Development Of Radiation-induced Lung Injury Research Articles

STING facilitates the development of radiation-induced lung injury via regulating the PERK/eIF2α pathway

STING facilitates the development of radiation-induced lung injury via regulating the PERK/eIF2α pathway

Emetine dihydrochloride alleviated radiation-induced lung injury through inhibiting EMT.

Radiation-induced lung injury (RILI), divided into early radiation pneumonia (RP) and late radiation-induced pulmonary fibrosis (RIPF), is a common serious disease after clinical chest radiotherapy or nuclear accident, which seriously threatens the life safety of patients. There has been no effective prevention or treatment strategy till now. Epithelial-mesenchymal transition (EMT) is a key step in the occurrence and development of RILI. In this study, we demonstrated that emetine dihydrochloride (EDD) alleviated RILI through inhibiting EMT. We found that EDD significantly attenuated EMT-related markers, reduced Smad3 phosphorylation expression after radiation. Then, for the first time, we observed EDD alleviated lung hyperaemia and reduced collagen deposit induced by irradiation, providing protection against RILI. Finally, it was found that EDD inhibited radiation-induced EMT in lung tissues. Our study suggested that EDD alleviated RILI through inhibiting EMT by blocking Smad3 signalling pathways. In summary, our results indicated that EDD is a novel potential radioprotector for RILI.

The occurrence and development of radiation-induced lung injury after interstitial brachytherapy and stereotactic radiotherapy in SD rats

BackgroundTo compare the severity of radiation-induced lung injury (RILI) after the right lung of SD rats received interstitial brachytherapy and stereotactic radiotherapy (SBRT).MethodsRILI rat model was established using interstitial brachytherapy and SBRT methods, respectively. CT scan was performed to analyze the lung volume and the CT value difference between the left and right lungs in rats. Then the lung tissues were analyzed through H&E staining, peripheral blood was extracted to detect the expression levels of serum inflammatory cytokines, pro-fibrotic cytokines, and fibrotic-inhibiting cytokines by ELISA.ResultsThe difference between right and left lung CT values was significantly elevated in the SBRT group when compared with the control group and the interstitial brachytherapy group (P < 0.05). The IFN-γ expression in the interstitial brachytherapy group was significantly different from that in the SBRT group at week 1, 4, 8 and 16. Besides, the expressions of IL-2, IL-6 and IL-10 in SBRT group were significantly higher than that of interstitial brachytherapy group (P < 0.05). The TGF-β expression in interstitial brachytherapy group reached its peak with the increase of time from week 1 to week 16, and it was significantly lower than SBRT group (P < 0.05). The mortality rate in the SBRT group was 16.7%, which was significantly higher than that in the interstitial brachytherapy group.ConclusionThe treatment method of interstitial brachytherapy is considered as an effective and safe tool by reducing the side effects of radiotherapy and increasing the radiation dose of radiotherapy.

METTL3 Mediates Epithelial-Mesenchymal Transition by Modulating FOXO1 mRNA N6 -Methyladenosine-Dependent YTHDF2 Binding: A Novel Mechanism of Radiation-Induced Lung Injury.

The biological roles of epithelial-mesenchymal transition (EMT) in the pathogenesis of radiation-induced lung injury (RILI) have been widely demonstrated, but the mechanisms involved have been incompletely elucidated. N6 -methyladenosine (m6 A) modification, the most abundant reversible methylation modification in eukaryotic mRNAs, plays vital roles in multiple biological processes. Whether and how m6 A modification participates in ionizing radiation (IR)-induced EMT and RILI remain unclear. Here, significantly increased m6 A levels upon IR-induced EMT are detected both in vivo and in vitro. Furthermore, upregulated methyltransferase-like 3 (METTL3) expression and downregulated α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) expression are detected. In addition, blocking METTL3-mediated m6 A modification suppresses IR-induced EMT both in vivo and in vitro. Mechanistically, forkhead box O1 (FOXO1) is identified as a key target of METTL3 by a methylated RNA immunoprecipitation (MeRIP) assay. FOXO1 expression is downregulated by METTL3-mediated mRNA m6 A modification in a YTH-domain family 2 (YTHDF2)-dependent manner, which subsequently activates the AKT and ERK signaling pathways. Overall, the present study shows that IR-responsive METTL3 is involved in IR-induced EMT, probably by activating the AKT and ERK signaling pathways via YTHDF2-dependent FOXO1 m6 A modification, which may be a novel mechanism involved in the occurrence and development of RILI.

Gene Signatures for Latent Radiation-Induced Lung Injury Post X-ray Exposure in Mouse.

To investigate the X-ray-specific sensitive genes and potential signaling pathways involved in the latent period of radiation-induced lung injury (RILI) in mouse models. Mice were randomized into groups for whole thoracic irradiation with a single fraction of 20Gy X-ray or 12.5Gy carbon heavy ion. Lungs were harvested 3weeks after the irradiation, whole RNA was extracted and detected with the genome-wide transcriptional microarrays. Differentially expressed genes (DEGs) were calculated for each group and the X-ray-specific sensitive genes were determined, followed by the gene enrichment analysis of those DEGs exploring the potentially relevant signaling pathways and biological processes in latent RILI. Three weeks after irradiation, gene expression levels varied between groups. 76 up-regulated DEGs were determined with mice in the X-ray group and gene ontology enrichment analysis for biological process (GO-BP) obtained several processes which were associated with radiation reaction, mitotic, immune cell chemotaxis or metastasis, immune factors, p53 apoptosis, and tissue remodeling. KEGG signaling pathway enrichment analysis showed that those 76 up-regulated DEGs were enriched in p53, IL-17, FoXO, melanoma, and non-small-cell lung cancer signaling pathways. By comparing the DEGs in X-ray and heavy ion groups, X-ray-specific sensitive genes were determined, the top 10 genes were Adamts9, Aacs, Col6a2, Fdps, Mdk, Mcam, Stbd1, Lbh, Ak3, and Emid1. The expression level of the top 10 genes was found to be significantly higher in the X-ray group than in the control and heavy ion groups. Our research determined the X-ray-specific sensitive gene set in mice lungs after exposure to radiation. The gene set could be used as a genetic marker to suggest the latency of RILI. The enrichment analysis results suggested that the relevant signaling pathways were potentially involved in the development of RILI. Further validation of those genes and signaling pathways is needed to confirm these findings.

Potential Functions of the tRNA-Derived Fragment tRF-Gly-GCC Associated With Oxidative Stress in Radiation-Induced Lung Injury.

ObjectiveTransfer RNA-derived small RNAs (tsRNAs) are a novel type of non-coding RNA with various regulatory functions. They are associated with oxidative stress in various diseases, but their potential functions in radiation-induced lung injury (RILI) remain uncertain.MethodsTo explore the role of tsRNAs in RILI, we used X-rays to irradiate human bronchial epithelial cells and examined the expression profile of altered tsRNAs by RNA sequencing and bioinformatics analysis. Sequencing results were verified by qRT-PCR. tsRNA functions were explored using several methods, including CCK-8, reactive oxygen species (ROS) assays, cell transfection, and western blotting.ResultsEighty-six differentially expressed tRNA-derived fragments (tRFs) were identified: 64 were upregulated, and 22 were downregulated. Among them, the regulation of tRF-Gly-GCC, associated with oxidative stress, may be mediated by the inhibition of cell proliferation, promotion of ROS production, and apoptosis in the occurrence and development of RILI. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that the underlying molecular mechanism may involve the PI3K/AKT and the FOXO1 signaling pathways.ConclusionOur findings provide new insights into the molecular mechanisms underpinning RILI, advancing the clinical prevention and treatment of this disease.

Exploration of radiation-induced lung injury, from mechanism to treatment: a narrative review.

Background and ObjectiveRadiation-induced lung injury (RILI) is often found in thoracic tumor patients after thoracic radiation therapy, and influences patient quality of life. However, systematic exploration of RILI, including its molecular biological mechanisms and standardized treatment, has not yet been fully elucidated. The main objective of the narrative review was to describe the available evidence concerning RILI, from the biological mechanism to the clinical management. The underlying causes of RILI are multifactorial, including gene-level changes, the influence of signaling pathways, the convergence of various cells, as well as the expression of cytokines and chemokines. Based on the various mechanisms of RILI, several novel treatment strategies have been proposed and gradually applied in clinical practice.MethodsPubMed was used to collect articles about RILI from 1995 to 2021. The papers included clinical trials, reviews, as well as systematic reviews and meta-analyses. Based on the mechanism, diagnosis, and treatment, we synthesized and analyzed these papers to form a clearly logical and normative suggestion to guide clinical application.Key Content and FindingsRILI is a constantly developing and changing process including radiation pneumonitis and radiation lung fibrosis. Different kinds of inflammatory and immune cells such as macrophages, fibroblasts, and T cells play key roles in the development of RILI, and transforming growth factor-β (TGF-β), interleukin-4 (IL-4), IL-13, and interferon-γ (IFN-γ) are also participants in this process. At present, glucocorticoids are mainly therapeutic drugs for the early stage of RILI, and drugs treatment should abide early period, sufficient doses, and the individual principles. Other novel drugs such as Azithromycin also have been tried in clinical application. radiation dose, combination therapy modality, the condition of the tumor, and the age and underlying conditions of patients all effect the occurrence of RILI. Importantly, RILI has a relatively higher incidence in patients who received radiotherapy combined with other treatments, especially immunotherapy.ConclusionsThe occurrence of RILI after radiotherapy will greatly affect the prognosis and quality of life of patients. In clinical practice, early intervention, active treatment, and more effective therapeutic drugs should be found.

Radiation Induces Pulmonary Fibrosis by Promoting the Fibrogenic Differentiation of Alveolar Stem Cells.

The lung is a radiosensitive organ, which imposes limits on the therapeutic dose in thoracic radiotherapy. Irradiated alveolar epithelial cells promote radiation-related pneumonitis and fibrosis. However, the role of lung stem cells (LSCs) in the development of radiation-induced lung injury is still unclear. In this study, we found that both LSCs and LSC-derived type II alveolar epithelial cells (AECII) can repair radiation-induced DNA double-strand breaks, but the irradiated LSCs underwent growth arrest and cell differentiation faster than the irradiated AECII cells. Moreover, radiation drove LSCs to fibrosis as shown with the elevated levels of markers for epithelial-mesenchymal transition and myofibroblast (α-smooth muscle actin (α-SMA)) differentiation in in vitro and ex vivo studies. Increased gene expressions of connective tissue growth factor and α-SMA were found in both irradiated LSCs and alveolar cells, suggesting that radiation could induce the fibrogenic differentiation of LSCs. Irradiated LSCs showed an increase in the expression of surfactant protein C (SP-C), the AECII cell marker, and α-SMA, and irradiated AECII cells expressed SP-C and α-SMA. These results indicated that radiation induced LSCs to differentiate into myofibroblasts and AECII cells; then, AECII cells differentiated further into either myofibroblasts or type I alveolar epithelial cells (AECI). In conclusion, our results revealed that LSCs are sensitive to radiation-induced cell damage and may be involved in radiation-induced lung fibrosis.

KATP Channel Blocker Glibenclamide Prevents Radiation-Induced Lung Injury and Inhibits Radiation-Induced Apoptosis of Vascular Endothelial Cells by Increased Ca2+ Influx and Subsequent PKC Activation.

Radiation-induced lung injury (RILI) is a common and severe side effect of thoracic radiotherapy, which compromises patients' quality of life. Recent studies revealed that early vascular injury, especially microvascular damage, played a central role in the development of RILI. For this reason, early vascular protection is essential for RILI therapy. The ATP-sensitive K+ (KATP) channel is an ATP-dependent K+ channel with multiple subunits. The protective role of the KATP channel in vascular injury has been demonstrated in some published studies. In this work, we investigated the effect of KATP channel on RILI. Our findings confirmed that the KATP channel blocker glibenclamide, rather than the KATP channel opener pinacidil, remitted RILI, and in particular, provided protection against radiation-induced vascular injury. Cytology experiments verified that glibenclamide enhanced cell viability, increased the potential of proliferation after irradiation and attenuated radiation-induced apoptosis. Involved mechanisms included increased Ca2+ influx and PKC activation, which were induced by glibenclamide pretreatment. In conclusion, the KATP channel blocker glibenclamide remitted RILI and inhibited the radiation-induced apoptosis of vascular endothelial cells by increased Ca2+ influx and subsequent PKC activation.

Metabolic Risk Factors for the Development of Radiation Pneumonitis in Stage III Non-Small Cell Lung Cancer (NSCLC)

Radiation pneumonitis (RP) is a dose-limiting toxicity associated with radiotherapy in the treatment of non-small cell lung cancer (NSCLC). Several studies have linked diabetes as a risk factor for development of radiation-induced lung injury and statin medications as a protective parameter. We aimed to further investigate these findings and explore other metabolic risk factors for the development of RP. After IRB approval, we retrospectively evaluated 162 patients with stage III NSCLC treated with definitive chemoradiation at our institution between 2001 and 2014. Chemotherapy was delivered as couplet therapy with the majority of patients receiving carboplatin-paclitaxel (141/162, 87.2%). Patient information including age, gender, comorbidities, medications, serum glucose values, toxicities, disease, and treatment parameters were collected from patients’ medical records. Serum glucose values spanning from 90 days prior to 90 days post-radiation therapy (n=2870) were collected. We then categorized values into pre-, during, and post-radiotherapy; defined as within 90 days prior to, during, and within 90 days post-radiotherapy. Patients were then stratified into groups achieving serum glucose values ≥150, ≥175, and ≥200 mg/dL at each of the preceding time points. Chi-Squared analyses were used to analyze the effect categorical variables had on the development of RP in our study population. Parameters achieving significance were selected for analysis against pulmonary toxicity grade. We further used a binary logistic regression model to assess how continuous variables may affect the development of RP. The median prescribed dose was 72 Gy (range= 54-84). The overall rate of RP was 17.9% (n=29). In our dataset, 3/46 (6.5%) diabetic patients developed RP vs. 22/101 (21.8%) patients without diabetes (p=0.022). The rate of radiation-induced lung injury in insulin users was 3/42 (7.1%) vs. 22/105 (21.0%) in non-users (p=0.044). Both diabetes (2.2% vs. 14.9%, p=0.022) and insulin (2.4% vs. 14.3%, p=0.036) were associated with lower incidence of RP requiring medical intervention (grade ³2). Diabetes was further correlated with reduced incidence of grade ³3 RP (0% vs. 9.1%, p=0.037). Serum glucose values at the aforementioned time points were not associated with RP. Neither statins nor other diabetic medications correlated with RP risk. Gross tumor volume (GTV), planning target volume (PTV), radiation technique, radiation dose, dose per fraction, and treatment duration were not significant predictors of our primary endpoints. Finally, patients who developed RP had similar rates of mortality, loco-regional recurrence, and distant metastasis to those without. In contrast to prior data, we identify both diabetes and insulin therapy as protective factors against the development of RP in the treatment of NSCLC. These results should be confirmed by subsequent high-quality prospective studies.

Establishment and effect evaluation of a variety of radiation-induced lung injury animal models

Radiation-induced lung injury (RILI) is a common complication after radiotherapy for thoracic tumors. It is one of the most urgent problems to explore the optimal animal model and evaluate the effect for basic research and drug intervention along with the extensive studies of radiation pneumonia. By reviewing the literatures published in recent 10 years, the selection of irradiation site, determination of irradiation dosage, irradiation method and effect evaluation were statistically compared among different RILI animal models, aiming to explore a stable method of establishing the animal model with RILI and identify a definite effect mechanism. It provides a reliable approach for basic research and drug development to prevent and mitigate the incidence and development of RILI. Key words: Radiation-induced lung injury; Animal model; Dose volume relationship; Model evaluation

Naringenin Ameliorates Radiation-Induced Lung Injury by Lowering IL-1β Level.

Radiation-induced lung injury (RILI) is the main complication of radiotherapy for thoracic malignancies. Since naringenin, a potent immune-modulator, has been found to relieve bleomycin-induced lung fibrosis by restoring the balance of disordered cytokines, we sought to determine whether naringenin would mitigate RILI and to investigate the underlying mechanism. Animals received fractionated irradiation in the thoracic area to induce RILI. Enzyme-linked immunosorbent assay and MILLIPLEX assays were used for serum and bronchoalveolar lavage fluid for cytokine analyses, hematoxylin and eosin staining for pathologic changes, and Masson trichrome staining for determination of lung fibrosis. Interleukin (IL)-1β was found significantly elevated after thoracic irradiation and it triggered production of profibrotic tumor growth factor β both in vivo and in vitro, suggesting the vital role of in IL-1β in the development of RILI. Furthermore, we found that naringenin was able to ameliorate RILI through downregulation of IL-1β and restoration of the homeostasis of inflammatory factors. Our results demonstrated that naringenin could serve as a potent immune-modulator to ameliorate RILI. More importantly, we suggest that a new complementary strategy of maintaining the homeostasis of inflammatory factors combined with radiation could improve the efficacy of thoracic radiotherapy.

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

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. 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 β-galactosidase activity in lung and primary pneumocytes. 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. 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.

Abstract 1803: A truncated Plasminogen Activator Inhibitor-1 protein protects from pulmonary fibrosis mediated by irradiation in a murine model

Abstract Background: Fibrosis can develop as a late side effect of radiation exposure in a variety of tissues, including lung and skin. Late radiation injury and fibrosis are characterized by parenchymal cell depletion, inflammation, senescence, fibroblast proliferation, and excessive deposition of collagen. Plasminogen activator inhibitor 1 (PAI-1) is a critical mediator of cellular senescence and fibrin stabilization, and its expression is increased in experimental fibrosis models. We sought to determine if inhibition of PAI-1 signaling with a recombinant truncated protein (rPAI-123) would protect from the development of radiation-induced lung injury. Methods: C57Bl/6 mice received intraperitoneal injections of rPAI-123 (5.4 μg/kg/day) or vehicle (PBS) for 18 weeks beginning two days prior to radiation exposure. Cohorts of mice treated with rPAI-123 or vehicle were exposed to thoracic irradiation in 5 daily fractions of 6 Gy (RT), and followed for survival (n = 8 per group) and tissue collection (n = 3 per each time point). Histologic changes in irradiated lungs were evaluated by Masson-Trichrome staining at 19 weeks after RT. Senescence was assessed with staining for beta-galactosidase activity in lung tissue and primary pneumocytes. To define the roles of rPAI-123 in the fibroproliferative response, cell proliferation (MTT assay) and collagen deposition (Hydroxyproline assay) were examined in a mouse fibroblast cell line in vitro. Results: Administration of rPAI-123 increased C57Bl/6 mice survival from 37.5% to 62.5% at 19 weeks after radiation exposure. At 19 weeks after irradiation, hydroxyproline content was markedly decreased in mice received rPAI-123 compared to mice received vehicle (RT+rPAI-123: 56.2±10.79, RT+vehicle: 84.97±2.98, μg/lung respectively, p = 0.001 between RT+vehicle and RT+rPAI-123). C57Bl/6 mice exposed to RT+vehicle had dense foci of subplueral fibrosis at 19 weeks, whereas the lungs of mice exposed to RT+rPAI-123 were largely devoid of fibrotic foci. Cellular senescence in response to radiation was significantly decreased by rPAI-123 treatment in primary type2 pneumocyte culture (2-fold reduction at 5 days after RT, p = 0.036), and in lung tissues (&amp;gt;2-fold reduction at 4, 8, and 16 weeks after RT, p&amp;lt;0.001 at each time point). Treatment of NIH-3T3 fibroblasts with rPAI-123 resulted in decreased collagen production, but had no effect on proliferation.Conclusions: These studies identify that rPAI-123 has a novel protection mechanism against radiation-induced fibrosis in murine lungs due to its ability to reduce senescence in type2 pneumocytes, and the potential to be an effective therapy option for radiation induced fibrosis. Citation Format: Eunjoo Chung, Ayla White, Bradley T. Scroggins, Grace B. McKay-Corkum, Mary Jo Mulligan-Kehoe, Deborah E. Citrin. A truncated Plasminogen Activator Inhibitor-1 protein protects from pulmonary fibrosis mediated by irradiation in a murine model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1803. doi:10.1158/1538-7445.AM2015-1803

Abstract 3308: Enhanced expression of galectin-3 isassociated with the alternative activation of macrophages and development oflung fibrosis following radiation

Abstract Radiotherapy is a major treatment approach to non-operable lung cancer patients. Unfortunately, radiation-induced lung injury occurs in 5-20% of lung cancer patients following radiotherapy. Radiation-induced lung fibrosis contributes to an increased risk of death or a decreased quality of life. The mechanism(s) for the development of radiation-induced lung fibrosis is not fully understood, and no interventions to prevent/ameliorate it have proven successful. Macrophages are the first line of defense for airways and alveolar surfaces and play a key role in the initiation and orchestration of inflammatory responses. Two distinct macrophage subsets have been identified; classically activated macrophages (M1) and alternatively activated macrophages (M2). Increased numbers of activated M2 macrophages have been seen in human fibrotic conditions and rodent models of fibrosis. However, the mechanism(s) driving the persistent activation of M2 macrophages and how this leads to the development of fibrosis remains undefined. Recent studies indicate that galectin-3 (gal-3) is potentially a key regulator of M2 macrophage activation; inhibition of gal-3 expression prevents M2 macrophage activation. In this paper, we show elevated numbers of Ym-1 positive alveolar macrophages in the irradiated lung. Increased expression of gal-3 cells was found in the fibrotic lung, which was associated with fibrotic lesions. M2 macrophages were the majority of cells that expressed gal-3. In our in vitro studies, we found that knocking-down gal-3 using Mission lentiviral transduction particles prevented radiation-induced increases in the expression of YM-1 in Raw 264.7 macrophage. Mouse recombinant gal-3 stimulated mouse lung fibroblast cell proliferation and expression of α-SMA. These data suggest that gal-3 appears to regulate alternative activation of M2 macrophage and play an important role in the development of radiation-induced lung injury. Citation Format: Weiling Zhao, Jing-Hua Zhang, Ingrid J. Lopes. Enhanced expression of galectin-3 isassociated with the alternative activation of macrophages and development oflung fibrosis following radiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3308. doi:10.1158/1538-7445.AM2015-3308

Role of matrix metalloproteinases in radiation-induced lung injury in alveolar epithelial cells of Bama minipigs.

Radiation-induced lung injury (RILI) is a common complication associated with thoracic radiotherapy. The aim of the present study was to investigate the effects of a single 15-Gy dose of right-thoracic lung irradiation on the expression levels of matrix metalloproteinases (MMPs) and other proteins in the alveolar epithelial type II (AE2) cells of Bama minipigs. All minipigs received either right-thoracic irradiation or sham irradiation under anesthesia, and were sacrificed at 4, 8, 12 or 24 weeks after irradiation. Collagen deposition was measured using Massons trichrome staining. Surfactant protein A (SP-A), transforming growth factor β1 (TGFβ1), MMP2, MMP9, vimentin and E-cadherin protein expression levels were evaluated using western blot analysis, and the MMP2 and MMP9 gelatinase activities were tested using gelatin zymography. SP-A and α-smooth muscle actin (α-SMA) co-localization was visualized using double immunofluorescence staining. At each time-point following irradiation, a significant increase in TGFβ1, α-SMA, MMP2, MMP9 and vimentin protein expression levels and MMP2 and MMP9 gelatinase activity were observed in the irradiated lungs compared with the sham-irradiated controls. By contrast, SP-A and E-cadherin protein expression levels decreased in a time-dependent manner post-irradiation. SP-A and α-SMA co-localization was observed in irradiated alveolar epithelial cells. These data demonstrate that E-cadherin, SP-A, MMP2 and MMP9 may function as sensitive predictors of RILI. Epithelial-mesenchymal transition (EMT) occurs in the irradiated lungs of Bama minipigs, and MMP2 and MMP9 may contribute to EMT in AE2 cells by regulating TGFβ1. Therefore, EMT may serve a crucial function in the development of RILI.

Hyperpolarized 3 He pulmonary functional magnetic resonance imaging prior to radiation therapy

Radiation-induced lung injury (RILI) is the primary dose-limiting toxicity for radiation therapy of the lung, and although the effects of radiation dose on RILI development have been well characterized, the influence of chronic obstructive pulmonary disease (COPD) on the development of RILI and other outcomes is not well understood. The purpose of this small pilot study was to evaluate the relationship between hyperpolarized (3)He magnetic resonance imaging (MRI) measurements of COPD with RILI and 12-month survival in lung cancer patients undergoing radical radiotherapy and to evaluate the feasibility of pulmonary functional MRI as an image guidance∕planning tool for radiation therapy. Fifteen non-small cell and small cell lung cancer patients underwent pulmonary function tests, x-ray computed tomography (CT), and hyperpolarized (3)He MRI prior to radical radiation therapy (≥60 Gy). Conventional thoracic (1)H and hyperpolarized (3)He MRI were acquired to generate ventilation defect percent and the apparent diffusion coefficient for the ipsilateral and contralateral lungs independently. CT was acquired postradiation therapy and qualitatively evaluated for radiological evidence of RILI and 12-month survival was reported. Hyperpolarized (3)He MRI measurements of COPD classified 10∕15 subjects with contralateral lung COPD (CLC), and five subjects without COPD [contralateral lung normal (CLN)]. Of the 10 subjects with CLC, only four had a previous clinical diagnosis of COPD. CT images were acquired postradiation therapy for 13 subjects, and for eight (62%) of these there was qualitative evidence of RILI, including 5∕9 CLC and 3∕4 CLN subjects. The one-year survival was 2∕10 for CLC and 3∕5 for CLN subjects. In this small pilot study, we report the use of (3)He MRI to stratify lung cancer patients based on MRI evidence of COPD and showed that comorbid COPD was present in the majority of lung cancer subjects stratified for radiation therapy. Lung cancer patients with imaging evidence of COPD did not have an increased incidence of RILI compared to patients without COPD. However, preliminary data presented here indicated that one-year survival in COPD subjects was lower than expected based on previously published survival rates, which may have implications for radiation therapy in lung cancer patients with comorbid COPD.

Does Pre-Treatment SPECT-Based Respiratory Ventilation and Perfusion Phenotype Predict for Development of Radiation-Induced Lung Injury?

Does Pre-Treatment SPECT-Based Respiratory Ventilation and Perfusion Phenotype Predict for Development of Radiation-Induced Lung Injury?

Irradiation of Varying Volumes of Rat Lung to Same Mean Lung Dose: a Little to a Lot or a Lot to a Little?

To investigate whether irradiating small lung volumes with a large dose or irradiating large lung volumes with a small dose, given the same mean lung dose (MLD), has a different effect on pulmonary function in laboratory animals. WAG/Rij/MCW male rats were exposed to single fractions of 300 kVp X-rays. Four treatments, in decreasing order of irradiated lung volume, were administered: (1) whole lung irradiation, (2) right lung irradiation, (3) left lung irradiation, and (4) irradiation of a small lung volume with four narrow beams. The irradiation times were chosen to accumulate the same MLD of 10, 12.5, or 15 Gy with each irradiated lung volume. The development of radiation-induced lung injury for < or =20 weeks was evaluated as increased breathing frequency, mortality, and histopathologic changes in the irradiated and control rats. A significant elevation of respiratory rate, which correlated with the lung volume exposed to single small doses (> or =5 Gy), but not with the MLD, was observed. The survival of the rats in the whole-lung-irradiated group was MLD dependent, with all events occurring between 4.5 and 9 weeks after irradiation. No mortality was observed in the partial-volume irradiated rats. The lung volume irradiated to small doses might be the dominant factor influencing the loss of pulmonary function in the rat model of radiation-induced lung injury. Caution should be used when new radiotherapy techniques that result in irradiation of large volumes of normal tissue are used for the treatment of lung cancer and other tumors in the thorax.

Small Molecular Inhibitor of Transforming Growth Factor-β Protects Against Development of Radiation-Induced Lung Injury

To determine whether an anti-transforming growth factor-beta (TGF-beta) type 1 receptor inhibitor (SM16) can prevent radiation-induced lung injury. One fraction of 28 Gy or sham radiotherapy (RT) was administered to the right hemithorax of Sprague-Dawley rats. SM16 was administered in the rat chow (0.07 g/kg or 0.15 g/kg) beginning 7 days before RT. The rats were divided into eight groups: group 1, control chow; group 2, SM16, 0.07 g/kg; group 3, SM16, 0.15 g/kg; group 4, RT plus control chow; group 5, RT plus SM16, 0.07 g/kg; group 6, RT plus SM16, 0.15 g/kg; group 7, RT plus 3 weeks of SM16 0.07 g/kg followed by control chow; and group 8, RT plus 3 weeks of SM16 0.15 g/kg followed by control chow. The breathing frequencies, presence of inflammation/fibrosis, activation of macrophages, and expression/activation of TGF-beta were assessed. The breathing frequencies in the RT plus SM16 0.15 g/kg were significantly lower than the RT plus control chow from Weeks 10-22 (p <0.05). The breathing frequencies in the RT plus SM16 0.07 g/kg group were significantly lower only at Weeks 10, 14, and 20. At 26 weeks after RT, the RT plus SM16 0.15 g/kg group experienced a significant decrease in lung fibrosis (p = 0.016), inflammatory response (p = 0.006), and TGF-beta1 activity (p = 0.011). No significant reduction was found in these measures of lung injury in the group that received SM16 0.7 g/kg nor for the short-course (3 weeks) SM16 at either dose level. SM16 at a dose of 0.15 g/kg reduced functional lung damage, morphologic changes, inflammatory response, and activation of TGF-beta at 26 weeks after RT. The data suggest a dose response and also suggest the superiority of long-term vs. short-term dosing.