Exogenous Growth Hormone Exacerbates Post-Irradiation Atherosclerosis in Susceptible Epicardial Coronary Arteries.

Cardiac exposure to ionizing radiation can damage both the microvasculature and coronary arteries, as well as increase the long-term risk of heart disease, myocardial fibrosis, and conduction abnormalities. Therapeutic agents capable of promoting recovery from radiation injury to the heart are limited. Growth hormone is linked to improved cardiac function following injury. Here, we leveraged a cynomolgus macaque model to determine the long-term outcomes of recombinant human growth hormone (rhGH) therapy on the heart following low-dose ionizing radiation. Macaques were exposed to 2 Gy radiation, treated with rhGH for one month, and assessed after 2 years. Overall, plasma lipid profile, cardiac function, and coronary artery disease were similar between rhGH and placebo treated animals. However, a subgroup of rhGH-treated animals exhibited more extensive atherosclerotic plaques in the coronary arteries. Together, these findings indicate that transient human growth hormone therapy subsequent to a single low dose of ionizing radiation involving the heart does not result in long-term changes to plasma cholesterol but may promote exacerbated coronary artery disease in a subset of individuals.

Vitamin D heritability and effect of pregnancy status in Vervet monkeys (Chlorocebus aethiops sabaeus) under conditions of modest and high dietary supplementation.

OBJECTIVES: The two objectives of the current study were to: 1) investigate the genetic contributions to variations in serum vitamin D concentrations under two dietary conditions (a standard monkey biscuit diet vs. a diet designed to model typical American consumption); and 2) explore the interaction of vitamin D with pregnancy status using a cohort of pedigreed female vervet/African green monkeys. METHODS: This study includes 185 female (≥3.5 years) vervet/African green monkeys (Chlorocebus aethiops sabaeus) from a multi-generational, pedigreed breeding colony. The 25(OH)D3 concentrations were first measured seven to eight weeks after consuming a "typical American" diet (TAD), deriving 37, 18, and 45% of calories from fat, protein sources, and carbohydrates, and supplemented with vitamin D to a human equivalent of 1,000 IU/day. Vitamin D concentrations were assessed again when animals were switched to a low-fat, standard biscuit diet (LabDiet 5038) for 8 months, which provided a human equivalent of approximately 4,000 IU/day of vitamin D. All statistical analyses were implemented in SOLAR. RESULTS: Pregnancy was associated with reduced 25(OH)D3 concentrations. Heritability analyses indicated a significant genetic contribution to 25(OH)D3 concentrations in the same monkeys consuming the biscuit diet (h(2) =0.66, P=0.0004) and TAD (h(2) =0.67, P=0.0078) diets, with higher 25(OH)D3 concentrations in animals consuming the biscuit diet. Additionally, there was a significant genotype-by-pregnancy status interaction on 25(OH)D3 concentrations (P<0.05) only among animals consuming the TAD diet. DISCUSSION: These results support the existence of a genetic contribution to differences in serum 25(OH)D3 concentrations by pregnancy status and emphasize the role of diet (including vitamin D supplementation) in modifying genetic signals as well as vitamin D concentrations.

Effects of long-term sertraline treatment and depression on coronary artery atherosclerosis in premenopausal female primates.

OBJECTIVES: Major depressive disorder and coronary heart disease often co-occur in the same individuals. Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed for depression and other disorders, but their effects on coronary heart disease risk remain unclear. We determined the effects of an SSRI on coronary artery atherosclerosis (CAA) in an established nonhuman primate model used to clarify the association between depression and CAA. METHODS: Forty-two adult female cynomolgus macaques consuming a Western diet were characterized during an 18-month pretreatment phase and assigned to SSRI (sertraline hydrochloride 20 mg/kg, per os, once a day) or placebo balanced on pretreatment depression, body weight (BW), and iliac artery atherosclerosis extent measured via biopsy. After 18 months, CAA extent was measured using histomorphometry. RESULTS: Before and during treatment, depressed monkeys had lower BW, body mass index, and plasma high-density lipoprotein cholesterol, and higher heart rates during the pretreatment (p < .01) but not the treatment phase (p = .17). There were no pretreatment differences between the sertraline and placebo groups. Sertraline reduced anxious behavior but had no effect on BW, body mass index, heart rate, plasma lipids, or depression. CAA, analyzed by a 2 (depressed, nondepressed) × 2 (placebo, sertraline) × 3 (coronary arteries) analysis of covariance adjusted for pretreatment iliac atherosclerosis, was greater in depressed than in nondepressed monkeys (p < .036), and in sertraline than in placebo-treated monkeys (p = .040). The observed CAA extent in depressed monkeys treated with sertraline was 4.9 times higher than that in untreated depressed monkeys, and 6.5 times higher than that in nondepressed monkeys, on average. CONCLUSIONS: Depressed animals developed more CAA, and long-term treatment with sertraline resulted in more extensive CAA.

Psychosocial stress increases risk for type 2 diabetes in female cynomolgus macaques consuming a western diet.

Chronic psychosocial stress is associated with increased risk of many chronic diseases including type 2 diabetes mellitus. However, it is difficult to establish a causal relationship between stress and diabetes in human studies because stressors often are self-reported and may be distant in time from metabolic consequences. Macaques are useful models of the effects of chronic psychosocial stress on health and may develop obesity and diabetes similar to human beings. Thus, we studied the relationships between social subordination stress - a well-validated psychological stressor in macaques - and body composition and carbohydrate metabolism in socially housed, middle-aged female cynomolgus monkeys (Macaca fascicularis; n = 42). Following an 8-week baseline phase, the monkeys were fed a Western diet for 36 months (about equivalent to 10 human years). Social status was determined based on the outcomes of agonistic interactions (X¯= 33.3 observation hours/monkey). Phenotypes collected included plasma cortisol, body composition, circulating markers of glucose metabolism, activity levels, and heart rate variability measured as RMSSD (root of mean square of successive differences) and SDDN (standard deviation of beat to beat interval) after 1.5- and 3-years on diet. Mixed model analyses of variance revealed that aggression received, submissions sent, and cortisol were higher, and RMSSD and SDNN were lower in subordinates than dominants (social status: p < 0.05). After 3 years of Western diet consumption, fasting triglyceride, glucose and insulin concentrations, calculated insulin resistance (HOMA-IR), body weight and body fat mass increased in all animals (time: all p's < 0.05); however, the increase in fasting glucose and HOMA-IR was significantly greater in subordinates than dominants (time x social status: p's < 0.05). Impaired glucose metabolism, (glucose > 100 mg/dl) incidence was significantly higher in subordinates (23%) than dominants (0%) (Fisher's exact test, p < 0.05). These findings suggest that chronic psychosocial stress, on a Western diet background, significantly increases type 2 diabetes risk in middle-aged female primates.

Distribution, size, shape, growth potential and extent of abdominal aortic calcified deposits predict mortality in postmenopausal women.

BACKGROUND: Aortic calcification is a major risk factor for death from cardiovascular disease. We investigated the relationship between mortality and the composite markers of number, size, morphology and distribution of calcified plaques in the lumbar aorta. METHODS: 308 postmenopausal women aged 48-76 were followed for 8.3 ± 0.3 years, with deaths related to cardiovascular disease, cancer, or other causes being recorded. From lumbar X-rays at baseline the number (NCD), size, morphology and distribution of aortic calcification lesions were scored and combined into one Morphological Atherosclerotic Calcification Distribution (MACD) index. The hazard ratio for mortality was calculated for the MACD and for three other commonly used predictors: the EU SCORE card, the Framingham Coronary Heart Disease Risk Score (Framingham score), and the gold standard Aortic Calcification Severity score (AC24) developed from the Framingham Heart Study cohorts. RESULTS: All four scoring systems showed increasing age, smoking, and raised triglyceride levels were the main predictors of mortality after adjustment for all other metabolic and physical parameters. The SCORE card and the Framingham score resulted in a mortality hazard ratio increase per standard deviation (HR/SD) of 1.8 (1.51-2.13) and 2.6 (1.87-3.71), respectively. Of the morphological x-ray based measures, NCD revealed a HR/SD >2 adjusted for SCORE/Framingham. The MACD index scoring the distribution, size, morphology and number of lesions revealed the best predictive power for identification of patients at risk of mortality, with a hazard ratio of 15.6 (p < 0.001) for the 10% at greatest risk of death. CONCLUSIONS: This study shows that it is not just the extent of aortic calcification that predicts risk of mortality, but also the distribution, shape and size of calcified lesions. The MACD index may provide a more sensitive predictor of mortality from aortic calcification than the commonly used AC24 and SCORE/Framingham point card systems.

The unique value of primate models in translational research. Nonhuman primate models of women's health: introduction and overview.

This special issue of AJP is focused on research using nonhuman primates as models to further the understanding of women's health. Nonhuman primates play a unique role in translational science by bridging the gap between basic and clinical investigations. The use of nonhuman primates in biomedical research challenges our resolve to treat all life as sacred. The scientific community has responded by developing ethical guidelines for the care and the use of primates and clarifying the responsibility of investigators to insure the physical and psychological well-being of nonhuman primates used in research. Preclinical investigations often involve the use of animal models. Rodent models have been the mainstay of biomedical science and have provided enormous insight into the workings of many mammalian systems that have proved applicable to human biological systems. Rodent models are dissimilar to primates in numerous ways, which may limit the generalizability to human biological systems. These limitations are much less likely in nonhuman primates and in Old World primates, in particular, Macaques are useful models for investigations involving the reproductive system, bioenergetics, obesity and diabetes, cardiovascular health, central nervous system function, cognitive and social behavior, the musculoskeletal system, and diseases of aging. This issue considers primate models of polycystic ovary syndrome; diet effects on glycemic control, breast and endometrium; estrogen, reproductive life stage and atherosclerosis; estrogen and diet effects on inflammation in atherogenesis; the neuroprotective effects of estrogen therapy; social stress and visceral obesity; and sex differences in the role of social status in atherogenesis. Unmet research needs in women's health include the use of diets in nonhuman primate studies that are similar to those consumed by human beings, primate models of natural menopause, dementia, hypertension, colon cancer, and frailty in old age, and dedicated colonies for the study of breast cancer.

Coronary heart disease of females: lessons learned from nonhuman primates.

The cynomolgus monkey model has contributed to significant advances regarding the understanding of coronary artery atherosclerosis of females. There are currently 8 million women in the United States living with heart disease, necessitating further study and understanding of this leading cause of morbidity and mortality for postmenopausal women. Specifically, studies involving the monkey model have allowed greater understanding of the effect of the stage of reproductive life, time since menopause, and the extent of subclinical atherosclerosis as determinates of estrogen-mediated effects on arteries. Utilizing the commonalities among monkeys and human beings, these studies have shown that postmenopausal atherosclerosis is associated with the premenopausal reproductive timeframe. In addition, monkey studies have shown that estrogen deficiency during the premenopausal stage is extremely relevant regarding the progression of atherosclerosis. After several postmenopausal years, however, studies have shown that estrogen has no beneficial effects on atherosclerosis progression and may, in fact, be deleterious. Studies using the monkey model are currently underway to investigate further uses and possibilities of postmenopausal hormone therapy for treating menopausal symptoms while protecting the breast and uterus and inhibiting the progression of coronary artery atherosclerosis. These studies will hopefully clarify the role of estrogen and eliminate the need for the possibly harmful progestin effects through the use of a highly selective estrogen receptor modulator.

Social stress, visceral obesity, and coronary artery atherosclerosis: product of a primate adaptation.

Abdominal obesity is prevalent and often accompanied by an array of metabolic perturbations including elevated blood pressure, dyslipidemia, impaired glucose tolerance or insulin resistance, a prothrombotic state, and a proinflammatory state, together referred to as the metabolic syndrome. The metabolic syndrome greatly increases coronary heart disease (CHD) risk. Social stress also increases CHD although the mechanisms through which this occurs are not completely understood. Chronic stress may result in sustained glucocorticoid production, which is thought to promote visceral obesity. Thus, one hypothesis is that social stress may cause visceral fat deposition and the metabolic syndrome, which, in turn increases CHD. CHD is caused by coronary artery atherosclerosis (CAA) and its sequelae. Cynomolgus monkeys (Macaca fascicularis) are a well-established models of CAA. Social subordination may be stressful to cynomolgus monkeys and result in hypercortisolemia and exacerbated CAA in females. Herein is reviewed a body of literature which suggests that social stress increases visceral fat deposition in cynomolgus monkeys, that subordinate females are more likely than dominants to have visceral obesity, that females with visceral obesity have behavioral and physiological characteristics consistent with a stressed state, and that females with high ratios of visceral to subcutaneous abdominal fat develop more CAA. While these relationships have been most extensively studied in cynomolgus macaques, obesity-related metabolic disturbances are also observed in other primate species. Taken together, these observations support the view that the current obesity epidemic is the result of a primate adaptation involving the coevolution with encephalization of elaborate physiological systems to protect against starvation and defend stored body fat in order to feed a large and metabolically demanding brain. Social stress may be engaging these same physiological systems, increasing the visceral deposition of fat and its sequelae, which increase CHD risk.

Depressive behavior and coronary artery atherogenesis in adult female cynomolgus monkeys.

OBJECTIVE: To examine depressive behavior and early coronary artery atherogenesis in 36 socially housed female cynomolgus monkeys, an established model of atherogenesis and depression. Coronary heart disease (CHD) is caused by coronary artery atherosclerosis (CAA) and its sequelae which develop over a period of decades. Thus, in prospective studies of depression and CHD, CAA was likely present at baseline in most subjects who experienced cardiac events. Little is known about the relationship between depression and CAA. METHODS: The monkeys were free of atherosclerosis before being fed a diet containing moderate amounts of fat and cholesterol for 52 months. Depressed behavior and activity levels recorded in weekly 15-minute focal samples, telemetered 24-hour heart rate, plasma total (TPC) and high-density lipoprotein cholesterol (HDLC), luteal phase serum progesterone concentrations, basal cortisol, cortisol response to corticotrophin-releasing hormone (CRH), and CAA extent were assessed. RESULTS: Time spent in depressed behavior over 4 years was significantly associated with early CAA (r = .73, p < .001), as were activity level, 24-hour heart rate, TPC, HDLC, cortisol response to CRH, and mean peak progesterone (all p < or = 0.05). Depressed females had four times the CAA compared with nondepressed females. CONCLUSIONS: Depression in primates is associated with perturbations in multiple CHD risk factors and accelerated early atherogenesis. These data are consistent with the hypotheses that depression and CAA both stem from a common mechanism and that depression may cause CAA.

Soy-tibolone combination - effect on lipids in postmenopausal monkeys and women.

OBJECTIVES: To determine whether co-administration of soy during tibolone treatment would prevent tibolone-induced dyslipoproteinemia in postmenopausal monkeys and women. METHODS: Surgically postmenopausal cynomolgus monkeys (n = 18) were assigned randomly to one of four dietary regimens in a Latin Square crossover design, such that all animals received all diets for 14 weeks with a 4-week washout period: (1) casein/lactalbumin (CL); (2) tibolone (Tib, 1.25 mg/day women's equivalent); (3) soy (138 mg isoflavones/day women's equivalent); (4) Soy + Tib. Postmenopausal women on tibolone treatment were randomized to receive soy powder (52 g of soy protein containing 112 mg isoflavones) or placebo (containing 52 g of milk protein) daily in a crossover trial for 8 weeks with a 4-week washout period. RESULTS: Monkeys given Tib alone had approximately 14% increase in plasma LDL + VLDL-C; whereas those given soy combined with tibolone had significant ( approximately 22%) reductions. Tib treated monkeys had reductions in plasma HDL-C of about 48% vs. no reductions in Soy + Tib. In postmenopausal women using tibolone, soy reduced plasma LDL-C concentrations by approximately 10% from baseline without a change in HDL-C. CONCLUSIONS: Co-administration of soy during tibolone treatment improved the lipoprotein profile in both monkeys and women; however, the effects were more robust in monkeys.

Estrogen effects on arteries vary with stage of reproductive life and extent of subclinical atherosclerosis progression.

The past several years have been marked by confusion and controversy concerning whether estrogens are cardioprotective. The issue is of utmost public health importance because coronary heart disease (CHD) remains the leading cause of death among postmenopausal women. Fortunately, a unifying hypothesis has emerged that reproductive stage is a major determinant of the effect of estrogens on atherosclerosis progression, complications, and plaque vulnerability. PREMENOPAUSAL YEARS: Premenopausal atherosclerosis progression seems to be an important determinant of postmenopausal atherosclerosis and thus the risk for CHD. Clearly, plasma lipids/lipoproteins influence this progression; however, estradiol deficiency seems to be the major modulator. Both monkeys and women with premenopausal estrogen deficiency develop premature atherosclerosis, an effect that can be prevented in both species by estrogen-containing oral contraceptives. PERIMENOPAUSAL/EARLY POSTMENOPAUSAL YEARS: During this stage, there are robust estrogen benefits. Monkeys given estrogens immediately after surgical menopause have a 70% inhibition in coronary atherosclerosis progression. Estrogen treatment prevented progression of atherosclerosis of women in the Estrogen in the Prevention of Atherosclerosis Trial. A meta-analysis of women younger than 60 years given hormone therapy had reduced total mortality (relative risk = 0.61, 95% CI: 0.39-0.95). LATE POSTMENOPAUSAL YEARS: This stage is one in which there are no or possible deleterious estrogen effects. Monkeys lose CHD benefits of estrogens when treatment is delayed. The increase in CHD events associated with initiating hormone therapy 10 or more years after menopause seems to be related to up-regulation of the plaque inflammatory processes and plaque instability and may be down-regulated by statin pretreatment.

Consumption of Mediterranean versus Western Diet Leads to Distinct Mammary Gland Microbiome Populations.

Recent identification of a mammary gland-specific microbiome led to studies investigating bacteria populations in breast cancer. Malignant breast tumors have lower Lactobacillus abundance compared with benign lesions, implicating Lactobacillus as a negative regulator of breast cancer. Diet is a main determinant of gut microbial diversity. Whether diet affects breast microbiome populations is unknown. In a non-human primate model, we found that consumption of a Western or Mediterranean diet modulated mammary gland microbiota and metabolite profiles. Mediterranean diet consumption led to increased mammary gland Lactobacillus abundance compared with Western diet-fed monkeys. Moreover, mammary glands from Mediterranean diet-fed monkeys had higher levels of bile acid metabolites and increased bacterial-processed bioactive compounds. These data suggest that diet directly influences microbiome populations outside the intestinal tract in distal sites such as the mammary gland. Our study demonstrates that diet affects the mammary gland microbiome, establishing an alternative mechanistic pathway for breast cancer prevention.

Estradiol Treatment Initiated Early After Ovariectomy Regulates Myocardial Gene Expression and Inhibits Diastolic Dysfunction in Female Cynomolgus Monkeys: Potential Roles for Calcium Homeostasis and Extracellular Matrix Remodeling.

Background Left ventricular ( LV ) diastolic dysfunction often precedes heart failure with preserved ejection fraction, the dominant form of heart failure in postmenopausal women. The objective of this study was to determine the effect of oral estradiol treatment initiated early after ovariectomy on LV function and myocardial gene expression in female cynomolgus macaques. Methods and Results Monkeys were ovariectomized and randomized to receive placebo (control) or oral estradiol at a human-equivalent dose of 1 mg/day for 8 months. Monkeys then underwent conventional and tissue Doppler imaging to assess cardiac function, followed by transcriptomic and histomorphometric analyses of LV myocardium. Age, body weight, blood pressure, and heart rate were similar between groups. Echocardiographic mitral early and late inflow velocities, mitral annular velocities, and mitral E deceleration slope were higher in estradiol monkeys (all P<0.05), despite similar estimated LV filling pressure. MCP1 (monocyte chemoattractant protein 1) and LV collagen staining were lower in estradiol animals ( P<0.05). Microarray analysis revealed differential myocardial expression of 40 genes (>1.2-fold change; false discovery rate, P<0.05) in estradiol animals relative to controls, which implicated pathways associated with better calcium ion homeostasis and muscle contraction and lower extracellular matrix deposition ( P<0.05). Conclusions Estradiol treatment initiated soon after ovariectomy resulted in enhanced LV diastolic function, and altered myocardial gene expression towards decreased extracellular matrix deposition, improved myocardial contraction, and calcium homeostasis, suggesting that estradiol directly or indirectly modulates the myocardial transcriptome to preserve cardiovascular function.

Estrogen effects on arteries vary with stage of reproductive life and extent of subclinical atherosclerosis progression.

The past several years have been marked by confusion and controversy concerning whether estrogens are cardioprotective. The issue is of utmost public health importance because coronary heart disease (CHD) remains the leading cause of death among postmenopausal women. Fortunately, a unifying hypothesis has emerged that reproductive stage is a major determinant of the effect of estrogens on atherosclerosis progression, complications, and plaque vulnerability. PREMENOPAUSAL YEARS: Premenopausal atherosclerosis progression seems to be an important determinant of postmenopausal atherosclerosis and thus the risk for CHD. Clearly, plasma lipids/lipoproteins influence this progression; however, estradiol deficiency seems to be the major modulator. Both monkeys and women with premenopausal estrogen deficiency develop premature atherosclerosis, an effect that can be prevented in both species by estrogen-containing oral contraceptives. PERIMENOPAUSAL/EARLY POSTMENOPAUSAL YEARS: During this stage, there are robust estrogen benefits. Monkeys given estrogens immediately after surgical menopause have a 70% inhibition in coronary atherosclerosis progression. Estrogen treatment prevented progression of atherosclerosis of women in the Estrogen in the Prevention of Atherosclerosis Trial. A meta-analysis of women younger than 60 years given hormone therapy had reduced total mortality (relative risk = 0.61, 95% CI: 0.39-0.95). LATE POSTMENOPAUSAL YEARS: This stage is one in which there are no or possible deleterious estrogen effects. Monkeys lose CHD benefits of estrogens when treatment is delayed. The increase in CHD events associated with initiating hormone therapy 10 or more years after menopause seems to be related to up-regulation of the plaque inflammatory processes and plaque instability and may be down-regulated by statin pretreatment.

Relationships of depressive behavior and sertraline treatment with walking speed and activity in older female nonhuman primates.

Depression is the most common mental health problem in aging persons and is a leading risk factor for physical disability, especially in women. Though antidepressant drugs such as serotonin reuptake inhibitors (SSRI) are commonly prescribed, epidemiological evidence reveals mixed effects of long-term antidepressant use on physical function and activity, possibly depending on depressive status. The purpose of this preclinical trial was to determine the relationships of depressive behavior and the potential for an SSRI treatment to modulate walking speed and activity patterns in older adult female cynomolgus monkeys (Macaca fascicularis). We evaluated the effects of depression and a commonly prescribed SSRI, sertraline HCl (20 mg/kg/day p.o.), on (a) walking speed, (b) accelerometry-derived activity (counts) and sedentariness (daytime 60-s sedentary epochs), and (c) observed locomotor and sedentary behaviors (% time) in adult female depressed and nondepressed monkeys (n = 42; 17.2 ± 1.8 years) during an 18 month pre-treatment phase and an 18 month treatment phase using a longitudinal, stratified placebo-control study design. Monkeys that were depressed prior to treatment (19/42) subsequently had slower walking speeds (F D [1, 38] = 4.14; p ≤ 0.05) and tended to be more sedentary during the daytime (F D [1, 38] = 3.63; p ≤ 0.06). Sertraline did not affect depressive behaviors, walking speed, accelerometry-derived physical activity or sedentariness, or time observed in total locomotor or sedentary behavior (all p > 0.10). This study provides the first experimental demonstration of relationships between nonhuman primate behavioral depression and walking speed, activity, and sedentariness and provides evidence for a lack of an effect of SSRI treatment on these phenotypes.

Determination of plasma genistein fatty acid esters following administration of genistein or genistein 4'7-O-dioleate in monkeys.

Soy-derived isoflavone phytoestrogens, such as genistein (4',5,7-trihydroxyisoflavone), have been shown to protect low-density lipoprotein from oxidation. In addition, human plasma was previously shown to be capable of converting genistein into lipophilic fatty acid esters that accumulate in lipoproteins in vitro. We developed a method for the quantitation of genistein fatty acid esters in plasma. Furthermore, the method was utilized to measure genistein ester concentrations in monkey plasma following administration of genistein or genistein 4',7-O-dioleate. After extraction from plasma, genistein fatty acid esters were separated from unesterified genistein by Sephadex LH-20 column chromatography. The genistein ester fraction was hydrolyzed by saponification and purified by a second chromatography on Sephadex LH-20. The hydrolyzed genistein esters were measured by time-resolved fluoroimmunoassay. Adult female rhesus monkeys (n=10) received a subcutaneous injection of genistein (24 mg, n=2) or genistein 4',7-O-dioleate (71 mg, n=3) or an oral dose of genistein (24 mg, n=2) or genistein 4',7-O-dioleate (71 mg, n=3). Plasma was collected at 4, 8, and 24 h post-dosing. Following subcutaneous administration of genistein 4',7-O-dioleate, the plasma concentrations of genistein esters became elevated in two out of three monkeys with 8-h values exceeding 7.5 nmol/L and 24-h values above 12 nmol/L. Other treatments resulted in lower plasma values ranging between 2.7 and 6.1 nmol/L. The lower limit of detection for the method was 1.44 nmol/L. Subcutaneously administered genistein 4',7-O-dioleate was also converted to water-soluble conjugates, but oral administration did not elevate plasma genistein fatty acid ester levels. The results suggest that it may be possible to introduce intact genistein ester molecules into plasma by parenteral but not oral administration.

Low dose estrogens inhibit coronary artery atherosclerosis in postmenopausal monkeys.

OBJECTIVES: To determine if low dose conjugated equine estrogens (CEE) result in a reduction of coronary artery atherosclerosis progression, and to relate these findings to previous studies using the traditional dose. METHODS: Adult female monkeys (Macaca fascicularis) were fed an atherogenic diet for 10 months, to induce fatty streaks and small plaques comparable to those present in early postmenopausal women, and then ovariectomized and treated orally with: CEE (0.30 mg/day women's equivalent dose, n=28) or placebo (n=25) daily for 24 months. Body weight and estradiol were measured at 3, 6, 12 and 18 months and plasma lipids were measured at baseline and every 6 months. RESULTS: Despite the lack of effect on plasma lipid profiles, monkeys treated with low dose CEE had marked reductions in coronary artery atherosclerosis plaque extent (intimal area) in all three main coronary arteries: left anterior descending artery (52% less, 0.044 mm(2) versus 0.091 mm(2), p=0.04); left circumflex artery (62% less, 0.045 mm(2) versus 0.119 mm(2), p=0.006) and right circumflex artery (42% less, 0.018 mm(2) versus 0.031 mm(2), p=0.20). The overall mean coronary atherosclerosis extent was 52% lower in CEE treated animals (0.042 mm(2) versus 0.088 mm(2), p=0.02). CONCLUSION: Low dose CEE (0.30 mg/woman/day equivalent) was effective in reducing coronary atherosclerosis and the magnitude of the protection was comparable to previously reported studies using doses equivalent to 0.625 mg/woman/day. This study provides an experimental basis for the assumption that low dose CEE may be as effective as the traditional dose in inhibiting coronary atherosclerosis progression in early postmenopausal subjects.

Atherosclerosis and Vascular Biologic Responses to Estrogens: Histologic, Immunohistochemical, Biochemical, and Molecular Methods.

Atherogenesis is a multifactorial pathologic process influenced by genetics and environmental factors such as diet, exercise, stress, and other exposures. Estrogen receptors (ER) are expressed in cells of the arterial wall, suggesting that estrogen receptor ligands (estradiol, natural and pharmacologic ligands) may directly affect arterial biology and atherogenesis. Ligand bound estrogen receptor alpha and beta (ERα, ERß) can influence physiology through direct binding to estrogen response elements in the DNA, through interactions with other transcription factors such as NF-κB, or through rapid effects not dependent on gene expression changes but instead through non-nuclear membrane sites involving ERα, ERß, or G-coupled protein ER (GPER1). Elucidation of potential direct effects of estrogens on the artery wall requires careful evaluation of arterial biologic responses to estrogens. We have developed a comprehensive approach to understand the mechanisms of estrogen action which employs histologic measures of the size and other characteristics of atherosclerotic lesions, immunohistochemical assessments of cellular composition, evaluation of chemical, molecular, and genomic changes in the arterial environment, and determination of the relationships between arterial estrogen receptor expression and atherogenesis. This approach can provide important insights into the mechanisms of action of estrogen and other mediators of atherogenesis.

Vitamin D and conjugated equine estrogen: the association with coronary artery atherosclerosis in cynomolgus monkeys.

OBJECTIVE: To analyze vitamin D3 plasma concentrations among monkeys randomized to oral conjugated equine estrogen (CEE) versus control and the association with coronary artery atherosclerosis (CAA). METHODS: Surgically postmenopausal monkeys (N = 50) were fed an atherogenic diet containing a woman's equivalent of 1000 IU/day of vitamin D3. The monkeys were randomized at baseline to receive CEE (equivalent of 0.45 mg/d, n = 25) or placebo (n = 25). 25-hydroxyvitamin D3 (25OHD3) was measured at baseline and 20 months later. At 20 months, CAA evidence of coronary artery remodeling, and American Heart Association (AHA) severity scores were assessed. RESULTS: The percent change in 25OHD3 concentrations from baseline to 20 months postrandomization was inversely correlated with plaque area of the right coronary artery (P = 0.048), left circumflex artery (P = 0.039), left anterior descending artery (P = 0.017), and AHA severity score (AHA LADmax) (P = 0.016). Those with increased 25OHD3 concentrations who were taking CEE also had significantly lower AHA scores compared with those who were not taking CEE and did not have an increase in 25OHD3 (P = 0.01). CONCLUSIONS: Monkeys with increases in 25OHD3 concentrations had significantly less severe CAA. Those with increases in 25OHD3 with CEE were associated with significantly decreased AHA lesion scores, decreased plaque, and greater coronary artery remodeling. If these findings are present in women, achieving higher 25OHD3 concentrations (or being a vitamin D supplementation "responder") may be associated with cardioprotection, and further studies to evaluate a synergistic effect with CEE and vitamin D on cardiovascular health are needed.

Sertraline inhibits increases in body fat and carbohydrate dysregulation in adult female cynomolgus monkeys.

Selective serotonin reuptake inhibitor (SSRI) antidepressants are widely prescribed for depression and other disorders. SSRIs have become one of the most commonly used drugs in the United States, particularly by women. Acute effects on body composition and carbohydrate metabolism have been reported, but little is known regarding the effects of chronic SSRI use. We evaluated the effects of chronic administration of a commonly prescribed SSRI, sertraline HCl, on body weight and composition, fat distribution, carbohydrate metabolism, as well as activity, in adult female depressed and nondepressed cynomolgus monkeys (Macaca fascicularis; n=42) using a placebo-controlled, longitudinal, randomized study design. Phenotypes were evaluated prior to and after 18 months of oral sertraline (20mg/kg) or placebo. Over the 18 month treatment period, the placebo group experienced increases in body weight, body fat (visceral and subcutaneous) fasting insulin concentrations, and homeostasis model assessment of insulin resistance scores (HOMA-IR). Sertraline treatment prevented increases in body weight, fat, insulin, and HOMA-IR (all p<0.05), without significantly altering activity levels. Sertraline treatment altered adiponectin in an unusual way - reducing circulating adiponectin in depressed monkeys without affecting fat mass or body weight. Deleterious effects on adiponectin, a potentially insulin-sensitizing and atheroprotective protein, may result in adverse effects on cardiovascular health despite otherwise beneficial effects on body composition and carbohydrate metabolism.