HIF-1α/COX-2 expression and mouse brain capillary remodeling during prolonged moderate hypoxia and subsequent re-oxygenation

Abstract

Dynamic microvascular remodeling maintains an optimal continuous supply of oxygen and nutrients to the brain to account for prolonged environmental variations. The objective of this study was to determine the relative time course of capillary regression during re-oxygenation after exposure to prolonged moderate hypoxia and expression of the primary signaling factors involved in the process. Four-month old male C57BL/6 mice were housed and maintained in a hypobaric chamber at 290 Torr (0.4 atm) for 21 days and allowed to recover at normoxia (room air) for up to 21 days. The mice were either decapitated or perfused in-situ and brain samples collected were either homogenized for Western blot analysis or fixed and embedded in paraffin for immunohistochemistry. Hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and erythropoietin (EPO) expression were increased during hypoxic exposure and diminished during subsequent re-oxygenation. However, cyclooxygenase-2 (COX-2) and angiopoietin-2 (Ang-2) were both elevated during hypoxia as well as subsequent re-oxygenation. Significantly increased capillary density at the end of the 3rd week of hypoxia regressed back toward normoxic baseline as the duration of re-oxygenation continued. In conclusion, elevated COX-2 and Ang-2 expression during hypoxia where angiogenesis occurs and re-oxygenation, when micro-vessels regress, identifies these proteins as vascular remodeling molecules crucial for angioplasticity.

Keywords: Ang-2; Brain capillary remodeling; EPO; VEGF.

Fig. 1. Change in body weight in 4-month old mice during hypoxia and recovery from hypoxia (re-oxygenation)

Solid lines indicate change in body weight during 21 days of hypoxia followed by 21 days of re-oxygenation. Dashed lines indicate weight change in normoxic littermate control during the experimental periods. *P < 0.05 compared with initial normoxic value. ^♣P < 0.05 compared with corresponding normoxic value. Values are mean ± SD; n = 16 for normoxia; 10 for 1, 4, 7, 14, and 21 days of hypoxia and day 1 re-oxygenation; 9 for 4, 7, 14 days of re-oxygenation and 5 for 21 days of re-oxygenation.

Fig. 2. Hematocrit and arterial oxygen saturation during normoxia (N), chronic hypoxia (H), and re-oxygenation (R) in 4-month old mice

A. Change in hematocrit value: *P < 0.05 compared with normoxia; ^♣P < 0.05 compared to 21 days of hypoxia. Values are mean ± SD; n = 16 for normoxia, 8 for 21 day hypoxia, 6 for 1 and 4 day recovery, 9 for 7, 14 and 21 day recovery. B. Arterial oxygen saturation: Values are mean ± SD; n = 5 mice in each case.*P < 0.05 compared with normoxic value. Error bars indicated at normoxia, and 1, 4, 7, 14, and 21 days of hypoxia and re-oxygenation.

Fig. 3. HIF-1α and COX-2 expression in mouse cerebral cortex during hypoxia and reoxygenation

A: HIF-1α and COX-2 protein expression in normoxia, hypoxia and reoxygenation. B: Optical density ratio in normoxia (0), and 1, 4, 7, 14, and 21 days of hypoxia and consecutive 21 days (22 – 42 days) of re-oxygenation at room air. *P < 0.05 compared with normoxic control values of each category. Values are mean ± SD. n = 3 mice per time point.

Fig. 4. VEGF and Ang-2 expression in mouse cerebral cortex during hypoxia and reoxygenation

A: VEGF and Ang-2 protein expression in normoxia, hypoxia and re-oxygenation. B: Optical density ratio in normoxia (0), and 1, 4, 7, 14, and 21 days of hypoxia and consecutive 21 days (22 – 42 days) of re-oxygenation. *P < 0.05 compared with normoxic control values of each category. Values are mean ± SD. n = 4 mice per time point.

Fig. 5. EPO protein expression in mouse cerebral cortex during hypoxia and reoxygenation

A: EPO protein expression in normoxia, hypoxia and re-oxygenation. B: Optical density ratio in normoxia (0), and 1, 4, 7, 14, and 21 days of hypoxia and consecutive 21 days (22 – 42 days) of re-oxygenation. *P < 0.05 compared with normoxic baseline value. Values are mean ± SD. n = 3 mice per time point.

Fig. 6. Microvascular density in mice cerebral cortex during re-oxygenation after 3 weeks of hypoxia

A: composite photomicrograph of GLUT-1–stained sections spanning part of the parietal cortex of mice at normoxia, 21 days of hypoxia and 21 days of re-oxygenation B: Capillary density (N/mm2) of GLUT-1-stained sections of the mice during normoxia (0), 21 days of hypoxia (21H), and 7, 14 and 21 days of re-oxygenation in room air (R). *p < 0.05 compared with corresponding normoxic control. Values are mean ± SD, n = 5 mice per time point in each group.