Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction Omid Azimzadeh,* ,† Wolfgang Sievert, ‡ Hakan Sarioglu, § Juliane Merl-Pham, § Ramesh Yentrapalli, † Mayur V. Bakshi, † Dirk Janik, ∥ Marius Ueffing, §,⊥ Michael J. Atkinson, †,# Gabriele Multhoff, ‡ and Soile Tapio † † Helmholtz Zentrum Mü nchen - German Research Centre for Environmental Health, Institute of Radiation Biology, Ingolstaedter Landstrasse 1, D-85764 Neuherberg, Germany ‡ Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universitä t Mü nchen, Munich, Germany and Clinical Cooperation Group (CCG) ‘‘Innate Immunity in Tumor Biology’’, Helmholtz Zentrum Mü nchen, Deutsches Forschungszentrum fü r Gesundheit und Umwelt, Ismaninger Strasse 22, D-81675 Munich, Germany § Helmholtz Zentrum Mü nchen, German Research Centre for Environmental Health, Research Unit Protein Science, Ingolstaedter Landstr. 1, D-85764 Neuherberg, Germany ∥ Helmholtz Zentrum Mü nchen, German Research Centre for Environmental Health, Institute of Pathology, Ingolstaedter Landstr. 1, D-85764 Neuherberg, Germany ⊥ Centre of Ophthalmology, University Medical Centre, Rö ntgenweg 11, D-72076 Tuebingen, Germany # Chair of Radiation Biology, Technical University of Munich, Ismaninger Strasse 22, D-81675 Munich, Germany * S Supporting Information ABSTRACT: Epidemiological data from radiotherapy patients show the damaging effect of ionizing radiation on heart and vasculature. The endothelium is the main target of radiation damage and contributes essentially to the development of cardiac injury. However, the molecular mechanisms behind the radiation-induced endothelial dysfunction are not fully understood. In the present study, 10-week-old C57Bl/6 mice received local X-ray heart doses of 8 or 16 Gy and were sacrificed after 16 weeks; the controls were sham-irradiated. The cardiac microvascular endothelial cells were isolated from the heart tissue using streptavidin-CD31-coated microbeads. The cells were lysed and proteins were labeled with duplex isotope-coded protein label methodology for quantification. All samples were analyzed by LC−ESI−MS/MS and Proteome Discoverer software. The proteomics data were further studied by bioinformatics tools and validated by targeted transcriptomics, immunoblotting, immunohistochemistry, and serum profiling. Radiation-induced endothelial dysfunction was characterized by impaired energy metabolism and perturbation of the insulin/IGF-PI3K-Akt signaling pathway. The data also strongly suggested premature endothelial senescence, increased oxidative stress, decreased NO availability, and enhanced inflammation as main causes of radiation-induced long-term vascular dysfunction. Detailed data on molecular mechanisms of radiation-induced vascular injury as compiled here are essential in developing radiotherapy strategies that minimize cardiovascular complications. KEYWORDS: ionizing radiation, proteomics, ICPL, insulin, PI3K, PPAR alpha, endothelial cell, endothelial cell dysfunction, heart, cardiovascular disease ■ INTRODUCTION Epidemiological studies indicate that high and moderate local doses (>0.5 Gy) of ionizing radiation to the heart increase the risk of cardiovascular disease (CVD). Adverse effects are observed in patients after thoracic radiotherapy for breast cancer, Hodgkin’s disease, or a number of childhood cancers. 1−4 Because of novel postoperative methods, 5 the heart dose from left- tangential radiotherapy has been reduced. However, certain parts of the heart still receive >20 Gy if the cancer is localized on the left thoracic side of the patient. 6 Because breast cancer is one of the most common tumors and the age of this patient group is relatively low, there is a high estimated risk of developing radiation-induced CVD later in life. 5 Therefore, deeper insight into the molecular mechanisms of CVD following irradiation of the heart is urgently needed. Human and animal data indicate the important role of vascular injury and endothelial dysfunction in the pathogenesis of radiation-induced CVD. 7−12 Endothelial dysfunction refers to a complex pathological condition that is characterized by a series of Received: November 3, 2014 Published: January 15, 2015 Article pubs.acs.org/jpr © 2015 American Chemical Society 1203 DOI: 10.1021/pr501141b J. Proteome Res. 2015, 14, 1203−1219