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.

Mitochondria are a major source of reactive oxygen species (ROS) and oxidative stress, key contributors to aging and neurodegenerative disorders. We report that gonadal hormones influence brain mitochondrial ROS production in both females and males. Initial experiments showed that estrogen decreases mitochondrial superoxide production in a receptor-mediated manner, as measured by MitoSOX fluorescence in differentiated PC-12 cells. We then assessed in vivo effects of gonadal hormones on brain mitochondrial oxidative stress in female and male rats. Brain mitochondria were isolated to measure a functional indicator of ROS, i.e., activity of the ROS-sensitive mitochondrial enzyme, aconitase. Gonadectomy of both males and females caused a decrease in aconitase activity, suggesting endogenous gonadal hormones influence mitochondrial ROS production in the brain. In vivo treatment of gonadectomized animals with testosterone or dihydrotestosterone (DHT) had no effect, but estrogen replacement significantly increased aconitase activity in brain mitochondria from both female and male rats. This indicates estrogen decreases brain mitochondrial ROS production in vivo. Sex hormone treatments did not affect protein levels of brain mitochondrial uncoupling proteins (UCP-2, 4, and 5). However, estrogen did increase the activity, but not the levels, of manganese superoxide dismutase (MnSOD), the mitochondrial enzyme that catalyzes superoxide radical breakdown, in brain mitochondria from both female and male rats. Thus, in contrast to the lack of effect of androgens on mitochondrial ROS, estrogen suppression of mitochondrial oxidative stress may influence neurological disease incidence and progression in both females and males.

IMPORTANCE Few stroke survivors meet recommended cardiovascular goals, particularly among racial/ethnic minority populations, such as Black or Hispanic individuals, or socioeconomically disadvantaged populations. OBJECTIVE To determine if a chronic care model-based, community health worker (CHW), advanced practice clinician (APC; including nurse practitioners or physician assistants), and physician team intervention improves risk factor control after stroke in a safety-net setting (ie, health care setting where all individuals receive care, regardless of health insurance status or ability to pay). DESIGN, SETTING, AND PARTICIPANTS This randomized clinical trial included participants recruited from 5 hospitals serving low-income populations in Los Angeles County, California, as part of the Secondary Stroke Prevention by Uniting Community and Chronic Care Model Teams Early to End Disparities (SUCCEED) clinical trial. Inclusion criteria were age 40 years or older; experience of ischemic or hemorrhagic stroke or transient ischemic attack (TIA) no more than 90 days prior; systolic blood pressure (BP) of 130 mm Hg or greater or 120 to 130 mm Hg with history of hypertension or using hypertensive medications; and English or Spanish language proficiency. The exclusion criterion was inability to consent. Among 887 individuals screened for eligibility, 542 individuals were eligible, and 487 individuals were enrolled and randomized, stratified by stroke type (ischemic or TIA vs hemorrhagic), language (English vs Spanish), and site to usual care vs intervention

Activation of inflammatory mechanisms contributes to cerebrovascular pathophysiology. Male gender is associated with increased stroke risk, yet little is known about the effects of testosterone in the cerebral circulation. Therefore, we explored the impact of testosterone treatment on cerebrovascular inflammation with both in vivo and in vitro models of inflammation. We hypothesized that testosterone would augment the expression of two vascular markers of cellular inflammation, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Using four groups of male rats [intact, orchiectomized (ORX), and ORX treated with either testosterone (ORXT) or the testosterone metabolite 17beta-estradiol (ORXE)], we determined effects of the sex hormones on cerebrovascular inflammation after intraperitoneal LPS injection. Western blot analysis showed that induction of inflammatory markers was increased in cerebral blood vessels from ORXT rats compared with intact or ORX rats. In contrast, in cerebral blood vessels from ORXE rats, there was a significant decrease in endotoxin-induced COX-2 and iNOS protein levels. Confocal microscopy of cerebral blood vessels from ORXT rats showed increased COX-2 and iNOS immunoreactivity in both endothelial and smooth muscle cells after LPS treatment. In vitro incubation with LPS also induced COX-2 in pial vessels isolated from the four animal treatment groups, with the greatest induction observed in ORXT vessels compared with the ORX and ORXE groups. Production of PGE2, a principal COX-2-derived prostaglandin end product, was also greatest in cerebral vessels isolated from ORXT rats. In conclusion, testosterone increases cerebrovascular inflammation; this effect may contribute to stroke differences between men and women.