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Effects of Adrenomeduline and Ramp2 on the Lung of Mice Exposed to Total Body Radiation

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Effects of Adrenomeduline and Ramp2 on the Lung of Mice Exposed to Total Body Radiation Volume 18, No 3 International Journal of Radiation Research, July 2020 Effects of adrenomeduline and ramp2 on the lung of mice exposed to total body radiation M.K. Ozbilgin1*, G.Z. Karaman1, S. Gencur1, E. Gumustepe2, C. Kurtman2 ¹Department of Histology and Embryology, Celal Bayar University School of Medicine, Manisa, Turkey 2Department of Radiation Oncology, Ankara University School of Medicine, Ankara, Turkey ABSTRACT Background: Adrenomedullin (AM) and its receptor, receptor activity- ► Original article modifying protein (RAMP) 2 have pleiotropic regulatory functions in normal tissue and cancer tissue. AM is produced and secreted both numerous stromal cells and tumor cells. This study aims to investigate a possible role of AM and RAMP2 in the radiation exposure in the normal lung tissue. Materials *Corresponding authors: and Methods: Four groups with 6 male adult Swiss Albino mice per group Mahmut Kemal Ozbilgin, MD, were investigated. The mice were subjected to a 500 cGy single-dose E-mail: radiation exposure in the total body radiation device and lung tissues were [email protected] collected. 1, 2, and 7 days after radiation exposure, with 1 reference group which was not exposed to radiation. Results: The general histology and the Revised: June 2019 immunohistochemistry of the tissue samples prepared with anti- AM, anti– Accepted: August 2019 RAMP2, and monoclonal antibodies were investigated, yielding a statistically Int. J. Radiat. Res., July 2020; significant increase for AM on day 3 and for RAMP2 on day 1 after radiation 18(3): 571-578 exposure. Conclusion: The observed increase of AM and RAMP2 DOI: 10.18869/acadpub.ijrr.18.3.571 concentrations in the normal tissue matrix after radiation exposure may play a role in the side effects of radiotherapy. Keywords: AM, RAMP2, radiation, lung, carcinogens. INTRODUCTION and angiogenesis. Adrenomedullin (AM) is a member of the calcitonin peptide superfamily, Radiotherapy is widely used for cancer such as the calcitonin gene-related peptide, and treatment in order to eliminate malignant cells. first isolated from human pheochromocytoma however, it also causes major health hazards in (2). It is a circulating hormone and functions also the normal tissues, so it calls as a double-edged as a local paracrine and autocrine mediator with sword (1). Although the irradiation may lead to multiple biological activities such as natriuretic (3) (4) (5) Downloaded from ijrr.com at 21:09 +0330 on Wednesday October 6th 2021 [ DOI: 10.18869/acadpub.ijrr.18.3.571 ] the cancer cells die, the complete effects of the , diuretic bronchodilator effect , regulation radiotherapy on the nearby normal tissue, of brain functions(6) and anti-inflammatory, especially in the microenvironment of the cancer anti-oxidative, anti-fibrotic properties (7). AM is tissue, are not yet fully understood. The tumor also involved in many human pathologies and microenvironment enables and supports also cancer. AM levels in many cancer patients neoplastic cells to acquire adaptive benefits such as a bronchial neuroendocrine tumor were from the surrounding environment, including increased compared with healthy patients (8). the infiltrating tumor-associated immune cells, Receptor activity-modifying protein 2 vasculature and fibroblasts. (RAMP2) is a single-pass transmembrane There are many molecules within the tumor protein that heterodimerizes with several family microenvironment such as AM which has been B G-protein coupled receptors to alter their shown to be involved in tumor survival and function. RAMP2 has been primarily progression by promoting cellular proliferation characterized in association with calcitonin Ozbilgin et al. / Evaluation of am-ramp2 after radiation receptor-like receptor (Calcrl, CLR), forming the radiotherapy), group 1 (24 hours later, after canonical receptor complex for the endocrine radiotherapy), group 2 (72 hours later, after peptide AM (9). RAMP2 has been identified as an radiotherapy) and group 3 (7 days later, after essential mediator of cardiovascular radiotherapy). Except for the 6 subjects in the homeostasis. Conversely, the overexpression of control group, mice were exposed to whole body RAMP2 suppresses tumor cell adhesion to ionizing radiation at the same day with the endothelial cells, tumor metastasis, and device present in the Department of Radiation improved survival (10). RAMP2 causes vascular Oncology of Ankara University School of abnormalities and is embryonically lethal. Early Medicine, whole body irradiation applied with a after the induction, pronounced edema with source -to-axis distance of 100 cm and anterior enhanced vascular leakage occurred. AM-RAMP2 250 cGy and posterior 250 cGy; total 500 cGy system causes the disruption of actin formation, dose in a single fraction. This radiation leading to vascular and organ damage at the treatment dose could make lung injury(14). For chronic stage after the gene deletion (11). In adult obtaining sedation before the procedure, RAMP2+/- mice, reduced RAMP2 expression led intramuscular ketamine injection at a dose of 45 to vascular hyperpermeability and impaired –50 mg/kg was performed. The animals were neovascularization. Conversely, endothelial cells sacrificed, which are control group and on day 1, overexpressing RAMP2 had enhanced capillary 3 and 7 after radiotherapy and thorax region formation, firmer tight junctions, and reduced was dissected and lungs were completely vascular permeability (12). The vascular removed. All lung samples were first washed in a endothelial adrenomedullin-RAMP2 system is solution containing 10% formol and then placed essential for the vascular integrity and organ in screw-cap sampling containers containing homeostasis(11). Deletion of RAMP2 in mice 10% formol, with separate boxes used for every results in massive cutaneous edema and animal. The tissue samples taken after the mid-gestational death(13) and hemorrhage in procedure were embedded into paraffin animals (12). following the routine light microscopy paraffin In this study, we investigated the changes of tissue method at the Department of Histology AM and RAMP2 expression in the lung tissue on and Embryology of Celal Bayar University School day 1,3 and 7 days after 500 cGy single-dose of Medicine. Slides of 5 μm were evaluated with radiation exposure. the indirect immunohistochemistry method. Immunohistochemistry MATERIALS AND METHODS Formalin-fixed, paraffin-embedded sections were used for immunohistochemical staining. The study was approved by the Animal Tissue samples were stored at 60°C overnight Ethical Committee of the faculty of Medical and then dewaxed with xylene for 30 minutes. Medicine affiliated to Ankara University and was After dehydration of the sections with ethanol, conducted in accordance with the they were washed with distilled water. Downloaded from ijrr.com at 21:09 +0330 on Wednesday October 6th 2021 [ DOI: 10.18869/acadpub.ijrr.18.3.571 ] recommendations outlined in Guidelines for Subsequently, the samples were treated with 2% Care and Use of Experimental Animals. A total of trypsin (ab970; Abcam, Cambridge, UK) at 37°C 24 healthy male adult Swiss Albino mice, for 15 minutes and incubated in 3% H2O2 weighing 30–40 g each, were obtained from solution for 15 minutes to inhibit endogenous Ankara University Experimental Animal peroxidase activity. Then the sections were Laboratory and used as subjects. The subjects incubated with anti–adrenomedulline primary were isolated from stress and noise and fed with antibody (sc-1414; Santa Cruz, California) in a water and food ad libitum at 25°C in a cycle of 12 1/100 dilution and anti-RAMP2 Polyclonal hours of dark and 12 hours of light before being (Catalogue No. AR5423; Boster Biological included in the study. The animals were divided Technology Ltd., China) for 18 hours at +4°C. into 4 groups as control group (no They were then given an additional 3 times for 5 572 Int. J. Radiat. Res., Vol. 18 No. 3, July 2020 Ozbilgin et al. / Evaluation of am-ramp2 after radiation -minute washes in phosphate-buffered saline then used. (PBS), followed by incubation with biotinylated IgG and administration of streptavidin Statistical analysis peroxidase (Histostain Plus kit, Zymed 87-9999; For the statistical analysis, we used the Zymed, San Francisco, California, USA). After software SPSS 19.0 (IBM SPSS Statistics). The washing the secondary antibody with PBS 3 numerical variables are given with mean, times for 5 minutes, the sections were stained standard deviation, median, minimum, and with diaminobenzidine (DAB) (DAB, K007; DBS, maximum values. The Shapiro-Wilk test was Pleasanton, California, USA) to detect the used for the normality tests. One-way ANOVA immunoreactivity, and then stained with tests were used for comparisons between the 4 Mayer’s hematoxylin (72804E; Microm, groups for parametric variables, and the Tukey Walldorf, Germany) for counterstaining. They test was used for post-hoc tests. Additionally, we were covered with a mounting medium (01730; used the Kruskal-Wallis test for comparisons Surgipath, Cambridge, UK) and observed with between the 4 groups for non- parametric light microscopy (Olympus BX-40, Tokyo, Japan). variables and the Mann-Whitney U test with Immunostaining for AM and RAMP2 were Bonferroni correction for post-hoc tests. evaluated semi-quantitatively using H-score Statistical comparisons with a p value < 0.05 are analysis. The immunostaining intensity was assumed as statistically sign ificant. This categorized by the following scores: 0 (no
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