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

Tissue hypoxia has been identified as being a particularly important stimul us for triggering angiogenesis. Here we report early effects of hypoxia/reo xygenation (H/R) on the protein expression profiles and localization patter ns of the VEGF and Angiopoietin-Tie systems in adult rat myocardium. Wester n blot as well as immunohistochemical analyses were performed on hearts obt ained 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 fr om 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 signific ant and interesting relationship which came to light was the surprisingly c oincident yet opposite temporal trends between Ang-1 and Ang-2 protein leve ls. In the 1 h hypoxia group, there was significant induction of Ang-2 expr ession (31.3% compared to its baseline control) in contrast to relatively m ild Ang-1 expression (23.8% compared to its baseline control). Thereafter A ng-1 displayed a progressive increase in expression, parallel to a progress ive decrease in Ang-2 expression, becoming most pronounced in the 4 h hypox ia group (Ang-1, 50% and Ang-2, 12.6% compared to respective baseline contr ol values). This suggests that despite their being antagonists at the recep tor level, regulation of Ang-1 and Ang-2 protein levels in response to hypo xia runs much deeper and seems to indicate modulatory control at the transc riptional and/or translational level. Two additional groups of rats were sa crificed 7 days after 4 h hypoxia + 24 h reoxygenation, or after a 28 h per iod of time-matched normoxia. Left ventricular tissue sections were used to determine capillary density (CD) by using anti-CD31 immunohistochemistry a nd computer-assisted morphometry. CD was significantly increased in the 4 h hypoxia group compared to control (1814 +/- 56 vs. 1642 +/- 43 counts/mm(2 )) confirming that modulation of angiogenic factors and their receptors by H/R is capable of stimulating capillary proliferation in the myocardium. Ou r 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 sy stem. The stimulation of myocardial angiogenesis by H/R may constitute a po tential basis for a possible more long-lived adaptive response to stress af forded by preconditioning stimuli.