Decreased VEGF expression and microvascular density, but increased HIF-1 and 2α accumulation and EPO expression in chronic moderate hyperoxia in the mouse brain

Abstract

Normal brain function is dependent on continuous and controlled oxygen delivery. Chronic moderate hypoxia leads to angiogenesis, suggesting a modulatory role for oxygen in determining capillary density. The objective of this study was to determine physiologic and brain angiogenic adaptational changes during chronic moderate normobaric hyperoxia in mice. Four-month old C56BL/6J mice were kept in a normobaric chamber at 50% O(2) for up to 3 weeks. Normoxic littermates were kept in the same room outside the chamber. Freshly collected or fixed brain specimens were analyzed by RT-PCR, Western blot analysis and immunohistochemistry. Results show accumulation of hypoxia inducible factors 1 and 2α (HIF-1 and 2α), and increased expression of erythropoietin (EPO), cyclooxygenase-2 (COX-2) and angiopoietin-2 (Ang-2). Conversely, vascular endothelial growth factor (VEGF), and VEGF receptor-2 (KDR/Flk-1), Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and prolylhydroxylase-2 (PHD-2) expressions were decreased. VEGF mRNA level was diminished but there was no change in HIF-1α mRNA and von Hippel Lindau E3 ubiquitin ligase (VHL) protein expression. Microvascular density was significantly diminished by the end of the 3rd week of hyperoxia. Overall, our results are: (1) increased expression of the potent neuroprotective molecule, EPO; (2) diminished expression of the potent angiogenic factor, VEGF; and (3) decreased microvascular density. We can, therefore, conclude that brain microvascular density can be controlled by HIF-independent mechanisms, and that brain capillary density is a continuously adjusted variable with tissue oxygen availability as one of the controlling modulators.

Fig. 1. Change in body weight, hematocrit and arterial oxygen saturation during chronic moderate hyperoxia

Error bars (SD) are indicated at normoxia (0), and 1, 4, 7, 14, and 21 days of hyperoxia. A, change in gross body weight during hyperoxia; and B, relative change in % of body weight during hyperoxic exposure; n = 10 for normoxia and 15 for all hyperoxic durations. C, hematocrit values; n = 11 in normoxia (0) and 4 day of hyperoxia, and n= 12 in 7, 14 and 21 day of hyperoxia. D, arterial oxygen saturation; n = 6 in all cases. Values are mean ± SD; *P < 0.05 compared to normoxic value of each category.

Fig. 2. HIF-1α and HIF-2α accumulation in cerebral cortex of mouse in chronic hyperoxia

A, HIF-1 and 2α accumulation. B, optical density (OD) ratio of HIF-1 and 2α normalized to β-actin in normoxia (0), and 1, 4, 7, 14, and 21 days of hyperoxia. *p < 0.05 compared with normoxic control. Each value represents the mean ± SD from 5 mice.

Fig. 3. Expression of VEGF and Flk-1 in mouse cerebral cortex during hyperoxia

A, VEGF and Flk-1 protein expression in normoxic control and hyperoxia. B, optical density ratio in normoxia (0), and 1, 4, 7, 14, and 21 days of hyperoxia. *P < 0.05 compared with normoxic control values of each category. Values are mean ± SD. n = 5 mice per time point.

Fig. 4. HIF-1 α and VEGF RT- PCR, PHD-2 and VHL protein expressions in mouse cerebral cortex during hyperoxia

A, transcriptional activation of HIF-1α and VEGF m-RNA in normoxia and hyperoxia normalized to β-actin m-RNA. n = 4 in normoxia (0), 1 and 4 days of hyperoxia and 3 in 7 days of hyperoxia. B, PHD-2 and VHL protein expression in normoxic control and hyperoxia. C, optical density ratio of PHD-2 and VHL in normoxia (0), and 1, 4, 7, 14, and 21 days of hyperoxia. n = 3 mice per time point. Values are mean ± SD. *P < 0.05 from normoxic control values of each category.

Fig. 5. Expression of PGC-1α and EPO in mouse cerebral cortex during hyperoxia

A, PGC-1α and EPO protein expression in normoxic control and hyperoxia. B, Optical density ratio in normoxia (0), and 1, 4, 7, 14, and 21 days of hyperoxia. *P < 0.05 compared with normoxic control values of each category. Values are mean ± SD. n = 5 mice per time point.

Fig.6. Expression of COX-2 and Ang-2 in mouse cerebral cortex during hyperoxia

A, COX-2 and Ang-2 protein expression in normoxic control and hyperoxia. B, optical density ratio in normoxia (0), and 1, 4, 7, 14, and 21 days of hyperoxia. *P < 0.05 compared with normoxic control values of each category. Values are mean ± SD. n = 5 mice per time point.

Fig.7. Microvascular density in mouse cerebral cortex in prolonged hyperoxia

A, composite photomicrograph of GLUT-1–stained sections spanning part of the parietal cortex of a mouse in normoxia and 21 days of hyperoxia. B, capillary density (number per mm²) of GLUT-1-stained sections at normoxia (0), and 7, 14 and 21 days of hyperoxia. Values are mean ± SD, *p < 0.05 compared with normoxic control. n = 6 mice per time point.