quercetin is capable of mitigating radiation induced skin fibrosis and should be further explored as a therapy for radiation fibrosis.

Transforming Growth Factor Alpha is a Critical Mediator of Radiation Lung Injury

Radiation fibrosis of the lung is a late toxicity of thoracic irradiation. Epidermal growth factor (EGF) signaling has previously been implicated in radiation lung injury. We hypothesized that TGF-α, an EGF receptor ligand, plays a key role in radiation-induced fibrosis in lung. Mice deficient in transforming growth factor (TGF-α–/–) and control C57Bl/6J (C57-WT) mice were exposed to thoracic irradiation in 5 daily fractions of 6 Gy. Cohorts of mice were followed for survival (n ≥ 5 per group) and tissue collection (n = 3 per strain and time point). Collagen accumulation in irradiated lungs was assessed by Massons trichrome staining and analysis of hydroxyproline content. Cytokine levels in lung tissue were assessed with ELISA. The effects of TGF-α on pneumocyte and fibroblast proliferation and collagen production were analyzed in vitro. Lysyl oxidase (LOX) expression and activity were measured in vitro and in vivo. Irradiated C57-WT mice had a median survival of 24.4 weeks compared to 48.2 weeks for irradiated TGF-α–/– mice (P = 0.001). At 20 weeks after irradiation, hydroxyproline content was markedly increased in C57-WT mice exposed to radiation compared to TGF-α–/– mice exposed to radiation or unirradiated C57-WT mice (63.0, 30.5 and 37.6 μg/lung, respectively, P = 0.01). C57-WT mice exposed to radiation had dense foci of subpleural fibrosis at 20 weeks after exposure, whereas the lungs of irradiated TGF-α –/– mice were largely devoid of fibrotic foci. Lung tissue concentrations of IL-1β, IL-4, TNF-α, TGF-β and EGF at multiple time points after irradiation were similar in C57-WT and TGF-α–/– mice. TGF-α in lung tissue of C57-WT mice rose rapidly after irradiation and remained elevated through 20 weeks. TGF-α–/– mice had lower basal LOX expression than C57-WT mice. Both LOX expression and LOX activity were increased after irradiation in all mice but to a lesser degree in TGF-α–/– mice. Treatment of NIH-3T3 fibroblasts with TGF-α resulted in increases in proliferation, collagen production and LOX activity. These studies identify TGF-α as a critical mediator of radiation-induced lung injury and a novel therapeutic target in this setting. Further, these data implicate TGF-α as a mediator of collagen maturation through a TGF-β independent activation of lysyl oxidase.

Inhibition of radiation-induced skin fibrosis with imatinib.

Abstract Purpose: Dermal fibrosis is a disabling late toxicity of radiotherapy. Several lines of evidence suggest that overactive signaling via the Platelet-derived growth factor receptor-beta (PDGFR-β) and V-abl Abelson murine leukemia viral oncogene homolog 1 (cAbl) may be etiologic factors in the development of radiation-induced fibrosis. We tested the hypothesis that imatinib, a clinically available inhibitor of PDGFR-β, Mast/stem cell growth factor receptor (c-kit) and cAbl, would reduce the severity of dermal fibrosis in a murine model. Materials and methods: The right hind legs of female C3H/HeN mice were exposed to 35 Gy of X-rays. Cohorts of mice were maintained on chow formulated with imatinib 0.5 mg/g or control chow for the duration of the experiment. Bilateral hind limb extension was measured serially to assess fibrotic contracture. Immunohistochemistry and biochemical assays were used to evaluate the levels of collagen and cytokines implicated in radiation-induced fibrosis. Results: Imatinib treatment significantly reduced hind limb contracture and dermal thickness after irradiation. Immunohistochemical studies demonstrated a substantial reduction in PDGFR-β phosphorylation. We also observed reduced Transforming Growth factor-β (TGF-β) and collagen expression in irradiated skin of imatinib-treated mice, suggesting that imatinib may suppress the fibrotic process by interrupting cross-talk between these pathways. Conclusions: Taken together, these results support that imatinib may be a useful agent in the prevention and treatment of radiation-induced dermal fibrosis.

IL-13 is a therapeutic target in radiation lung injury

Pulmonary fibrosis is a potentially lethal late adverse event of thoracic irradiation. Prior research indicates that unrestrained TGF-β1 and/or type 2 cytokine-driven immune responses promote fibrosis following radiation injury, but the full spectrum of factors governing this pathology remains unclear. Interleukin 13 (IL-13) is a key factor in fibrotic disease associated with helminth infection, but it is unclear whether it plays a similar role in radiation-induced lung fibrosis. Using a mouse model, we tested the hypothesis that IL-13 drives the progression of radiation-induced pulmonary fibrosis. Irradiated lungs from wild-type c57BL/6NcR mice accumulated alternatively-activated macrophages, displayed elevated levels of IL-13, and extensive fibrosis, whereas IL-13 deficient mice were resistant to these changes. Furthermore, plasma from irradiated wild-type mice showed a transient increase in the IL-13 saturated fraction of the circulating decoy receptor IL-13Rα2. Finally, we determined that therapeutic neutralization of IL-13, during the period of IL-13Rα2 saturation was sufficient to protect mice from lung fibrosis. Taken together, our results demonstrate that IL-13 is a major regulator of radiation-induced lung injury and demonstrates that strategies focusing on IL-13 may be useful in screening for timely delivery of anti-IL-13 therapeutics.

Abstract 852: Enhanced expression of secretory clusterin/apolipoprotein J (sCLU) in pulmonary alveolar stem cells after ionizing radiation exposure

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ionizing radiation is commonly used in the treatment of broad range of malignancies. However radiation induces DNA damage and oxidative stress in malignant and normal cells and stimulates inflammation in irradiated tissues. These vents may lead to tumor cell death, but also to a range of side effects including self-limited acute toxicities, mild chronic symptoms, or severe organ dysfunction. Recently, we reported that accelerated senescence of type II pneumocytes, the alveolar stem cell, results in depletion of type II pneumocytes and precedes pulmonary fibrosis. Targeted isolated depletion of type II pneumocytes is known to result in fibrosis. To understand the underlying mechanisms between RIPF and senescence, we investigated the role of several molecules which were markedly changed in irradiated lungs with fibrogenic exposures. Secretory Clusterin/Apolipoprotein J (sCLU) is a heterodimeric disulfide-linked glycoprotein encoded by a single gene. sCLU expression is implicated in physiological processes including development, lipid transportation, differentiation, cellular senescence, and in many age-related diseases including neurodegeneration, vascular damage, diabetes and tumorigenesis. It was recently proposed that sCLU is a sensitive cellular biosensor of oxidative stress that functions to protect cells from the deleterious effects of free radicals and their derivatives. We investigated sCLU expression in primary murine pneumocytes after exposure to irradiation. Increased sCLU expression following irradiation was confirmed by immunocytochemical assays and quantitative real-time PCR in primary pneumocyte cultures. At 5 days after irradiation, sCLU expression colocalized with senescence-associated beta-galactosidase (SA-beta-gal) activity. To determine the temporal relationship between sCLU expression and SA-beta-gal activity, sCLU expression and SA-beta-galactosidase activity was measured in murine lung tissues at 2 and 4 weeks after radiation exposures. As observed in pneumocyte cultures, the expression of sCLU mRNA was significantly increased in murine lungs after fibrogenic irradiation, with staining colocalizing to cells staining positive for SA-beta-gal activity. IGF-1, known to induce sCLU expression, was positively correlated with the levels of sCLU in irradiated lungs. Inhibition of IGF-1 signaling reduced sCLU expression in primary pneumocytes exposed to fibrogenic irradiation. Collectively, these data suggest that sCLU may provide a marker or target of type II pneumocyte senescence after irradiation. Citation Format: Eunjoo Chung, Jason Horton, Ayla White, Bradly Scroggins, Kathryn Hudak, Deborah Citrin. Enhanced expression of secretory clusterin/apolipoprotein J (sCLU) in pulmonary alveolar stem cells after ionizing radiation exposure. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 852. doi:10.1158/1538-7445.AM2014-852

Biomarkers of radiation injury and response

Radiation injury is a continuum of complex biological responses. At the time of emergency, multiple methods of technologies are used to monitor radiation exposure levels and measure cellular damage. Identifying markers of exposure and finding unique biomarker profiles for toxicity in exposed individuals are complex tasks. The most useful markers are those that are most specific and quantitatively predictive of radiation-induced severity. In this chapter, we covered some of the available biomarkers including DNA/RNA, protein products, physiological markers, and metabolites. The objectives of this chapter are to describe common types of ionizing radiation, discuss the biological consequences and toxicities of exposure, provide information on different types biomarkers for monitoring radiation exposure and consider use of some promising biomarkers, especially as tools for diagnosis, prognosis and as targets for therapy.