Article pubs.acs.org/jpr
Radiation-Induced Endothelial Inflammation Is Transferred via the Secretome to Recipient Cells in a STAT-Mediated Process Jos Philipp,† Omid Azimzadeh,† Vikram Subramanian,† Juliane Merl-Pham,‡ Donna Lowe,§ Daniela Hladik,† Nadine Erbeldinger,∥ Svetlana Ktitareva,∥ Claudia Fournier,∥ Michael J. Atkinson,† Ken Raj,§ and Soile Tapio*,†
J. Proteome Res. 2017.16:3903-3916. Downloaded from pubs.acs.org by WESTERN UNIV on 09/24/18. For personal use only.
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Helmholtz Zentrum München - German Research Center for Environmental Health GmbH, Institute of Radiation Biology, D-85764 Neuherberg, Germany ‡ Helmholtz Zentrum München - German Research Centre for Environmental Health, Research Unit Protein Science, D-80939 Munich, Germany § Biological Effects Department, Centre for Radiation, Chemicals and Environmental Hazards, Public Health England, OX11 0RQ Chilton, United Kingdom ∥ GSI Helmholtz Zentrum für Schwerionenforschung, 64291 Darmstadt, Germany S Supporting Information *
ABSTRACT: Radiation is the most common treatment of cancer. Minimizing the normal tissue injury, especially the damage to vascular endothelium, remains a challenge. This study aimed to analyze direct and indirect radiation effects on the endothelium by investigating mechanisms of signal transfer from irradiated to nonirradiated endothelial cells by means of secreted proteins. Human coronary artery endothelial cells (HCECest2) undergo radiation-induced senescence in vitro 14 days after exposure to 10 Gy X-rays. Proteomics analysis was performed on HCECest2 14 days after irradiation with X-ray doses of 0 Gy (control) or 10 Gy using label-free technology. Additionally, the proteomes of control and radiation-induced secretomes, and those of nonirradiated HCECest2 exposed for 24 h to secreted proteins of either condition were measured. Key changes identified by proteomics and bioinformatics were validated by immunoblotting, ELISA, bead-based multiplex assays, and targeted transcriptomics. The irradiated cells, their secretome, and the nonirradiated recipient cells showed similar inflammatory response, characterized by induction of interferon type I-related proteins and activation of the STAT3 pathway. These data indicate that irradiated endothelial cells may adversely affect nonirradiated surrounding cells via senescence-associated secretory phenotype. This study adds to our knowledge of the pathological background of radiation-induced cardiovascular disease. KEYWORDS: senescence-associated secretory phenotype, X-ray irradiation, MHC-I class, proteomics, STAT, cardiovascular disease
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INTRODUCTION The vascular endothelium is a monolayer of cells lining all blood vessels in the body.1 Well-functioning endothelial cells act in a paracrine, endocrine, and autocrine manner to modulate blood fluidity, inflammation, immune response, and vascular tone.2 With aging, endothelial cells may enter senescence, an early pathophysiological state hallmarking cardiovascular disease (CVD).3 In spite of losing their replicative potential, senescent endothelial cells stay in a metabolically active state.4,5 They secrete a defined pattern of proteins comprising proinflammatory cytokines, chemokines, growth factors, and proteases, altogether called “senescence-associated secretory phenotype” (SASP).6 SASP is known to influence cell differentiation, cancer growth, cancer invasion, and promotion of endothelial cell invasion.4,7−9 It has been suggested that the SASP operates by activating interferon-related pathways, © 2017 American Chemical Society
associated with the release of interferon-inducible (IFI) proteins.10 SASP is known to spread inflammatory response and senescence to surrounding tissues11,12 and to contribute to age-dependent diseases such as CVD.5,13 High and moderate doses of ionizing radiation are able to induce premature endothelial senescence in vitro14−19 and in vivo.20 Thus, increased expression of intercellular and vascular adhesion molecules (ICAM1, ICAM2, VCAM1) and enhanced levels of senescence markers p16 and p21 have been found in endothelial cells isolated from murine heart 3−6 months after local irradiation with 8−16 Gy.20,21 A simultaneous increase in the levels of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin 1 alpha (IL-1a), and interleukin 6 (IL-6) was detected in the serum of these mice. Received: July 31, 2017 Published: August 29, 2017 3903
DOI: 10.1021/acs.jproteome.7b00536 J. Proteome Res. 2017, 16, 3903−3916
Article
Journal of Proteome Research
Figure 1. Workflow showing the experimental design of the study. Cells were not passaged, but media of the cells were changed every other day except over the weekend when 25% more media were provided to the cells. This was done by mixing conditioned media with fresh media with serum in a 1:1 relation. HCECest2 cells were irradiated with the dose of 10 Gy (X-ray) and isolated at day 14 for further analyses. For the secretome analysis, on the last transfer of media (day 13), the conditioned media was mixed 1:1 with serum-free media; the irradiated cells were grown for further 24 h in the serum-free media that was collected at day 14. For the recipient cell analysis, the serum-free conditioned media from the control or irradiated cells was diluted 1:1 with serum-containing media and transferred to the nonirradiated recipient HCECest2 cells cultured in parallel. The recipient cells were further cultured for 24 h and collected at day 14. All steps included a nonirradiated control.
STAT3 pathway was found in irradiated donor cells as well as nonirradiated recipient cells exposed to radiation-induced secretome.
This indicates that local irradiation is able to cause systemic pro-inflammatory alteration in the blood and thereby possibly influence the neighboring nonirradiated cells and tissues.20 Indeed, local irradiation has been shown to induce systemic out-of-field (“bystander”) effects by activating the innate immune system to produce pro-inflammatory cytokines, leading to chronic inflammation.22 Partial lung radiation in rats induced increased expression of pro-inflammatory cytokines and ROS in the shielded lung volume adjacent but external to the targeted field.23−25 The term “bystander effect” was coined in the early 1990s to describe effects occurring in cells that are not directly irradiated.26 Studies investigating effects of radiation-induced SASP on neighboring “bystander” cells are scarce. Proteins secreted from radiation-induced senescent breast cancer cell line (MCF7) were analyzed by proteomics and by cytokine microarrays.27,28 These studies indicated that human umbilical vein endothelial cells (HUVEC) exposed to the radiationinduced secretome showed increased cell proliferation, invasion, migration, and wound healing activity. Xiao et al. showed immediate activation of the p38 pathway in HUVEC cocultured with irradiated macrophages.29 In order to elucidate factors that lead to radiation-induced CVD it is important to know in detail how endothelial cells respond to radiation and how they communicate with the surrounding nonirradiated cells after the radiation injury. The goal of this study was to investigate molecules and biological pathways involved in this communication. We performed nonbiased label-free proteomics analysis of (i) irradiated endothelial cells, (ii) their secretome, and (iii) nonirradiated recipient (bystander) endothelial cells that were exposed to the radiation-induced secretome. Significant activation of the
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EXPERIMENTAL SECTION
Materials
Ammonium bicarbonate (NH4HCO3) was obtained from Sigma (St. Louis, MO). Acetonitrile (ACN), formic acid (FA), and trifluoroacetic acid (TFA) were obtained from Roth (Karlsuhe, Germany). Iodoacetamide, tris- (hydroxymethyl) aminomethane (Tris), and sequencing-grade trypsin were obtained from Promega (Madison, WI). Cyano-4-hydroxycinnamic acid was obtained from Bruker Daltonik (Bremen, Germany). All solutions were prepared using HPLC grade water from Roth (Karlsuhe, Germany). Cell Culture and Irradiation
Human telomerase-immortalized coronary artery endothelial cells (HCECest2) tested negative for mycoplasma were cultured at 37 °C with 5% CO2 as described previously.19 The cells (1.8 million per plate) were seeded and grown in Human MesoEndo Endothelial Cell Medium containing fetal bovine serum (Cell Applications), exposed in a confluent state to X-ray doses of 0 Gy (control) or 10 Gy using AGO HS320/ 250 X-ray cabinet (250 kV, 13 mA, 1.5 mm Al, 1.2 mm Cu, 3 keV/μm), and cultivated for 14 days before harvesting, a time point at which these cells have reached a radiation-induced senescent status.30 Cells were not passaged, but media of the cells were changed every other day except over the weekend when 25% more media were provided to the cells. This was done by mixing conditioned medium with fresh medium with serum in a 1:1 relation. 3904
DOI: 10.1021/acs.jproteome.7b00536 J. Proteome Res. 2017, 16, 3903−3916
Article
Journal of Proteome Research
target of 105 and a maximum injection time of 50 ms. Intensity threshold was set to 1 × 104 and unassigned charges, and charges of +1 and >8 were excluded.
For the secretome analysis, on the last medium transfer (day 13), the media was changed to serum-free media to remove albumin and isolated 24 h later by centrifugation at 10 000g for 10 min to remove all cells and cell debris. To study the signal transfer to nonirradiated HCECest2, these were grown in the secreted medium from either control or irradiated cells diluted 1:1 with MesoEndo Endothelial Cell Medium with serum for 24 h before harvesting.6 After all cells were harvested, they were washed once using Hanks Balanced Salt Solution with 1.5 mM Mg2+ and 1 mM Ca2+ (HBSS++) (Cell Applications, San Diego, U.S.A.) before storing at −70 °C. All media were stored at −70 °C. The workflow is shown in Figure 1.
Label-Free Proteomic Analysis
The acquired spectra were loaded to the Progenesis QI software (version 2.0, Nonlinear Dynamics) for label-free quantification and analyzed as described previously.32,33 Briefly, profile data of the MS and MS/MS scans were transformed to peak lists with respective peak m/z values, intensities, abundances (areas under the peaks), and m/z width. After reference selection, the retention times of the other samples were aligned by automatic alignment to a maximal overlay of all features. After exclusion of all features with only one charge or more than seven charges, all remaining MS/MS spectra were exported as Mascot generic file and used for peptide identification with Mascot (version 2.5.1) in the Ensembl Human protein database (release 83, 31 286 148 residues, 83 462 sequences). Search parameters used were: 10 ppm peptide mass tolerance and 20 mmu fragment mass tolerance, one missed cleavage allowed, carbamidomethylation was set as fixed modification, methionine oxidation and asparagine or glutamine deamidation were allowed as variable modifications. A Mascot-integrated decoy database search calculated an average false discovery of 2.00 or 2.00 or
