Miller, Virginia M.

Objective Preeclampsia, a hypertensive pregnancy disorder, increases the risk for cardiovascular disease including coronary artery calcification (CAC) as women age. Controversy exists regarding the relationship between bone mineral density (BMD) and CAC in menopausal women. This study was designed to determine and compare the relationship between BMD and CAC in postmenopausal women with defined histories of either normotensive or preeclamptic pregnancies. Methods Women who gave birth in Olmsted County, MN between 1976 and 1982 were identified from their medical record as having a preeclamptic pregnancy (PE, n=40) and were age/parity‐matched with women having normotensive pregnancies (NP, n=40). The study was approved by the IRB at Mayo Clinic and all participants gave written informed consent. Quantitative Computed Tomography was used to assess vertebral (T7‐9) BMD and CAC. Statistical analyses included linear regression with general estimating equation (GEE) to correct for within‐subject correlations. Results Women averaged 35 years since the index pregnancy and were 60 years of age at the time of the study. CAC was significantly greater in women of the PE group (median (25 th , 75 th percentiles: 0 (0, 0.3) NP and 0 (0, 25) PE, P=0.026). There were no significant differences in cortical, trabecular or central BMD between PE and NP groups. However, there was a positive correlation between cortical BMD and CAC in the PE group (ρ=0.336, P=0.042) and a negative correlation between central BMD and CAC in the NP group (ρ=−0.392, P=0.017). Conclusions Although CAC was greater in women with a history PE, BMD did not differ from women with a history of NP. Associations between cortical and central BMD with CAC differed by pregnancy history suggesting that there may be underlying differences in calcium metabolism associated with PE. Support or Funding Information This work was funded by grants from the NIH AG44170, AG034676, UL1 TR000135 and the Mayo Foundation.

Orexin A (OxA) is a hypothalamic neuropeptide involved in regulation of feeding behavior, metabolism, sleep‐wakefulness and emotion. Plasma levels of this peptide increase with age in both men and women. However, little is known about changes in circulating OxA levels during age‐associated, sex‐ specific hormonal changes, such as menopause, or the effect of menopausal hormone therapy (MHT) on OxA levels. No studies have assessed changes of OxA levels after discontinuation of MHT. The objective of this study was to assess plasma OxA during and three years after cessation of either oral conjugated equine estrogen (o‐CEE, n = 17), transdermal 17β‐estradiol (t‐E2; n= 20), or placebo pills and patches (n = 22) in healthy recently menopausal women (> 6 months < 3 years from their menses) who participated in the Kronos Early Estrogen Prevention Study. The plasma OxA was measured by enzyme‐linked immune absorbent assay prior to randomization to treatment (Baseline) and at 12 months, 24 months, 36 months, 48 months after randomization and at 36 months after cessation of study MHT. Data are presented as median with 25 th and 75 th percentile. Baseline plasma levels of OxA did not differ among groups (o‐CEE: 3.02 (2.2–4.4) ng/mL, t‐E2=4.09 (3.6–4.5) ng/mL, Placebo= 3.15 (2.5–6.2) ng/mL. During MHT treatment, women randomized to o‐CEE had higher OxA levels when compared to their baseline values at 24, 36 and 48 months of exposure (4.4 ng/mL (2.5–6.3), 4.1 ng/mL(2.5–7.3), 4.2 ng/mL(3.1–6.5), respectively). Changes of OxA levels in the t‐E2 and placebo groups were variable and did not show a consistent trend over the MHT treatment period. Three years after cessation of MHT, only the o‐CEE group showed significantly higher Ox A levels than during the treatment phase of the trial (treatment 3 year discontinuation median – 48 month treatment median =1.369 ng/mL). Our study suggests that the formulation of MHT, in particular o‐CEE (contain metabolites of estrogen) increases plasma OxA. This is the first study to assess Ox A levels for a period of more than 6 months under MHT and after years of discontinuation. If plasma levels of OxA reflect those in the brain, these results may explain in part the beneficial effects of o‐CEE on mood and other menopausal symptoms in women of KEEPS. An increase in OxA driven by o‐CEE could be a mechanism by which MHT helps women regulate energy metabolism and emotional behavior responses. Support or Funding Information This project was supported by the Mayo Clinic Office of Health Disparities Research and NIH AG 44170. In addition DC was supported by the CTSA Grant Number TL1 TR000137 from the National Center for Advancing Translational Science, a component of the National Institutes of Health.

Women who have experienced hypertensive pregnancy disorders (HPD) are at an increased risk for developing cardiovascular disease later in life, including fatal and non‐fatal stroke. Cerebrovascular reactivity is related to stroke in individuals with carotid artery disease, and it is possible that this measure may be altered in women with a history of HPD prior to overt cerebrovascular disease. It is unknown if cerebrovascular reactivity is altered in postmenopausal women decades after experiencing a hypertensive pregnancy. Therefore, the goal of this study was to determine if cerebral blood flow velocity and cerebrovascular reactivity are altered in women with a history of HPD in comparison to women with no history of HPD. We studied a cohort of postmenopausal women approximately 35 years after they experienced an HPD (n=27, age at testing: 58±1) and a group of age‐ and parity‐matched women with no history of HPD (n=30, age at testing: 59±1). We measured middle cerebral artery velocity (MCAv, via transcranial Doppler), continuous blood pressure (finger photoplethysmography), and end‐tidal CO 2 (nasal cannula) in these women at baseline (room air) and during stepped hypercapnia at 2, 4, and 6% inhaled CO 2 for 3 minutes at each condition. Women with a history of HPD had a higher body mass index than women without a history of HPD (29±1 vs. 26±1 kg/m 2 , respectively; p<0.05). At baseline and during 6% CO 2 , MCAv was lower in women with a history of HPD (baseline: 60±4 vs. 71±3 cm/s, respectively; 6% CO 2 : 82±6 vs. 99±5 cm/s, respectively; p<0.05 for both conditions) compared to women without a history of HPD. Cerebrovascular reactivity, the slope defining the association between MCAv and end‐tidal CO 2 across the entire breathing trial, was also lower in women with a history of HPD (2.0±0.2 vs. 2.7±0.2 cm/s/mmHg, respectively; p<0.05). Body mass index was not correlated with MCAv or cerebrovascular reactivity in either group (p>0.05). In conclusion, resting MCAv and cerebrovascular reactivity to CO 2 is lower in women with a history of HPD in comparison with women who have never experienced HPD. Our findings suggest that HPD may have long‐term effects on cerebrovascular regulation in women who have suffered from these conditions. Support or Funding Information NIH HL083947, NIA 1P50AG044170‐01, and CTSA UL1TR000135

The disruption of the cerebral microvascular endothelium and increases in capillary permeability are characteristic of many brain pathologies, including Alzheimer's disease, small vessel disease, ischemia/hypoxia, and hemorrhagic stroke. This study was designed to investigate whether microvesicles (MVs) released from activated human brain microvascular endothelial cells (HBMECs) alter permeability of endothelial monolayers. Male donor HBMECs were cultured to confluence in flasks, then treated for 24 hrs with medium alone (control) or with medium plus either tumor necrosis factor (TNF a , 20 ng/ml), thrombin (THR, 2 U/ml), angiotensin II (AngII, 10 nM), or hydrogen peroxide (H 2 O 2 , 0.8 mM). MVs released into the medium were isolated by differential centrifugation, labeled with annex in V plus antibodies against endothelial adhesion molecules which have modified expression during inflammation (PECAM‐1, ICAM‐1, E‐select in, integrin aV, or MCAM); and then analyzed by FACS. Total number of MVs released (MVs/ml) by each of the agonists (TNF a , 615.8±13.9 MV/μL; THR, 113.8±4.8 MV/μL; and H 2 O 2, 152.8±23.5 MV/μL) were significantly greater (P<0.05) compared the number released from cells treated medium alone (82.2±5.4 MV/μL). Each agonist treatment released distinct types of MV identified by FACS. Permeability (% control) of the HBMEC monolayer assessed using FITC‐dextran transport across the monolayers increased after 90 minute exposure to all agonist treatments: 199.5%, 234.1%, 126.8%, and 588.3% for TNF a , THR, AngII, and H 2 O 2 , respectively. MV derived from the treated cells were labeled with PKH67 and applied to monolayers of (control) HBMECs cultures. Within 30 minutes of exposure punctate structures with PKH67 labeling were observed within the cytoplasm, and after 60 min, similar structures also associated with nuclei and some of which were co‐labeled with the lysosome marker lysotracker red. Co‐localization of labeled MVs with early endosomes, (labeled with anti‐EEA‐1 Ab/Alexa Fluor 647) was absent at these time points. Within these parameters, no differences in vesicle‐trafficking were observed between MVs generated from cells exposed to either medium, TNF a , or H 2 O 2 . These results suggest that treatment of HBMECs with agonists associated with inflammation and thrombosis increase release of distinct MVs which in turn increase paracellular permeability. Capillary endothelial permeability may be disturbed by populations of MVs released from proximal activated cells or MV derived from activated cells and carried in the blood. Support or Funding Information Supported by the Aurora Fnd, NIH HL88988 and Mayo Clinic

Abstract Objective This study aimed to determine long-term cardiometabolic effects of hormone therapies initiated within 3 years of onset of menopause after a 14-year follow-up study of participants of the Kronos Early Estrogen Prevention Study (KEEPS). Methods KEEPS was a multisite clinical trial that recruited recently menopausal women with good cardiovascular health for randomization to oral conjugated equine estrogens (Premarin, 0.45 mg/d) or transdermal 17β-estradiol (Climara, 50 μg/d) both with micronized progesterone (Prometrium, 200 mg/d) for 12 d/mo, or placebo pills and patch for 4 years. KEEPS continuation recontacted KEEPS participants 14 years after randomization and 10 years after the completion of the 4-year clinical trial to attend in-person clinic visits. Results Participants of KEEPS continuation (n = 299 of the 727 KEEPS participants; 41%) had an average age of 67 years (range, 58-73 y). Measurements of systolic and diastolic blood pressures, waist-to-hip ratio, fasting levels of glucose, insulin, lipid profiles, and homeostasis model assessment of insulin resistance were not different among the treatment groups at either KEEPS baseline or at KEEPS continuation visits, or for change between these two visits. The frequency of self-reported diabetes ( P = 0.007) and use of diabetes medications was higher in the placebo than the oral conjugated equine estrogens ( P = 0.045) or transdermal 17β-estradiol ( P = 0.02) groups, but these differences were not supported by the laboratory measurements of glycemia or insulin resistance. Conclusions There was no evidence of cardiovascular and/or metabolic benefits or adverse effects associated with 4 years use of oral or transdermal forms of hormone therapy by recently menopausal women with good cardiovascular health after 10 years.

AbstractBackgroundWomen have higher rates of hypertension at the time of menopausal transition than men. Although elevated systolic and diastolic blood pressures (SBP and DBP) are risk factors for white matter (WM) injury both in women and men, WM hyperintensities (WMH), a marker of WM injury, are more common in women than in men after the age of 60. Thus, women may be vulnerable to the effects of elevated BP on WM integrity during menopausal transition. We investigated the association of BP in recently postmenopausal women with good cardiovascular health with WMH volume assessed 13 years later.MethodWomen (n = 212; median age = 67; range 58‐72), who were previously enrolled in a multi‐site randomized menopausal hormone therapy trial, Kronos Early Estrogen Prevention Study (KEEPS), participated in the present observational KEEPS Continuation Study. SBP and DBP were measured at KEEPS baseline (median age = 54 years; range 44‐59). Participants who were on antihypertensive medications at KEEPS baseline were excluded from the analysis (n = 34). Approximately 9 years after the end of KEEPS menopausal hormones versus placebo 4‐year interventions, WM integrity was assessed with automated WMH volume measurements from 3D‐FLAIR MRI. Associations of baseline SBP and DBP with logWMH volume measured 13 years later were tested by linear regression analyses. Model 1 was adjusted for age, study site, and total intracranial volume. Model 2 was adjusted for covariates of model 1, triglycerides, low density lipoproteins, and waist/hip ratio.ResultThe median SBP was 116 (range 83‐158) and DBP was 74 (range 56‐98). Higher SBP was associated with greater WMH volume (model 1:p = 0.005; model 2:p = 0.005). Similarly, higher DBP was associated with greater WMH volume (model 1:p = 0.008; model 2:p = 0.006) (Figure 1). Neither menopausal hormone therapies versus placebo, nor having high/low SBP (≥120/<120) modified these associations. Voxel‐based analyses demonstrate the association of BPs with the spatial distribution of WMHs (Figure 2).ConclusionHigher BPs in recently menopausal women were associated with greater WM injury more than a decade later after adjusting for CVD risk factors. Early postmenopausal stage is a critical time for BP control in women, which may reduce the risk of WM injury later in life.

Cardiovascular (CV) disease (CVD) is the major health burden and cause of death for women. Marked disparities exist in CVD diagnosis, prevention, and treatment between women and men – as well as lack of female-specific data. Population, physiologic, translational, and clinical trial studies of sex and gender differences in CVD [[Unable to Display Character: &#8211;]] even when only women are studied[[Unable to Display Character: &#8211;]] often do not collect relevant data specific to women that could inform study outcomes. The ISIS CVD Network of the Society for Women’s Health Research compiled an inventory of items specific for women across the lifespan, together with references for methods and strategies to gather and evaluate this information; some items comprise robust measures, others are in development. The objective is to enhance usefulness of CVD research data in understanding sex and gender differences, thereby optimizing healthcare delivery and outcomes for women. Included are hormonal variables (menstrual cycle phase, hormone levels) oral contraceptive use, pregnancy history/complications, polycystic ovary syndrome (PCOS) components, measures of menopause, and menopausal hormone therapy, variables generally not collected in research studies, but essential to determine their role as sex-specific contributors to CV health and disease. Clear associations exist between reproductive health and CV health and disease. For example 25-33% of women experience complications of pregnancy that may precede and predispose to CVD. Vascular complications during pregnancy, antecedent risk factors and subsequent clinical CVD can be ascertained using medical records, birth registries, and/or maternal recall. Evaluating compilations of patient data with known hormonal or menopausal status using reference standards and patient data for PCOS could inform relationships to subsequent CV outcomes. Variables predominant among women that preferentially disadvantage them should be considered; e.g. psychosocial issues and elderly age. Depressive disorders are twice as common among women as men. They adversely affect CVD outcomes in women, yet the effect of reproductive life cycle and of hormonal fluctuations on depression and etiologic contributions of depression to CVD are inadequately explored. In addition to traditional CVD risk factors, diabetes mellitus, chronic inflammatory disorders, oxidative stress, vasomotor dysfunction, coronary microvascular disorders, and other novel risk variables that preferentially impact women should be explored. Along with increased enrollment of women in CVD research studies and analysis of clinical and genetic studies by sex, improvements and expansion of study design must include these understudied uniquely or predominantly female characteristics. This will enhance the quality and quantity of evidence-based medicine to guide CVD care in women and men thereby setting the stage for personalized approach to medicine.

AbstractBackgroundElevated systolic blood pressure (SBP) and diastolic blood pressure (DBP) are risk factors for white matter (WM) injury associated with cognitive decline and dementia. White matter hyperintensities (WMH) on MRI, markers of WM injury, increase with aging and are more common in women than in men after the age of 60. We examined SBP and DBP in relation to WMH volume and diffusion MRI measures of WM integrity in postmenopausal women.MethodWomen (n=144) with median age of 67 (range 58‐72) and of good cardiovascular health who were previously enrolled in the multi‐site randomized clinical trial, Kronos Early Estrogen Prevention Study (KEEPS), participated in the present observational KEEPS Continuation Study. SBP, DBP, and MRI data were collected approximately 9 years after the end of KEEPS menopausal hormones versus placebo 4‐year interventions. WM integrity was assessed with WMH volume measurements (log transformed) on structural MRI and with measures of WM microstructure on diffusion MRI. Multiple linear regression analyses, adjusted for age, were used to test for associations of SBP and DBP with MRI measures.ResultThe median SBP was 128 (range 86‐180) and DBP was 77 (range 47‐106). Higher SBP (r=0.332;p=0.015) and DBP (r=0.346;p=0.007) were associated with higher WMH volumes. Among the WM tracts, higher SBP was associated with lower fractional anisotropy (FA) in lateral orbitofrontal gyrus WM (r=0.198;p=0.039) and sagittal stratum (r=0.226;p=0.043), and higher DBP was associated with lower FA in the superior occipital gyrus WM (r=0.370;p=0.045). Higher SBP and DBP were associated with higher mean diffusivity (MD) in the external capsule [(r=0.311;p=0.007);(r=375;p<0.001)] and the fronto‐occipital fasciculus [(r=0.286;p=0.016); (r=0.303;p=0.008)]. Furthermore, higher DBP was associated with higher MD in the anterior corona radiata (r=0.233;p=0.039), internal capsule (r=0.303;p=0.042), and the middle (r=0.275; p=0.03) and superior frontal gyrus (r=0.269;p=0.03) WM.ConclusionElevated SBP and DBP measurements are associated with greater WM injury in postmenopausal women. In particular, the frontal lobe WM and its connecting tracts may be more vulnerable to elevation in SBP and DBP. Whether BP control reduces the risk for WM injury, a risk factor for dementia, in post‐menopausal women warrants study.

AbstractBackgroundResearch on menopausal hormone therapy (MHT) for the reduction/prevention of cognitive decline or Alzheimer’s disease has been inconsistent. The Women’s Health Initiative Memory Study (WHIMS) revealed elevated incident cognitive impairment for women starting MHT after age >65 with oral conjugated equine estrogens (o‐CEE) treatment, especially among women with diabetes. Subsequently, the Cognitive and Affective substudy of the Kronos Early Estrogen Prevention Study (KEEPS‐Cog) found that MHT initiated soon after menopause caused neither cognitive benefit nor harm. Waist‐to‐hip ratio (WHR) is a measure of central adiposity and a surrogate marker for insulin resistance/prediabetes. To explore effects of MHT in insulin‐resistant states, in younger women, we analyzed WHR as a predictor of cognitive response to MHT in KEEPS‐Cog.MethodsKEEPS‐Cog randomized women less than 36‐months postmenopause to oCEE, transdermal 17‐β‐estradiol (tE2) or placebo for 48 months. Cognitive tests, completed at baseline, 18, 36, and 48 months post‐randomization were summarized as factor scores representing four cognitive domains: verbal learning and memory (VLM), auditory attention and working memory (AAWM), visual attention and executive function (VAEF), and speeded language and mental flexibility (SLMF). WHR, measured at screening and months 12, 36, and 48 was modeled as a time‐varying moderator of MHT on cognitive function over time. Education was included as a time‐invariant covariate.ResultsWHR decreased throughout the study in both the tE2 and oCEE groups compared to placebo. WHR was also inversely associated with all cognitive parameters at baseline. The inverse relationship of WHR on SLMF was significant at baseline and month 18, but decreased at month 48. The inverse relationship of WHR on VLM increased significantly over time. Models including interaction effects were significant for oCEE x WHR effects at months 18, 36, and 48 on SLMF, indicating better performance in MHT vs placebo arms. No other interaction effects were significant.ConclusionsWHR decreased with MHT, and was consistently associated with VLM through 4 years of MHT. Further, women on oCEE performed better on SLMF tasks at a given WHR. Differences between KEEPS‐Cog and WHIMS findings may be related to the timing of MHT administration, and effects on central adiposity/insulin resistance/prediabetes.

Abstract Women with premenopausal bilateral oophorectomy (PBO) are at increased risk for physical function (PF) declines. This study investigated the relationships of field-based physical activity measures with clinical PF and strength parameters in post-menopausal women with and without PBO. Women with (n=21; age=64±4 years; BMI=32±8 kg.m-2) and without (n=15; age=67±6 years; BMI=28±6 kg.m-2) PBO performed PF and strength tests (walking speed, distance walked, short physical performance battery (SPBB), leg and chest strength), and wore ankle accelerometers for 7 days (daily step count and loading index [the cumulative sum of each step’s skeletal loading]). Age, BMI, step count and loading index were entered into stepwise multiple regression to identify significant predictors of PF and strength parameters. Step count was a predictor of SPBB score in both groups. In women without PBO, step count was a predictor of walking speed; loading index was a predictor of leg strength; step count and loading index were predictors of distance; and step count and age were predictors of chest strength. For PBO women, loading index and BMI were predictors of walking speed and distance; BMI was a predictor of leg strength; and there were no predictors of chest strength. These data suggest while field-based physical activity was strongly and positively associated with clinical PF and strength measures for women without PBO, BMI was a dominant negative factor for PF in women with PBO. Future work will include a larger sample size and additional confounders to further elucidate underlying factors of reduced PF and mobility after PBO.