Gender differences in hypoxic acclimatization in cyclooxygenase-2-deficient mice

Abstract

The aim of this study was to determine the effect of cyclooxygenase-2 (COX-2) gene deletion on the adaptive responses during prolonged moderate hypobaric hypoxia. Wild-type (WT) and COX-2 knockout (KO) mice of both genders (3 months old) were exposed to hypobaric hypoxia (~0.4 ATM) or normoxia for 21 days and brain capillary densities were determined. Hematocrit was measured at different time intervals; brain hypoxia-inducible factor -1α (HIF-1α), angiopoietin 2 (Ang-2), brain erythropoietin (EPO), and kidney EPO were measured under normoxic and hypoxic conditions. There were no gender differences in hypoxic acclimatization in the WT mice and similar adaptive responses were observed in the female KO mice. However, the male KO mice exhibited progressive vulnerability to prolonged hypoxia. Compared to the WT and female KO mice, the male COX-2 KO mice had significantly lower survival rate and decreased erythropoietic and polycythemic responses, diminished cerebral angiogenesis, decreased brain accumulation of HIF-1α, and attenuated upregulation of VEGF, EPO, and Ang-2 during hypoxia. Our data suggest that there are physiologically important gender differences in hypoxic acclimatization in COX-2-deficient mice. The COX-2 signaling pathway appears to be required for acclimatization in oxygen-limiting environments only in males, whereas female COX-2-deficient mice may be able to access COX-2-independent mechanisms to achieve hypoxic acclimatization.

Keywords: Capillary density; hypoxia‐induced angiogenesis; hypoxic adaptation; prolonged hypoxia; sex differences.

Figure 1

Overall survival in the wild‐type (WT) and the COX‐2 knockout (KO) mice during 21‐day hypoxic exposure. (A) In males, the KO group had a significantly lower survival rate compared to the WT group (KO: 57%, 8/14; WT: 100%, 21/21). (B) In females, the survival rate was similar in the WT and the KO groups (WT: 100%, 21/21; KO: 94%, 15/16). * indicates significant difference from the WT group, Wilcoxon survival analysis, P < 0.05.

Figure 2

(A) Body weights of age‐matched wild‐type (WT) and COX‐2 knockout (KO) mice before hypoxic or normoxic exposure. Values are mean ± SD, * indicates significant difference (t‐test, P < 0.05) from the WT group with same sex; n for each group is indicated on each bar. (B) Change in body weight (% of initial weight) in mice during normoxic or hypoxic exposure. WT‐N: WT normoxic, n = 13 each for males and females; KO‐N: KO normoxic, n = 10 each for males and females; WT‐H: WT hypoxic, n = 21 each for males and females; KO‐H: KO hypoxic, n = 8 and 15 for males and females, respectively. Values are mean ± SD, * indicates significant difference (t‐test, P < 0.05) from the WT group at the same time point with same exposure condition.

Figure 3

Change in hematocrit in the wild‐type (WT) and the COX‐2 knockout (KO) mice during hypoxic exposure. Values are mean ± SD. n of each group is indicated for each time point, normoxic, 1, 4, 7, 14, and 21 days of hypoxia. * indicates significant difference (t‐test, P < 0.05) from the corresponding normoxic baseline; † indicates significant difference (t‐test, P < 0.05) from the WT group at the same time point.

Figure 4

EPO expression in kidney in the wild‐type (WT) and the COX‐2 knockout (KO) mice under normoxia and 7‐day hypoxia. (A) Representative Western blot analysis of normoxic control and 7‐day hypoxia in male and female mice, respectively. (B) Optical density ratios of EPO normalized to β‐tubulin. Values are mean ± SD, n = 6 for each group. * indicates significant difference (t‐test, P < 0.05) from the corresponding normoxic baseline; † indicates significant difference (t‐test, P < 0.05) from the WT group with same exposure condition.

Figure 5

Microvascular density in cerebral cortex in the wild‐type (WT) and the COX‐2 knockout (KO) mice under normoxia and 21‐day hypoxia. (A) Representative images of GLUT‐1 immunohistostaining from WT and KO mice of both genders. (B) Capillary density (Number/mm²) as identified by the GLUT‐1 positive staining in brain cortex. Values are mean ± SD, n = 6 for each group. * indicates significant difference (t‐test, P < 0.05) from the corresponding normoxic baseline; † indicates significant difference (t‐test, P < 0.05) from the WT group at the same exposure condition.

Figure 6

Western blot analysis of HIF‐1α, VEGF, EPO, and ANG‐2 in cerebral cortex in the wild‐type (WT) and the COX‐2 knockout (KO) mice under normoxia and 7‐day hypoxia. (A) HIF‐1α. (B) VEGF. (C) EPO. (D) Ang‐2. For A to D, upper panel: Representative western blot analysis of normoxic control and 7‐day hypoxia in male and female mice, respectively. Lower panel: Optical density ratios of respective protein normalized to β‐tubulin or β‐actin. Values are mean ± SD, n = 4–8 for each group. * indicates significant difference (t‐test, P < 0.05) from the corresponding normoxic baseline; † indicates significant difference (t‐test, P < 0.05) from the WT group at the same exposure condition.