LOCALIZATION OF SEVERAL ANGIOGENIC FACTORS IN THE MOUSE MYOCARDIUM IN RESPONSE TO ENDURANCE TRAINING

Abstract

Introduction: Previous studies carried out in our laboratory showed the presence of hypertrophy associated with angiogenesis in the mouse myocardium in response to endurance training (I). Moreover, we analysed the expression of several angiogenic factors as VEGFR- 1/Flt-1, VEGFR-2/Flk-1, HIF-1a, iNOS and MMP-9 showing that the exercise differently regulates their expression levels in the angiogenic hearts according to the intensity and length of endurance training (II). The aim of this study was to examine the localization of VEGFR-1/Flt-1, VEGFR-2/Flk-1, HIF-1a, iNOS and MMP-9 in the same experimental model in order to observe in which cells these proteins are expressed in response to exercise. Methods: Hearts from sedentary and trained mice were fixed with formalin, embedded with paraffin and cut to obtain 5μm sections that were used to perform the immunohistochemical analyses. Sections were incubated with the monoclonal primary antibodies for VEGFR-1/Flt-1, VEGFR-2/Flk-1, HIF-1a, iNOS and MMP-9 at specific concentrations. Nonimmune mouse serum was substituted for negative controls. Results: We observed that VEGFR-1/Flt-1 and VEGFR-2/Flk-1 were specifically expressed by vascular endothelial cells of the myocardium. HIF-1 alpha was localized in nucleus of vascular endothelial cells and myocardiocytes. iNOS was expressed in cells of interstitial connective tissue and smooth muscle and MMP-9 localized in interstitium and vascular endothelial cells. We did not find any difference in the localization of these factors between sedentary and trained groups; instead we noted different expression levels between sedentary and trained groups and within the trained group in agreement with our previous data from immunoblotting analyses. Discussion: Our results suggest that both VEGFR-1/Flt-1 and VEGFR-2/Flk-1 might be involved in the proliferation of capillary endothelial cells in the cardiac angiogenesis induced by exercise. The expression of HIF-1a suggests that brief and transient episodes of hypoxia may occur in the heart during cardiac cycle in response to endurance training. This factor could modulate the gene expression of VEGF to increase the angiogenic response of heart to exercise. The expression of iNOS suggests the production of NO by the cells of interstitial tissue and smooth muscle in order to promote the vascular relaxation fundamental in the early steps of angiogenesis. The localization of MMP-9 in the vascular endothelial cells and interstitium suggests that endurance training can promote the expression and release of pro-enzymatic form of MMP-9; and a possible conversion of pro-MMP-9 in the active form in the interstitium.