Kathleen M. Gavin

Vascular Endothelial Estrogen Receptor α Is Modulated by Estrogen Status and Related to Endothelial Function and Endothelial Nitric Oxide Synthase in Healthy Women

CONTEXT AND OBJECTIVE Estrogen receptor alpha (ER alpha), a potent transcription factor expressed in vascular endothelial cells, plays a key role in regulating vascular function and health. We determined whether vascular endothelial cell expression of ER alpha is influenced by estrogen status and is related to vascular endothelial function in healthy women. METHODS ER alpha protein expression was measured (quantitative immunofluorescence) in endothelial cells from peripheral veins of 16 healthy, premenopausal women during the early follicular (EF) and late follicular (LF) phases of the menstrual cycle and 17 estrogen-deficient postmenopausal women. Endothelial-dependent dilation (EDD; brachial artery flow-mediated dilation) and endothelial nitric oxide synthase (eNOS) expression and activation were also measured in a subgroup of women. RESULTS In premenopausal women (n = 10), ER alpha expression was 30% lower (P < 0.001) during the EF (low estrogen) compared with the LF (high estrogen) phase of the menstrual cycle. In postmenopausal women, ER alpha expression was 33% lower (P < 0.001) compared with the LF phase of the menstrual cycle in premenopausal women. ER alpha expression was strongly related (r = 0.67; P < 0.001) to EDD, which was reduced in postmenopausal women. ER alpha abundance was positively related to expression of eNOS (r = 0.54; P = 0.009; n = 21) and ser1177 phosphorylated eNOS (r = 0.59; P = 0.006; n = 20). CONCLUSIONS These results provide the first evidence that expression of ER alpha in vascular endothelial cells is modulated by estrogen status and may be a key determinant of vascular endothelial function in healthy pre- and postmenopausal women. ER alpha expression may influence vascular endothelial function in women by affecting protein levels and activation of eNOS.

Ascorbic Acid Selectively Improves Large Elastic Artery Compliance in Postmenopausal Women

The compliance of large elastic arteries in the cardiothoracic region decreases with advancing age/menopause and plays an important role in the increased prevalence of cardiovascular diseases in postmenopausal women. We determined whether oxidative stress contributes to the reduced large elastic artery compliance of postmenopausal women. Carotid artery compliance was measured during acute intravenous infusions of saline (baseline control) and supraphysiological doses of the potent antioxidant ascorbic acid in premenopausal (n=10; 23±1; mean±SE) and estrogen-deficient postmenopausal (n=21; 55±1 years) healthy sedentary women. Carotid artery compliance was 56% lower in postmenopausal compared with premenopausal women during baseline control (P<0.0001). Ascorbic acid infusion increased carotid artery compliance by 26% in postmenopausal women (1.11±0.07 to 1.38±0.08 mm2/mm Hg×10−1; P<0.001) but had no effect in premenopausal women (2.50±0.25 versus 2.43±0.20 mm2/mm Hg×10−1). Carotid artery diameter, blood pressure, and heart rate were unaffected by ascorbic acid. In the pooled population, the change in arterial compliance with ascorbic acid correlated with baseline waist-to-hip ratio (r=0.56; P=0.001), plasma norepinephrine (r=0.58; P=0.001), and LDL cholesterol (r=0.54; P=0.001). These results suggest that oxidative stress may be an important mechanism contributing to the reduced large elastic artery compliance of sedentary, estrogen-deficient postmenopausal women. Increased abdominal fat storage, sympathetic nervous system activity, and LDL cholesterol may be mechanistically involved in oxidative stress–associated suppression of arterial compliance in postmenopausal women.

Oxidative stress explains differences in large elastic artery compliance between sedentary and habitually exercising postmenopausal women.

Objective: To determine whether oxidative stress contributes to differences in large elastic artery compliance between sedentary and habitually exercising postmenopausal women. Design: Carotid artery compliance was measured during acute intravenous infusions of saline (control) and supraphysiological doses of the potent antioxidant ascorbic acid (vitamin C) in sedentary (n = 15; 58 ± 1 years) and endurance exercise-trained (n = 11, 59 ± 1) healthy postmenopausal women. Results: Carotid artery compliance was 24% higher in the exercising versus sedentary women during control (P < 0.001). During ascorbic acid infusion, carotid artery compliance was increased by 28% in the sedentary women (1.29 ± 0.12 to 1.60 ± 0.12 mm2/mm Hg × 10−1, P < 0.001 vs control) but was unchanged in exercising women (1.60 ± 0.14 vs 1.48 ± 0.14 mm2/mm Hg × 10−1, P = 0.10), abolishing the habitual exercise-associated baseline difference. The change in compliance with ascorbic acid was most strongly related to maximal aerobic capacity (r = −0.64, P < 0.0001) and body fatness (r = 0.60, P < 0.0001) and was more modestly related to oxidized low-density lipoprotein, waist circumference, interleukin-6, total and low-density lipoprotein cholesterol (all r = 0.40 to 0.49, all P < 0.05), and high-density lipoprotein cholesterol (r = −0.48, P = 0.01). Carotid artery diameter, blood pressure, and heart rate were unaffected by ascorbic acid. Conclusions: These results indicate that the greater large elastic artery compliance in habitually exercising compared with sedentary estrogen-deficient postmenopausal women may be mediated by an absence of oxidative stress, perhaps related in part to more favorable cardiovascular risk factors.

De novo generation of adipocytes from circulating progenitor cells in mouse and human adipose tissue

White adipocytes in adults are typically derived from tissue resident mesenchymal progenitors. The recent identification of de novo production of adipocytes from bone marrow progenitor‐derived cells in mice challenges this paradigm and indicates an alternative lineage specification that adipocytes exist. We hypothesized that alternative lineage specification of white adipocytes is also present in human adipose tissue. Bone marrow from transgenic mice in which luciferase expression is governed by the adipocyte‐restricted adiponectin gene promoter was adoptively transferred to wild‐type recipient mice. Light emission was quantitated in recipients by in vivo imaging and direct enzyme assay. Adipocytes were also obtained from human recipients of hematopoietic stem cell transplantation. DNA was isolated, and microsatellite polymorphisms were exploited to quantify donor/recipient chimerism. Luciferase emission was detected from major fat depots of transplanted mice. No light emission was observed from intestines, liver, or lungs. Up to 35% of adipocytes in humans were generated from donor marrow cells in the absence of cell fusion. Nontransplanted mice and stromal‐vascular fraction samples were used as negative and positive controls for the mouse and human experiments, respectively. This study provides evidence for a nontissue resident origin of an adipocyte subpopulation in both mice and humans.—Gavin, K. M., Gutman, J. A., Kohrt, W. M., Wei, Q., Shea, K. L., Miller, H. L., Sullivan, T. M., Erickson, P. F., Helm, K. M., Acosta, A. S., Childs, C. R., Musselwhite, E., Varella‐Garcia, M., Kelly, K., Majka, S. M., Klemm, D. J., De novo generation of adipocytes from circulating progenitor cells in mouse and human adipose tissue. FASEB J. 30, 1096–1108 (2016). www.fasebj.org

Body composition and bone mineral density after ovarian hormone suppression with or without estradiol treatment.

Objective:Suppression of ovarian hormones in premenopausal women on gonadotropin-releasing hormone agonist (GnRHAG) therapy can cause fat mass (FM) gain and fat-free mass (FFM) loss. Whether this is specifically caused by a decline in serum estradiol (E2) is unknown. This study aims to evaluate the effects of GnRHAG with placebo (PL) or E2 add-back therapy on FM, FFM, and bone mineral density (BMD). Our exploratory aim was to evaluate the effects of resistance exercise training on body composition during the drug intervention. Methods:Seventy healthy premenopausal women underwent 5 months of GnRHAG therapy and were randomized to receive transdermal E2 (GnRHAG + E2, n = 35) or PL (GnRHAG + PL, n = 35) add-back therapy. As part of our exploratory aim to evaluate whether exercise can minimize the effects of hormone suppression, some women within each drug arm were randomized to undergo a resistance exercise program (GnRHAG + E2 + Ex, n = 12; GnRHAG + PL + Ex, n = 12). Results:The groups did not differ in mean (SD) age (36 [8] and 35 [9] y) or mean (SD) body mass index (both 28 [6] kg/m2). FFM declined in response to GnRHAG + PL (mean, −0.6 kg; 95% CI, −1.0 to −0.3) but not in response to GnRHAG + E2 (mean, 0.3 kg; 95% CI, −0.2 to 0.8) or GnRHAG + PL + Ex (mean, 0.1 kg; 95% CI, −0.6 to 0.7). Although FM did not change in either group, visceral fat area increased in response to GnRHAG + PL but not in response to GnRHAG + E2. GnRHAG + PL induced a decrease in BMD at the lumbar spine and proximal femur that was prevented by E2. Preliminary data suggest that exercise may have favorable effects on FM, FFM, and hip BMD. Conclusions:Suppression of ovarian E2 results in loss of bone and FFM and expansion of abdominal adipose depots. Failure of hormone suppression to increase total FM conflicts with previous studies of the effects of GnRHAG. Further research is necessary to understand the role of estrogen in energy balance regulation and fat distribution.

Regulation of Body Composition and Bioenergetics by Estrogens

Evidence points to an important role of estradiol (E2) in the regulation of body composition and bioenergetics. Basic and preclinical research shows that the disruption of E2 signaling through either genetic manipulation or surgical intervention accelerates fat accumulation, with a disproportionate increase in abdominal fat. Clinical evidence for the regulation of body composition and bioenergetics by E2 is less consistent. Evidence exists both for and against menopause as the mediator of changes in body composition. Thus, a need remains to better understand the metabolic actions of estrogens in women and the potential impact on health after the menopause.

Regulation of energy expenditure by estradiol in premenopausal women

Suppressing sex hormones in women for 1 wk reduces resting energy expenditure (REE). The effects of more chronic suppression on REE and other components of total energy expenditure (TEE), and whether the reduction in REE is specifically due to loss of estradiol (E2), are not known. We compared the effects of 5 mo of sex hormone suppression (gonadotropin releasing hormone agonist therapy, GnRHAG) with placebo (PL) or E2 add-back therapy on REE and the components of TEE. Premenopausal women received GnRHAG (leuprolide acetate 3.75 mg/mo) and were randomized to receive transdermal therapy that was either E2 (0.075 mg/d; n = 24; means ± SD, aged = 37 ± 8 yr, BMI = 27.3 ± 6.2 kg/m(2)) or placebo (n = 21; aged = 34 ± 9 yr, BMI = 26.8 ± 6.2 kg/m(2)). REE was measured by using a metabolic cart, and TEE, sleep EE (SEE), exercise EE (ExEE, 2 × 30 min bench stepping), non-Ex EE (NExEE), and the thermic effect of feeding (TEF) were measured by using whole room indirect calorimetry. REE decreased in GnRHAG+PL [mean (95% CI), -54 (-98, -15) kcal/d], but not GnRHAG+E2 [+6 (-33, +45) kcal/d] (difference in between-group changes, P < 0.05). TEE decreased in GnRHAG+PL [-128 (-214, -41) kcal/d] and GnRHAG+E2 [-96 (-159, -32) kcal/d], with no significant difference in between-group changes (P = 0.55). SEE decreased similarly in both GnRHAG+PL [-0.07 (-0.12, -0.03) kcal/min] and GnRHAG+E2 [-0.07 (-0.12, -0.02) kcal/min]. ExEE decreased in GnRHAG+PL [-0.46 (-0.79, -0.13) kcal/min], but not GnRHAG+E2 [-0.30 (-0.65, +0.06) kcal/min]. There were no changes in TEF or NExEE in either group. In summary, chronic pharmacologic suppression of sex hormones reduced REE and this was prevented by E2 therapy.

HYSTERECTOMY IS ASSOCIATED WITH LARGE ARTERY STIFFENING IN ESTROGEN-DEFICIENT POSTMENOPAUSAL WOMEN

Objective Hysterectomy, with or without oophorectomy, is associated with increased cardiovascular disease (CVD) risk due, in part, to an adverse CVD risk factor profile. Large artery stiffening, a biomarker of vascular aging, increases the risk for CVD. We determined whether hysterectomy with or without bilateral oophorectomy (BLO) is associated with arterial stiffening in healthy postmenopausal women. Methods We conducted a cross-sectional study including estrogen-deficient postmenopausal women who had a hysterectomy with ovarian preservation (n = 24; mean ± SE age, 59 ± 1 y) or with BLO (n = 21; 58 ± 2 y) and had no hysterectomy/no BLO (n = 58; 58 ± 1 y). Arterial stiffness (arterial compliance and &bgr; stiffness index) was measured by ultrasonography of the carotid artery. Results Carotid artery compliance was lower in women with hysterectomy alone and in women with hysterectomy with BLO compared with women with no hysterectomy (0.66 ± 0.03 and 0.71 ± 0.06 vs 0.89 ± 0.03 mm2/mm Hg × 10−1, respectively, both P < 0.05). There were no differences in traditional CVD risk factors (ie, adiposity, blood pressure and fasted lipids and lipoproteins, glucose, and insulin) between the groups. After adjustment for age, menopause duration, previous menopausal hormone therapy duration, parity, waist-to-hip ratio, systolic blood pressure, and sex hormone–binding globulin, hysterectomy status remained a significant predictor of arterial compliance. Conclusions These results indicate that hysterectomy status (with or without BLO) is associated with greater arterial stiffening in estrogen-deficient postmenopausal women. The greater arterial stiffening with hysterectomy was not related to an adverse CVD risk profile. Large artery stiffening may be an important mechanism by which hysterectomy increases the risk of CVD in postmenopausal women.

Hematopoietic-to-mesenchymal transition of adipose tissue macrophages is regulated by integrin β1 and fabricated fibrin matrices

ABSTRACT Some bona fide adult adipocytes arise de novo from a bone marrow-derived myeloid lineage. These studies further demonstrate that adipose tissue stroma contains a resident population of myeloid cells capable of adipocyte and multilineage mesenchymal differentiation. These resident myeloid cells lack hematopoietic markers and express mesenchymal and progenitor cell markers. Because bone marrow mesenchymal progenitor cells have not been shown to enter the circulation, we hypothesized that myeloid cells acquire mesenchymal differentiation capacity in adipose tissue. We fabricated a 3-dimensional fibrin matrix culture system to define the adipose differentiation potential of adipose tissue-resident myeloid subpopulations, including macrophages, granulocytes and dendritic cells. Our data show that multilineage mesenchymal potential was limited to adipose tissue macrophages, characterized by the acquisition of adipocyte, osteoblast, chondrocyte and skeletal muscle myocyte phenotypes. Fibrin hydrogel matrices stimulated macrophage loss of hematopoietic cell lineage determinants and the expression of mesenchymal and progenitor cell markers, including integrin β1. Ablation of integrin β1 in macrophages inhibited adipocyte specification. Therefore, some bona fide adipocytes are specifically derived from adipose tissue-resident macrophages via an integrin β1-dependent hematopoietic-to-mesenchymal transition, whereby they become capable of multipotent mesenchymal differentiation. The requirement for integrin β1 highlights this molecule as a potential target for controlling the production of marrow-derived adipocytes and their contribution to adipose tissue development and function.

Ovarian Hormones Regulate the Production of Adipocytes From Bone Marrow-Derived Cells

Sex differences in body fat distribution and menopause-associated shifts in regional adiposity suggest that sex hormones play an important role in regulating the differentiation and distribution of adipocytes, but the underlying mechanisms have not been fully explained. The aim of this study was to determine whether ovarian hormone status influences the production and distribution of adipocytes in adipose tissue arising from bone marrow-derived cells. Nine- to ten-week-old ovariectomized (OVX), surgery naïve (WT), and estrogen receptor alpha knockout (αERKO) mice underwent bone marrow transplantation from luciferase or green fluorescent protein expressing donors. A subset of OVX animals had estradiol (E2) added back. Eight-weeks posttransplant, whole body and gonadal fat BM-derived adipocyte production was highest in OVX and αERKO mice, which was attenuated in OVX mice by E2 add-back. All groups demonstrated the highest bone marrow derived adipocyte (BMDA) production in the gonadal adipose depot, a visceral fat depot in mice. Taken together, the loss of ovarian hormones increases the production of BMDAs. If translatable across species, production of BMDA may be a mechanism by which visceral adiposity increases in estrogen-deficient postmenopausal women.