Early effects of hypoxia/reoxygenation on VEGF, Ang-1, Ang-2 and their receptors in the rat myocardium: Implications for myocardial angiogenesis

Abstract

Tissue hypoxia has been identified as being a particularly important stimulus for triggering angiogenesis. Here we report early effects of hypoxia/reoxygenation (H/R) on the protein expression profiles and localization patterns of the VEGF and Angiopoietin-Tie systems in adult rat myocardium. Western blot as well as immunohistochemical analyses were performed on hearts obtained from rats exposed to various durations of in vivo systemic hypoxemic hypoxia followed by 24 h reoxygenation. The relative time course of protein expression in response to increasing durations of hypoxia, as indicated from our experiments, seems to suggest the involvement of the VEGF system and the Ang-Tie system in early angiogenesis. An apparent relationship between the expression profiles of Flk-1 and Ang-2 was observed. The most significant and interesting relationship which came to light was the surprisingly coincident yet opposite temporal trends between Ang-1 and Ang-2 protein levels. In the 1 h hypoxia group, there was significant induction of Ang-2 expression (31.3% compared to its baseline control) in contrast to relatively mild Ang-1 expression (23.8% compared to its baseline control). Thereafter Ang-1 displayed a progressive increase in expression, parallel to a progressive decrease in Ang-2 expression, becoming most pronounced in the 4 h hypoxia group (Ang-1, 50% and Ang-2, 12.6% compared to respective baseline control values). This suggests that despite their being antagonists at the receptor level, regulation of Ang-1 and Ang-2 protein levels in response to hypoxia runs much deeper and seems to indicate modulatory control at the transcriptional and/or translational level. Two additional groups of rats were sacrificed 7 days after 4 h hypoxia + 24 h reoxygenation, or after a 28 h period of time-matched normoxia. Left ventricular tissue sections were used to determine capillary density (CD) by using anti-CD31 immunohistochemistry and computer-assisted morphometry. CD was significantly increased in the 4 h hypoxia group compared to control (1814 ± 56 vs. 1642 ± 43 counts/mm²) confirming that modulation of angiogenic factors and their receptors by H/R is capable of stimulating capillary proliferation in the myocardium. Our study presents the first evidence for the Ang-Tie system's involvement in early stages of myocardial angiogenesis along with the VEGF-Flk-1-Flt-1 system. The stimulation of myocardial angiogenesis by H/R may constitute a potential basis for a possible more long-lived adaptive response to stress afforded by preconditioning stimuli.