PPM1A as a key target of the application of Jiawei­Maxing­Shigan decoction for the attenuation of radiation­induced epithelial­mesenchymal transition in type II alveolar epithelial cells.

Radiation­induced lung tissue injury is an important reason for the limited application of radiotherapy on thoracic malignancies. Previously, we reported that administration of Jiawei­Maxing­Shigan decoction (JMSD) attenuated the radiation­induced epithelial­mesenchymal transition (EMT) in alveolar epithelial cells (AECs) via TGF­ß/Smad signaling. The present study aimed to examine the role of protein phosphatase Mg2+/Mn2+­dependent 1A (PPM1A) in the anti­EMT activity of JMSD on AECs. The components in the aqueous extract of JMSD were identified by high­performance liquid chromatography coupled with electrospray mass spectrometry. Primary rat type II AECs were treated with radiation (60Co γ­ray at 8 Gy) and JMSD­medicated serum. PPM1A was overexpressed and knocked down in the AECs via lentivirus transduction and the effects of JMSD administration on the key proteins related to TGF­ß1/Smad signaling were measured by western blotting. It was found that radiation decreased the PPM1A expression in the AECs and JMSD­medicated serum upregulated the PPM1A expressions in the radiation­induced AECs. PPM1A overexpression increased the E­cadherin level but decreased the phosphorylated (p­)Smad2/3, vimentin and α­smooth muscle actin (α­SMA) levels in the AECs. By contrast, the PPM1A knockdown decreased the E­cadherin level and increased the p­Smad2/3, vimentin and α­SMA levels in the AECs and these effects could be blocked by SB431542 (TGF­ß1/Smad signaling inhibitor). JMSD administration increased the E­cadherin level and decreased the p­Smad2/3, vimentin and α­SMA levels in the AECs; however, these effects could be blocked by siPPM1A­2. In conclusion, PPM1A is a key target of JMSD administration for the attenuation of the radiation­induced EMT in primary type II AECs via the TGF­ß1/Smad pathway.

IGF-1 Receptor Signaling Regulates Type II Pneumocyte Senescence and Resulting Macrophage Polarization in Lung Fibrosis.

PURPOSE: Type II pneumocyte (alveolar epithelial cells type II [AECII]) senescence has been implicated in the progression of lung fibrosis. The capacity of senescent cells to modulate pulmonary macrophages to drive fibrosis is unexplored. Insulin-like growth factor-1 receptor (IGF-1R) signaling has been implicated as a regulator of senescence and aging. METHODS AND MATERIALS: Mice with an AECII-specific deletion of IGF-1R received thoracic irradiation (n ≥ 5 per condition), and the effect of IGF-1R deficiency on radiation-induced AECII senescence and macrophage polarization to an alternatively activated phenotype (M2) was investigated. IGF-1R signaling, macrophage polarization, and senescence were evaluated in surgically resected human lung (n = 63). RESULTS: IGF-1R deficient mice demonstrated reduced AECII senescence (senescent AECII/field; intact: 7.25% ± 3.5% [mean ± SD], deficient: 2.75% ± 2.8%, P = .0001), reduced accumulation of M2 macrophages (intact: 24.7 ± 2.2 cells/field, deficient: 15.5 ± 1.2 cells/field, P = .0086), and fibrosis (hydroxyproline content; intact: 71.9 ± 21.7 µg/lung, deficient: 31.7 ± 7.9, P = .0485) after irradiation. Senescent AECII enhanced M2 polarization in a paracrine fashion (relative Arg1 mRNA, 0 Gy: 1.0 ± 0.4, 17.5 Gy: 7.34 ± 0.5, P < .0001). Evaluation of surgical samples from patients treated with chemoradiation demonstrated increased expression of IGF-1 (unirradiated: 10.2% ± 4.9% area, irradiated: 15.1% ± 11.5%, P = .0377), p21 (unirradiated: 0.013 ± 0.02 histoscore, irradiated: 0.084 ± 0.09 histoscore, P = .0002), IL-13 (unirradiated: 13.7% ± 2.8% area, irradiated: 21.7% ± 3.8%, P < .0001), and M2 macrophages in fibrotic regions relative to nonfibrotic regions (unirradiated: 11.4 ± 12.2 CD163 + cells/core, irradiated: 43.1 ± 40.9 cells/core, P = .0011), consistent with findings from animal models of lung fibrosis. CONCLUSIONS: This study demonstrates that senescent AECII are necessary for the progression of pulmonary fibrosis and serve as a targetable, chronic stimuli for macrophage activation in fibrotic lung.

ß-Catenin/Lin28/let-7 regulatory network determines type II alveolar epithelial stem cell differentiation phenotypes following thoracic irradiation.

The contribution of type II alveolar epithelial stem cells (AEC II) to radiation-induced lung fibrosis (RILF) is largely unknown. Cell differentiation phenotypes are determined by the balance between Lin28 and lethal-7 microRNA (let-7 miRNA). Lin28 is activated by ß-catenin. The aim of this study was to track AEC II phenotypes at different phases of injury following thoracic irradiation and examine the expression of ß-catenin, Lin28 and let-7 to identify their role in AEC II differentiation. Results showed that coexpression of prosurfactant protein C (proSP-C, an AEC II biomarker) and HOPX (homeobox only protein X, an AEC I biomarker) or vimentin (a differentiation marker) was detected in AEC II post-irradiation. The protein expression levels of HOPX and proSP-C were significantly downregulated, but vimentin was significantly upregulated following irradiation. The expression of E-cadherin, which prevents ß-catenin from translocating to the nucleus, was downregulated, and the expression of ß-catenin and Lin28 was upregulated after irradiation (P < 0.05 to P < 0.001). Four let-7 miRNA members (a, b, c and d) were upregulated in irradiated lungs (P < 0.05 to P < 0.001), but let-7d was significantly downregulated at 5 and 6 months (P < 0.001). The ratios of Lin28 to four let-7 members were low during the early phase of injury and were slightly higher after 2 months. Intriguingly, the Lin28/let-7d ratio was strikingly increased after 4 months. We concluded that ß-catenin contributed to RILF by promoting Lin28 expression, which increased the number of AEC II and the transcription of profibrotic molecules. In this study, the downregulation of let-7d miRNA by Lin28 resulted in the inability of AEC II to differentiate into type I alveolar epithelial cells (AEC I).

Glucosamine protects against radiation-induced lung injury via inhibition of epithelial-mesenchymal transition.

Radiotherapy is one of the most important treatments for chest tumours. Although there are plenty of strategies to prevent damage to normal lung tissues, it cannot be avoided with the emergence of radiation-induced lung injury. The purpose of this study was to investigate the potential radioprotective effects of glucosamine, which exerted anti-inflammatory activity in joint inflammation. In this study, we found glucosamine relieved inflammatory response and structural damages in lung tissues after radiation via HE staining. Then, we detected the level of epithelial-mesenchymal transition marker in vitro and in vivo, which we could clearly observe that glucosamine treatment inhibited epithelial-mesenchymal transition. Besides, we found glucosamine could inhibit apoptosis and promote proliferation of normal lung epithelial cells in vitro caused by radiation. In conclusion, our data showed that glucosamine alleviated radiation-induced lung injury via inhibiting epithelial-mesenchymal transition, which indicated glucosamine could be a novel potential radioprotector for radiation-induced lung injury.

12-Lipoxygenase is a Critical Mediator of Type II Pneumocyte Senescence, Macrophage Polarization and Pulmonary Fibrosis after Irradiation.

Radiation-induced pulmonary fibrosis (RIPF) is a chronic, progressive complication of therapeutic irradiation of the thorax. It has been suggested that senescence of type II pneumocytes (AECIIs), an alveolar stem cell, plays a role in the development of RIPF through loss of replicative reserve and via senescent AECII-driven release of proinflammatory and profibrotic cytokines. Within this context, we hypothesized that arachidonate 12-lipoxygenase (12-LOX) is a critical mediator of AECII senescence and RIPF. Treatment of wild-type AECIIs with 12S-hydroxyeicosateraenoic acid (12S-HETE), a downstream product of 12-LOX, was sufficient to induce senescence in a NADPH oxidase 4 (NOX4)-dependent manner. Mice deficient in 12-LOX exhibited reduced AECII senescence, pulmonary collagen accumulation and accumulation of alternatively activated (M2) macrophages after thoracic irradiation (5 × 6 Gy) compared to wild-type mice. Conditioned media from irradiated or 12S-HETE-treated primary pneumocytes contained elevated levels of IL-4 and IL-13 compared to untreated pneumocytes. Primary macrophages treated with conditioned media from irradiated AECII demonstrated preferential M2 type polarization when AECIIs were derived from wild-type mice compared to 12-LOX-deficient mice. Together, these data identified 12-LOX as a critical component of RIPF and a therapeutic target for radiation-induced lung injury.

IGFBP7 contributes to epithelial-mesenchymal transition of HPAEpiC cells in response to radiation.

Radiation-induced lung injury (RILI) frequently occurs in patients with thoracic malignancies. In response to radiation, alveolar epithelial cells (AEC) undergo epithelial-mesenchymal transition (EMT) and contribute to the pathogenesis of RILI. Insulin-like growth factor binding protein 7 (IGFBP7) is reported as a downstream mediator of transforming growth factor-ß1 (TGF-ß1) pathway, which plays a crucial role in radiation-induced EMT. In the present study, the levels of IGFBP7 and TGF-ß1 were simultaneously increased in experimental RILI models and radiation-treated AEC (human pulmonary alveolar epithelial cells [HPAEpic]). The expression of IGFBP7 in radiation-treated HPAEpic cells was obviously inhibited by the specific inhibitor of TGF-ß receptor antagonist SB431542 and TGF-ß1 neutralizing antibody, and time-dependently enhanced by TGF-ß1 treatment. Moreover, IGFBP7 knockdown significantly attenuated the effects of radiation on morphology change, cell migration, expression of EMT-related markers (E-cadherin, α-SMA, and Vimentin), and phosphorylation of extracellular-signal-regulated kinase (ERK). The effects of IGFBP7 overexpression on the expression of EMT-related markers were partially reversed by the ERK inhibitor PD98059. In conclusion, IGFBP7, was enhanced by TGF-ß1, may be involved in radiation-induced EMT of AEC via the ERK signaling pathway, thus contributing to the pathogenesis of RILI.

Baicalin alleviates radiation-induced epithelial-mesenchymal transition of primary type II alveolar epithelial cells via TGF-ß and ERK/GSK3ß signaling pathways.

BACKGROUND: Radiation therapy is commonly used to treat thoracic malignancies. However, it may lead to severe lung pneumonitis and ultimately fibrosis. Irradiation has been reported to increase epithelial-mesenchymal transition (EMT) of type II alveolar epithelial cells (AEC), which play an important role in pulmonary fibrosis. The transforming growth factor-ß (TGF-ß) and ERK/glycogen synthase kinase 3ß (GSK3ß) pathways are critically involved in radiation-induced EMT. In the present study, we investigated whether baicalin was a novel therapeutic candidate for radiation-induced EMT in type II AEC. METHODS: Primary type II AEC were isolated and treated with 60Co γ-rays and a series doses of baicalin (2µM, 10µM and 50µM). The ultrastructure and morphology changes were observed by transmission electron microscopy and optical microscopy, respectively. Protein expression was determined by western blotting analysis. Immunofluorescence staining was performed to detect the nuclear translocation of Snail. RESULTS: After irradiation, type II AEC displayed a mesenchymal-like morphology accompanied by a decrease in E-cadherin expression, an increase in the expression of Vimentin and α-SMA. Nuclear translocation of Snail, the activation of TGF-ß/Smad pathway, and the inactivation of GSK3ß were prominent in radiation-treated cells. Baicalin significantly attenuated the effects of radiation on type II AEC. CONCLUSIONS: Baicalin may a useful radioprotective agent through suppressing the EMT of type II AEC.

Photobiomodulation therapy improves both inflammatory and fibrotic parameters in experimental model of lung fibrosis in mice.

Lung fibrosis (LF) is a chronic and progressive lung disease characterized by pulmonary parenchyma progressive lesion, inflammatory infiltration, and interstitial fibrosis. It is developed by excessive collagen deposition and other cellular matrix components, resulting in severe changes in the alveolar architecture. Considering the absence of effective treatment, the aim of this study was to investigate the effect of photobiomodulation therapy (PBMT) on the development of PF. For this purpose, we used C57BL6 mice subjected to induction of LF by bleomycin administration (1.5 U/kg) by orotracheal route and, after 14 days of the induction, mice were treated with PBMT applied to the thorax 1×/day for 8 days (wavelength 660 ± 20 nm, power 100 mW, radiant exposure 5 J/cm2, irradiance 33.3 mW/cm2, spot size 2.8cm2, total energy 15 J, time of irradiation: 150 s) and inflammatory and fibrotic parameters were evaluated with or without PBMT. Our results showed that PBMT significantly reduced the number of inflammatory cells in the alveolar space, collagen production, interstitial thickening, and static and dynamic pulmonary elastance. In addition, we observed reduced levels of IL-6 e CXCL1/KC released by pneumocytes in culture as well as reduced level of CXCL1/KC released by fibroblasts in culture. We can conclude that the PBMT improves both inflammatory and fibrotic parameters showing a promising therapy which is economical and has no side effects.

The Cellular and Molecular Mechanism of Radiation-Induced Lung Injury.

The lung is one of several moderately radiosensitive organs. Radiation-induced lung injury (RILI), including acute radiation pneumonitis and chronic radiation-induced pulmonary fibrosis, occurs most often in radiotherapy of lung cancer, esophageal cancer, and other thoracic cancers. Clinical symptoms of RILI include dry cough, shortness of breath, chest pain, fever, and even severe respiratory failure and death. The occurrence of RILI is a complex process that includes a variety of cellular and molecular interactions which ultimately leads to large fibroblast accumulation, proliferation, and differentiation, resulting in excessive extracellular matrix deposits, causing pulmonary fibrosis. The progress that has been made in recent years in the understanding of cellular and molecular mechanisms of RILI is summarized in this review.

Novel Double-Hit Model of Radiation and Hyperoxia-Induced Oxidative Cell Damage Relevant to Space Travel.

Spaceflight occasionally requires multiple extravehicular activities (EVA) that potentially subject astronauts to repeated changes in ambient oxygen superimposed on those of space radiation exposure. We thus developed a novel in vitro model system to test lung cell damage following repeated exposure to radiation and hyperoxia. Non-tumorigenic murine alveolar type II epithelial cells (C10) were exposed to >95% O2 for 8 h only (O2), 0.25 Gy ionizing γ-radiation (IR) only, or a double-hit combination of both challenges (O2 + IR) followed by 16 h of normoxia (ambient air containing 21% O2 and 5% CO2) (1 cycle = 24 h, 2 cycles = 48 h). Cell survival, DNA damage, apoptosis, and indicators of oxidative stress were evaluated after 1 and 2 cycles of exposure. We observed a significant (p < 0.05) decrease in cell survival across all challenge conditions along with an increase in DNA damage, determined by Comet analysis and H2AX phosphorylation, and apoptosis, determined by Annexin-V staining, relative to cells unexposed to hyperoxia or radiation. DNA damage (GADD45α and cleaved-PARP), apoptotic (cleaved caspase-3 and BAX), and antioxidant (HO-1 and Nqo1) proteins were increased following radiation and hyperoxia exposure after 1 and 2 cycles of exposure. Importantly, exposure to combination challenge O2 + IR exacerbated cell death and DNA damage compared to individual exposures O2 or IR alone. Additionally levels of cell cycle proteins phospho-p53 and p21 were significantly increased, while levels of CDK1 and Cyclin B1 were decreased at both time points for all exposure groups. Similarly, proteins involved in cell cycle arrest was more profoundly changed with the combination challenges as compared to each stressor alone. These results correlate with a significant 4- to 6-fold increase in the ratio of cells in G2/G1 after 2 cycles of exposure to hyperoxic conditions. We have characterized a novel in vitro model of double-hit, low-level radiation and hyperoxia exposure that leads to oxidative lung cell injury, DNA damage, apoptosis, and cell cycle arrest.

Geranylgeranylacetone alleviates radiation-induced lung injury by inhibiting epithelial-to-mesenchymal transition signaling.

Radiation-induced lung injury (RILI) involves pneumonitis and fibrosis, and results in pulmonary dysfunction. Moreover, RILI can be a fatal complication of thoracic radiotherapy. The present study investigated the protective effect of geranylgeranlyacetone (GGA), an inducer of heat shock protein (HSP)70, on RILI using a C57BL/6 mouse model of RILI developing 6 months subsequent to exposure to 12.5 Gy thoracic radiation. GGA was administered 5 times orally prior and subsequent to radiation exposure, and the results were assessed by histological analysis and western blotting. The results show that late RILI was alleviated by GGA treatment, possibly through the suppression of epithelial­to­mesenchymal transition (EMT) marker expression. Based on histological examination, orally administered GGA during the acute phase of radiation injury not only significantly inhibited pro­surfactant protein C (pro­SPC) and vimentin expression, but also preserved E­cadherin expression 6 months after irradiation­induced injury of the lungs. GGA induced HSP70 and inhibited EMT marker expression in L132 human lung epithelial cells following IR. These data suggest that the prevention of EMT signaling is a key cytoprotective effect in the context of RILI. Thus, HSP70­inducing drugs, such as GGA, could be beneficial for protection against RILI.

Truncated Plasminogen Activator Inhibitor-1 Protein Protects From Pulmonary Fibrosis Mediated by Irradiation in a Murine Model.

PURPOSE: To determine whether the delivery of recombinant truncated plasminogen activator inhibitor-1 (PAI-1) protein (rPAI-1(23)) would protect from the development of radiation-induced lung injury. METHODS AND MATERIALS: C57Bl/6 mice received intraperitoneal injections of rPAI-1(23) (5.4 µg/kg/d) or vehicle for 18 weeks, beginning 2 days before irradiation (IR) (5 daily fractions of 6 Gy). Cohorts of mice were followed for survival (n=8 per treatment) and tissue collection (n=3 per treatment and time point). Fibrosis in lung was assessed with Masson-Trichrome staining and measurement of hydroxyproline content. Senescence was assessed with staining for ß-galactosidase activity in lung and primary pneumocytes. RESULTS: Hydroxyproline content in irradiated lung was significantly reduced in mice that received rPAI-1(23) compared with mice that received vehicle (IR+vehicle: 84.97 µg/lung; IR+rPAI-1(23): 56.2 µg/lung, P=.001). C57Bl/6 mice exposed to IR+vehicle had dense foci of subpleural fibrosis at 19 weeks, whereas the lungs of mice exposed to IR+rPAI-1(23) were largely devoid of fibrotic foci. Cellular senescence was significantly decreased by rPAI-1(23) treatment in primary pneumocyte cultures and in lung at multiple time points after IR. CONCLUSIONS: These studies identify that rPAI-1(23) is capable of preventing radiation-induced fibrosis in murine lungs. These antifibrotic effects are associated with increased fibrin metabolism, enhanced matrix metalloproteinase-3 expression, and reduced senescence in type 2 pneumocytes. Thus, rPAI-1(23) is a novel therapeutic option for radiation-induced fibrosis.

Radiation induced lung injury: prediction, assessment and management.

Radiation induced lung injury has long been considered a treatment limiting factor for patients requiring thoracic radiation. This radiation induced lung injury happens early as well as late. Radiation induced lung injury can occur in two phases viz. early (<6 months) when it is called radiation pneumonitis and late (>6 months) when it is called radiation induced lung fibrosis. There are multiple factors that can be patient, disease or treatment related that predict the incidence and severity of radiation pneumonitis. Radiation induced damage to the type I pneumocytes is the triggering factor to initiate such reactions. Over the years, radiation therapy has witnessed a paradigm shift in radiation planning and delivery and successfully reduced the incidence of lung injury. Radiation pneumonitis is usually a diagnosis of exclusion. Steroids, ACE inhibitors and pentoxyphylline constitute the cornerstone of therapy. Radiation induced lung fibrosis is another challenging aspect. The pathophysiology of radiation fibrosis includes continuing inflammation and microvascular changes due to pro-angiogenic and pro- fibrogenic stimuli resembling those in adult bronchiectasis. General supportive management, mobilization of airway secretions, anti-inflammatory therapy and management of acute exacerbations remains the treatment option. Radiation induced lung injury is an inevitable accompaniment of thoracic radiation.

An experimental model-based exploration of cytokines in ablative radiation-induced lung injury in vivo and in vitro.

INTRODUCTION: Stereotactic ablative radiotherapy is a newly emerging radiotherapy treatment method that, compared with conventionally fractionated radiation therapy (CFRT), allows an ablative dose of radiation to be delivered to a confined area around a tumor. The aim of the present study was to investigate the changes of various cytokines that may be involved in ablative radiation-induced lung injury in vitro and in vivo. METHODS: In the in vivo study, ablative-dose radiation was delivered to a small volume of the left lung of C3H/HeJCr mice using a small-animal irradiator. The levels of 24 cytokines in the peripheral blood were tested at several time points after irradiation. For the in vitro study, three mouse cell types (type II pneumocytes, alveolar macrophages, and fibroblasts) known to play important roles in radiation-induced pneumonitis and lung fibrosis were analyzed using a co-culture system. RESULTS: In the in vivo study, we found obvious patterns of serum cytokine changes depending on the volume of tissue irradiated (2-mm vs. 3.5-mm collimator). Only the levels of 3 cytokines increased with the 2-mm collimator at the acute phase (1-2 weeks after irradiation), while the majority of cytokines were elevated with the 3.5-mm collimator. In the in vitro co-culture system, after the cells were given an ablative dose of irradiation, the levels of five cytokines (GM-CSF, G-CSF, IL-6, MCP-1, and KC) increased significantly in a dose-dependent manner. CONCLUSIONS: The cytokine levels in our radiation-induced lung injury model showed specific changes, both in vivo and in vitro. These results imply that biological studies related to ablative-dose small-volume irradiation should be investigated using the corresponding experimental models rather than on those simulating large-volume CFRT.

Biological effects of passive versus active scanning proton beams on human lung epithelial cells.

The goal was to characterize differences in cell response after exposure to active beam scanning (ABS) protons compared to a passive delivery system. Human lung epithelial (HLE) cells were evaluated at various locations along the proton depth dose profile. The dose delivered at the Bragg peak position was essentially identical (∼4 Gy) with the two techniques, but depth dose data showed that ABS resulted in lower doses at entry and more rapid drop-off after the peak. Average dose rates for the passive and ABS beams were 1.1 Gy/min and 5.1 Gy/min, respectively; instantaneous dose rates were 19.2 Gy/min and 2,300 Gy/min (to a 0.5 × 0.5 mm(2) voxel). Analysis of DNA synthesis was based on (3)H-TdR incorporation. Quantitative real-time polymerase chain reaction (RT-PCR) was done to determine expression of genes related to p53 signaling and DNA damage; a total of 152 genes were assessed. Spectral karyotyping and analyses of the Golgi apparatus and cytokines produced by the HLE cells were also performed. At or near the Bragg peak position, ABS protons resulted in a greater decrease in DNA synthesis compared to passively delivered protons. Genes with >2-fold change (P < 0.05 vs. 0 Gy) after passive proton irradiation at one or more locations within the Bragg curve were BTG2, CDKN1A, IFNB1 and SIAH1. In contrast, many more genes had >2-fold difference with ABS protons: BRCA1, BRCA2, CDC25A, CDC25C, CCNB2, CDK1, DMC1, DNMT1, E2F1, EXO1, FEN1, GADD45A, GTSE1, IL-6, JUN, KRAS, MDM4, PRC1, PTTG1, RAD51, RPA1, TNF, WT1, XRCC2, XRCC3 and XRCC6BP1. Spectral karyotyping revealed numerous differences in chromosomal abnormalities between the two delivery systems, especially at or near the Bragg peak. Percentage of cells staining for the Golgi apparatus was low after exposure to passive and active proton beams. Studies such as this are needed to ensure patient safety and make modifications in ABS delivery, if necessary.

Role of type II pneumocyte senescence in radiation-induced lung fibrosis.

BACKGROUND: Radiation is a commonly delivered therapeutic modality for cancer. The causes underlying the chronic, progressive nature of radiation injury in the lung are poorly understood. METHODS: C57Bl/6NCr mice were exposed to thoracic irradiation (n = 3 per dose and time point for tissue collection). Microarray analysis of gene expression from irradiated murine lung was performed using one-way analysis of variance with post hoc Scheffe analysis. Senescence and type II airway epithelial cell (AECII) count were assayed in irradiated murine lung tissue (n = 3 per condition). Irradiated mice were treated with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase (NOX), and fibrosis was assessed by collagen assays. All statistical tests were two-tailed. RESULTS: Gene expression in lung tissue from mice irradiated to 17.5 Gy clustered with that of aged unirradiated mice. Only fibrogenic exposures led to AECII senescence (0 Gy: 0.66% ± 0.67%; 5 Gy: 4.5% ± 1.19%; 17.5 Gy: 18.7% ± 3.05; P = .007) and depletion (0 Gy: 2.89 per alveolus ± 0.26; 5 Gy: 2.41 ± 0.19; 17.5 Gy: 1.6 ± 0.14; P < .001) at 30 weeks. Treatment of irradiated mice with DPI for 16 weeks markedly reduced collagen accumulation (5×6 Gy: 57.26 µg/lung ± 9.91; 5×6 Gy ± DPI: 36.54µg/lung ± 4.39; P = .03) and AECII senescence (5×6 Gy: 37.61% ± 4.82%; 5×6 Gy ± DPI: 12.38% ± 2.78; P < .001). CONCLUSIONS: These studies identify senescence as an important process in AECII in vivo and indicate that NOX is a critical mediator of radiation-induced AECII senescence and pulmonary fibrosis.

Effects of thoracic irradiation on pulmonary endothelial compared to alveolar type-II cells in fibrosis-prone C57BL/6NTac mice.

BACKGROUND/AIM: Thoracic irradiation results in an acute inflammatory response, latent period, and late fibrosis. Little is known about the mechanisms involved in triggering late radiation fibrosis. MATERIALS AND METHODS: Thoracic irradiated fibrosis prone C57BL/6NTac mice were followed for detectable mRNA transcripts in isolated lung cells and micro-RNA in whole-tissues, and the effect of administration of water-soluble oxetanyl sulfoxide MMS350 was studied. Marrow stromal cell motility in medium from fibrotic-phase explanted pulmonary endothelial and alveolar type-II cells was measured. RESULTS: RNA and micro-RNA expression in lung correlated with fibrosis. MMS350 reduced pro-fibrotic gene expression in both endothelial and alveolar type-II cells in irradiated mice. Conditioned medium from irradiated cells did not alter cell motility in vitro. CONCLUSION: These studies should facilitate identification of potential new drug targets for ameliorating irradiation-induced pulmonary fibrosis.

Oxidative stress mediates radiation lung injury by inducing apoptosis.

PURPOSE: Apoptosis in irradiated normal lung tissue has been observed several weeks after radiation. However, the signaling pathway propagating cell death after radiation remains unknown. METHODS AND MATERIALS: C57BL/6J mice were irradiated with 15 Gy to the whole thorax. Pro-apoptotic signaling was evaluated 6 weeks after radiation with or without administration of AEOL10150, a potent catalytic scavenger of reactive oxygen and nitrogen species. RESULTS: Apoptosis was observed primarily in type I and type II pneumocytes and endothelium. Apoptosis correlated with increased PTEN expression, inhibition of downstream PI3K/AKT signaling, and increased p53 and Bax protein levels. Transforming growth factor-ß1, Nox4, and oxidative stress were also increased 6 weeks after radiation. Therapeutic administration of AEOL10150 suppressed pro-apoptotic signaling and dramatically reduced the number of apoptotic cells. CONCLUSION: Increased PTEN signaling after radiation results in apoptosis of lung parenchymal cells. We hypothesize that upregulation of PTEN is influenced by Nox4-derived oxidative stress. To our knowledge, this is the first study to highlight the role of PTEN in radiation-induced pulmonary toxicity.

[Biological effect of (60)Cogamma ray on alveolar type II cells and interstitial cells of alveoliar septum in radiation pulmonary injury].

AIM: To explore the biological effect of (60)Cogamma ray on alveolar type II cells and interstitial cells of alveoliar septum. METHODS: Alveolar type II cells(AT-II) and interstitial cells including interstitial macrophages and fibroblasts were irradiated by 0, 3, 5, 7 Gy of gamma ray respectively. The effect of irradiation on AT-II proliferation was observed by argentation against nucleus. The activity of MMP-2, -9 in supernatants from AT-II and interstitial cells after irradiation was determined by zymography. The levels of TGF-beta1 and collagen type IV in supernatant from interstitial cells after irradiation were measured by ELISA. RESULTS: The nucleolus number of AT-II was increased with the increase of irradiation dose and group 7 Gy reached the highest level. The activity of MMP-2, -9 in supernatant from AT-II after irradiation increased at first and then decreased gradually. The activity of MMP-2, -9 and the content of TGF-beta1 in interstitial cells increased step by step, but collagen type IV decreased at first and then increased. CONCLUSION: AT-II, macrophages and fibroblasts are all involved in pulmonary invalid remodeling course in early radiation pulmonary injury, which is related to the initiation of pulmonary fibrosis in late period.