ABC | Volume 112, Nº2, February 2019

Original Article Maifrino et al Exercise effects on ovariectomized mice Arq Bras Cardiol. 2019; 112(2):180-188 differences in the percentage of apoptotic cells between the Control or LDL Knockout groups for each parameter used. However, the apoptosis process was greater in animals of LDL knockout groups, when compared with animals of the control groups, regardless if they were ovariectomized or not. This suggests that ovariectomy was not a major factor in the processes of apoptosis induction in the aorta. The high levels of LDL in the bloodstream may have been the apoptosis-inducing factor in the endothelium and the tunica media of the ascending aorta in LDL knockout groups. The endothelial dysfunction induced by LDL oxidation (ox-LDL) has been associated to the pathogenesis of atherosclerosis and other vascular disorders. It is known that the ox-LDL activates ROS release and has been associated to apoptosis and endothelium damage. 29 The apoptosis of vascular smooth muscle cells (VSMC) is associated with the occurrence of vascular diseases. In atherosclerosis, cell apoptosis induction has been associated with atherosclerotic plaque rupture, clotting , vessel remodeling , tunica media atrophy, aneurysm formation and calcification. 30 Furthermore, in various human diseases such as Marfan syndrome and cystic necrosis of the tunica media (CMN), the apoptotic processes results in higher breakage of the elastic fibers, abnormal extracellular matrix deposition and tunica media expansion. 31 In this environment, the release of interleukin IL1 α and IL8, as well as the chemoattractant protein expression of monocytes (MCP-1) occurs during the VSMC apoptosis, which have causes infiltrating macrophages in vivo , increasing the observed tissue damage. 32 The animals of the OT-C group showed a higher number of apoptotic cells compared to the OS-C and S-C groups (p < 0,05). Physical activity has been associated with increased apoptosis levels in rats’ thymocyte, mice skeletal muscle and lymphocytes. 33,34 Oxidative stress resulting from metabolism in physical activities has been largely associated to apoptosis. In patients with cardiovascular diseases, the deficiency in nitric oxide (NO) production, associated with oxidative stress, results in a decline of NO bioavailability, inducing apoptosis of endothelial cells and therefore, resulting in endothelial dysfunction. 35 The ovariectomized animals showed a higher percentage of apoptotic cells than the trained and sedentary control groups (OT-C and OS-C). This suggests that decreased hormone production may be related to a reduction in antioxidative effects on the body. 8-OHdG is one of the main oxidative products of DNA, which is considered a reliable marker of oxidative DNA damage. Thus, 8-OHdG has been widely used as a sensitive biomarker of oxidative stress. 36 The immunohistochemical analysis of 8-OHdG showed staining in all groups; however, the trained groups showed higher intensity staining. Goto et al., 9 found that high-intensity exercise increases 8-OHdG levels in the plasma, which explains the higher degree of staining in the trained groups. There were differences in MMP-2 expression, and the control group showed staining in the intima and adventitia layers, while the LDL KO group showed staining in the tunica media. According to Sakalihasan et al., 37 this occurs because the atherosclerotic lesion causes the migration of MMP-2 at the ends and at a lower quantity in normal tissue, for the tunica media, i.e., the formation of atherosclerotic plaques activates a set of chain reactions, which can increase the amount of MMP-2 present in the tissue. With regard to the MMP-9 expression, all groups showed tissue staining, but without a pattern. No evidence explaining this heterogeneous staining was found. Conclusion The experimental model analyzed shows histomorphometric changes with increased expression of 8-OHdG in trained groups. An increase in the apoptosis rate was observed in the trained groups and the LDL KO ovariectomized group. The groups stained with MMP-2 showed migration and its increased expression in the tunica media of LDL KO groups. However, the MMP-9 staining appeared in all groups, but did not follow a homogeneous pattern. Finally, studies on the expression of metalloproteinases in cardiac muscle tissues with atherosclerosis are very scarce, suggesting the need for further studies to investigate the issue. Thus, the results described herein suggest that moderate-intensity aerobic exercise in ovariectomized mice associated to an increase in LDL rates possibly increases oxidative stress and apoptosis induction. The evaluation of the parameters under study (MMPs, apoptosis and 8-OHdG) was performed by immunohistochemistry. However, other more sensitive technologies (such as molecular biology ones) could be used in these assessments, leading to more precise results and interpretations. Author contributions Conception and design of the research: Maifrino LBM, Souza LB, Quintana HT, Fonseca FLA; acquisition of data: Maifrino LBM, Lima NEA, Cardoso CG, Souza LB, Oliveira F, Fonseca FLA; analysis and interpretation of the data: Lima NEA, Cardoso CG, Souza LB, Tomé TC, Oliveira F, Reis BCAA; statistical analysis: Marques MR, Quintana HT, Oliveira F; obtaining funding: Maifrino LBM; writing of the manuscript: Maifrino LBM, Lima NEA, Marques MR, Souza LB, Oliveira F, Reis BCAA; critical revision of the manuscript for intellectual content: Maifrino LBM, Marques MR, Cardoso CG, Tomé TC, Reis BCAA, Fonseca FLA. Potential Conflict of Interest No potential conflict of interest relevant to this article was reported. Sources of Funding There were no external funding sources for this study. Study Association This study is not associatedwith any thesis or dissertationwork. Ethics approval and consent to participate This study was approved by the Ethics Committee on Animal Experiments of the Universidade de São Judas Tadeu under the protocol number 058/2007. 186