Mitochondrial reactive oxygen species (ROS) and endothelial dysfunction are key contributors to cerebrovascular pathophysiology. We previously found that 17␤-estradiol profoundly affects mitochondrial function in cerebral blood vessels, enhancing efficiency of energy production and suppressing mitochondrial oxidative stress. To determine whether estrogen specifically affects endothelial mitochondria through receptor mechanisms, we used cultured human brain microvascular endothelial cells (HBMECs). 17␤-Estradiol treatment for 24 h increased mitochondrial cytochrome c protein and mRNA; use of silencing RNA for estrogen receptors (ERs) showed that this effect involved ER␣, but not ER␤. Mitochondrial ROS were determined by measuring the activity of aconitase, an enzyme with an iron-sulfur center inactivated by mitochondrial superoxide. 17␤-Estradiol increased mitochondrial aconitase activity in HBMECs, indicating a reduction in ROS. Direct measurement of mitochondrial superoxide with MitoSOX Red showed that 17␤-estradiol, but not 17␣-estradiol, significantly decreased mitochondrial superoxide production, an effect blocked by the ER antagonist, 780 (fulvestrant). Selective ER agonists demonstrated that the decrease in mitochondrial superoxide was mediated by ER␣, not ER␤. The selective estrogen receptor modulators, raloxifene and 4-hydroxy-tamoxifen, differentially affected mitochondrial superoxide production, with raloxifene acting as an agonist but 4-hydroxy-tamoxifen acting as an estrogen antagonist. Changes in superoxide by 17␤-estradiol could not be explained by changes in manganese superoxide dismutase. Instead, ER␣-mediated decreases in mitochondrial ROS may depend on the concomitant increase in mitochondrial cytochrome c, previously shown to act as an antioxidant. Mitochondrial protective effects of estrogen in cerebral endothelium may contribute to sex differences in the occurrence of stroke and other age-related neurodegenerative diseases.

Duckles. Estrogen and progestagens differentially modulate vascular proinflammatory factors.

No abstract

In young adult females, estrogen treatment suppresses the cerebrovascular inflammatory response; this is mediated in part via NF-kappaB, a key regulator of inflammatory genes. To examine whether age modifies effects of estrogen on vascular inflammation in the brain, female rats, 3 and 12 mo of age, were ovariectomized; half were treated with estrogen for 4 wk. Cerebral blood vessels were isolated from the animals at 4 and 13 mo of age. Inflammation was induced by LPS, either injected in vivo or incubated with isolated vessels ex vivo. Basal levels of cytoplasmic NF-kappaB were significantly higher in cerebral vessels of young rats, but the ratio of nuclear to cytoplasmic levels was greater in middle-aged animals. LPS exposure increased nuclear NF-kappaB DNA binding activity, protein levels of inducible nitric oxide synthase and cyclooxygenase-2, and production of nitric oxide and PGE(2) in cerebral vessels. All effects of LPS were markedly greater in vessels from the older animals. Estrogen significantly inhibited the LPS-induced increase in NF-kappaB DNA binding activity in cerebral vessels from animals at both ages. In 4-mo-old rats, estrogen also significantly suppressed LPS induction of inducible nitric oxide synthase and cyclooxygenase-2 proteins, as well as production of nitric oxide and PGE(2). In contrast, in 13-mo-old females, estrogen did not significantly affect these indexes of cerebrovascular inflammation. Thus the protective, anti-inflammatory effect of estrogen on cerebral blood vessels that is observed in young adults may be attenuated in aged animals, which exhibit a greater overall cerebrovascular response to inflammatory stimuli.

No abstract