Hypertension disrupts the vascular clock in both sexes.

Blood pressure (BP) displays a circadian rhythm and disruptions in this pattern elevate cardiovascular risk. Although both central and peripheral clock genes are implicated in these processes, the importance of vascular clock genes is not fully understood. BP, vascular reactivity, and the renin-angiotensin-aldosterone system display overt sex differences, but whether changes in circadian patterns underlie these differences is unknown. Therefore, we hypothesized that circadian rhythms and vascular clock genes would differ across sex and would be blunted by angiotensin II (ANG II)-induced hypertension. ANG II infusion elevated BP and disrupted circadian patterns similarly in both males and females. In females, an impact on heart rate (HR) and locomotor activity was revealed, whereas in males hypertension suppressed baroreflex sensitivity (BRS). A marked disruption in the vascular expression patterns of period circadian regulator 1 (Per1) and brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1 (Bmal1) was noted in both sexes. Vascular expression of the G protein-coupled estrogen receptor (Gper1) also showed diurnal synchronization in both sexes that was similar to that of Per1 and Per2 and disrupted by hypertension. In contrast, vascular expression of estrogen receptor 1 (Esr1) showed a diurnal rhythm and hypertension-induced disruption only in females. This study shows a strikingly similar impact of hypertension on BP rhythmicity, vascular clock genes, and vascular estrogen receptor expression in both sexes. We identified a greater impact of hypertension on locomotor activity and heart rate in females and on baroreflex sensitivity in males and also revealed a diurnal regulation of vascular estrogen receptors. These insights highlight the intricate ties between circadian biology, sex differences, and cardiovascular regulation.NEW & NOTEWORTHY This study reveals that ANG II-induced hypertension disrupts the circadian rhythm of blood pressure in both male and female mice, with parallel effects on vascular clock gene and estrogen receptor diurnal patterns. Notably, sex-specific responses to hypertension in terms of locomotor activity, heart rate, and baroreflex sensitivity are revealed. These findings pave the way for chronotherapeutic strategies tailored to mitigate cardiovascular risks associated with disrupted circadian rhythms in hypertension.

High-plasma soluble prorenin receptor is associated with vascular damage in male, but not female, mice fed a high-fat diet.

Plasma soluble prorenin receptor (sPRR) displays sexual dimorphism and is higher in women with type 2 diabetes mellitus (T2DM). However, the contribution of plasma sPRR to the development of vascular complications in T2DM remains unclear. We investigated if plasma sPRR contributes to sex differences in the activation of the systemic renin-angiotensin-aldosterone system (RAAS) and vascular damage in a model of high-fat diet (HFD)-induced T2DM. Male and female C57BL/6J mice were fed either a normal fat diet (NFD) or an HFD for 28 wk to assess changes in blood pressure, cardiometabolic phenotype, plasma prorenin/renin, sPRR, and ANG II. After completing dietary protocols, tissues were collected from males to assess vascular reactivity and aortic reactive oxygen species (ROS). A cohort of male mice was used to determine the direct contribution of increased systemic sPRR by infusion. To investigate the role of ovarian hormones, ovariectomy (OVX) was performed at 32 wk in females fed either an NFD or HFD. Significant sex differences were found after 28 wk of HFD, where only males developed T2DM and increased plasma prorenin/renin, sPRR, and ANG II. T2DM in males was accompanied by nondipping hypertension, carotid artery stiffening, and aortic ROS. sPRR infusion in males induced vascular thickening instead of material stiffening caused by HFD-induced T2DM. While intact females were less prone to T2DM, OVX increased plasma prorenin/renin, sPRR, and systolic blood pressure. These data suggest that sPRR is a novel indicator of systemic RAAS activation and reflects the onset of vascular complications during T2DM regulated by sex.NEW & NOTEWORTHY High-fat diet (HFD) for 28 wk leads to type 2 diabetes mellitus (T2DM) phenotype, concomitant with increased plasma soluble prorenin receptor (sPRR), nondipping blood pressure, and vascular stiffness in male mice. HFD-fed female mice exhibiting a preserved cardiometabolic phenotype until ovariectomy revealed increased plasma sPRR and blood pressure. Plasma sPRR may indicate the status of systemic renin-angiotensin-aldosterone system (RAAS) activation and the onset of vascular complications during T2DM in a sex-dependent manner.

Sex differences in vascular aging and impact of GPER deletion.

Aging is a nonmodifiable risk factor for cardiovascular disease associated with arterial stiffening and endothelial dysfunction. We hypothesized that sex differences exist in vascular aging processes and would be attenuated by global deletion of the G protein-coupled estrogen receptor. Blood pressure was measured by tail-cuff plethysmography, pulse wave velocity (PWV) and echocardiography were assessed with high-resolution ultrasound, and small vessel reactivity was measured using wire myography in adult (25 wk) and middle-aged (57 wk) male and female mice. Adult female mice displayed lower blood pressure and PWV, but this sex difference was absent in middle-aged mice. Aging significantly increased PWV but not blood pressure in both sexes. Adult female carotids were more distensible than males, but this sex difference was lost during aging. Acetylcholine-induced relaxation was greater in female than male mice at both ages, and only males showed aging-induced changes in cardiac hypertrophy and function. GPER deletion removed the sex difference in PWV and ex vivo stiffness in adult mice. The sex difference in blood pressure was absent in KO mice and was associated with endothelial dysfunction in females. These findings indicate that the impact of aging on arterial stiffening and endothelial function is not the same in male and female mice. Moreover, nongenomic estrogen signaling through GPER impacted vascular phenotype differently in male and female mice. Delineating sex differences in vascular changes during healthy aging is an important first step in improving early detection and sex-specific treatments in our aging population.NEW & NOTEWORTHY Indices of vascular aging were different in male and female mice. Sex differences in pulse wave velocity, blood pressure, and large artery stiffness were abrogated in middle-aged mice, but the female advantage in resistance artery vasodilator function was maintained. GPER deletion abrogated these sex differences and significantly reduced endothelial function in adult female mice. Additional studies are needed to characterize sex differences in vascular aging to personalize early detection and treatment for vascular diseases.

New insights into arterial stiffening: does sex matter?

This review discusses sexual dimorphism in arterial stiffening, disease pathology interactions, and the influence of sex on mechanisms and pathways. Arterial stiffness predicts cardiovascular mortality independent of blood pressure. Patients with increased arterial stiffness have a 48% higher risk for developing cardiovascular disease. Like other cardiovascular pathologies, arterial stiffness is sexually dimorphic. Young women have lower stiffness than aged-matched men, but this sex difference reverses during normal aging. Estrogen therapy does not attenuate progressive stiffening in postmenopausal women, indicating that currently prescribed drugs do not confer protection. Although remodeling of large arteries is a protective adaptation to higher wall stress, arterial stiffening increases afterload to the left ventricle and transmits higher pulsatile pressure to smaller arteries and target organs. Moreover, an increase in aortic stiffness may precede or exacerbate hypertension, particularly during aging. Additional studies are needed to elucidate the mechanisms by which females are protected from arterial stiffness to provide insight into its mechanisms and, ultimately, therapeutic targets for treating this pathology.

Dihydrotestosterone induces arterial stiffening in female mice.

BACKGROUND: Androgens are important sex hormones in both men and women and are supplemented when endogenous levels are low, for gender transitioning, or to increase libido. Androgens also circulate at higher levels in women with polycystic ovarian syndrome, a condition that increases the risk for cardiovascular diseases including hypertension and arterial stiffness. Since our previous work shows an important role for the G protein-coupled estrogen receptor (GPER) in arterial stiffness, we hypothesized that other hormones including androgens may impact arterial stiffness in female mice via downregulation of GPER. METHODS: The impact of the non-aromatizable androgen dihydrotestosterone (DHT), the glucocorticoid dexamethasone, and the progestin medroxyprogesterone acetate (all 100 nM for 24 h) on GPER and ERα expression was assessed in cultured vascular smooth muscle cells using droplet digital PCR (ddPCR). To assess the in vivo impact of the DHT-induced downregulation of GPER, female ovary-intact C57Bl/6 mice at 15-16 weeks of age were treated with silastic capsules containing DHT for 4 weeks, one with a dosage expected to mimic human male DHT levels and another to double the expected human concentration (n = 8-9/group). RESULTS: In cultured vascular smooth muscle cells, GPER mRNA was decreased by DHT (P = 0.001) but was not impacted by dexamethasone or medroxyprogesterone. In contrast, ERα expression in cultured cells was significantly suppressed by all three hormones (P < 0.0001). In control mice or mice treated with a single or double dose of DHT, a dose-dependent increase in body weight was observed (control 22 ± 2 g, single dose 24 ± 2 g, double dose 26 ± 2 g; P = 0.0002). Intracarotid stiffness measured via pulse wave velocity showed a more than two-fold increase in both DHT-treated groups (control 1.9 ± 0.3 m/s, single dose 4.3 ± 0.8 m/s, double dose 4.8 ± 1.0 m/s). This increase in arterial stiffness occurred independent of changes in blood pressure (P = 0.59). Histological analysis of aortic sections using Masson's trichrome showed a significant decrease in collagen between the control group (24 ± 5%) and the double dose group (17 ± 3%, P = 0.007), despite no changes in aortic wall thickness or smooth muscle content. Lastly, ddPCR showed that in vivo DHT treatment decreased aortic expression of both GPER (control 20 ± 5, single dose 10.5 ± 5.6, double dose 10 ± 4 copies/ng; P = 0.001) and ERα (control 54 ± 2, single dose 24 ± 13, and double dose 23 ± 12 copies/ng; P = 0.003). CONCLUSIONS: These findings indicate that androgen promotes arterial stiffening and cardiovascular damage in female mice and is associated with decreased estrogen receptor expression. These data are important for transgender men, women using testosterone for fitness or reduced libido, as well as patients with polycystic ovarian syndrome.

The current study investigated the impact of other hormones on estrogen receptor expression and its impact on vascular health. In both cultured vascular cells and in vivo vascular tissue, dihydrotestosterone decreased the expression of estrogen receptors. Female mice treated with dihydrotestosterone also displayed increased body weight and arterial stiffness despite no change in blood pressure. These findings indicate that increases in testosterone may impact vascular health, which may be important clinically for transgender men, women using testosterone for fitness or reduced libido, as well as patients with polycystic ovarian syndrome.

Ovariectomy-Induced Arterial Stiffening Differs From Vascular Aging and Is Reversed by GPER Activation.

BACKGROUND: Arterial stiffness is a cardiovascular risk factor and dramatically increases as women transition through menopause. The current study assessed whether a mouse model of menopause increases arterial stiffness in a similar manner to aging and whether activation of the G-protein-coupled estrogen receptor could reverse stiffness. METHODS: Female C57Bl/6J mice were ovariectomized at 10 weeks of age or aged to 52 weeks, and some mice were treated with G-protein-coupled estrogen receptor agonists. RESULTS: Ovariectomy and aging increased pulse wave velocity to a similar extent independent of changes in blood pressure. Aging increased carotid wall thickness, while ovariectomy increased material stiffness without altering vascular geometry. RNA-sequencing analysis revealed that ovariectomy downregulated smooth muscle contractile genes. The enantiomerically pure G-protein-coupled estrogen receptor agonist, LNS8801, reversed stiffness in ovariectomy mice to a greater degree than the racemic agonist G-1. In summary, ovariectomy and aging induced arterial stiffening via potentially different mechanisms. Aging was associated with inward remodeling, while ovariectomy-induced material stiffness independent of geometry and a loss of the contractile phenotype. CONCLUSIONS: This study enhances our understanding of the impact of estrogen loss on vascular health in a murine model and warrants further studies to examine the ability of LNS8801 to improve vascular health in menopausal women.

Testosterone deficiency promotes arterial stiffening independent of sex chromosome complement.

BACKGROUND: Sex hormones and sex chromosomes play a vital role in cardiovascular disease. Testosterone plays a crucial role in men's health. Lower testosterone level is associated with cardiovascular and cardiometabolic diseases, including inflammation, atherosclerosis, and type 2 diabetes. Testosterone replacement is beneficial or neutral to men's cardiovascular health. Testosterone deficiency is associated with cardiovascular events. Testosterone supplementation to hypogonadal men improves libido, increases muscle strength, and enhances mood. We hypothesized that sex chromosomes (XX and XY) interaction with testosterone plays a role in arterial stiffening. METHODS: We used four core genotype male mice to understand the inherent contribution of sex hormones and sex chromosome complement in arterial stiffening. Age-matched mice were either gonadal intact or castrated at eight weeks plus an additional eight weeks to clear endogenous sex hormones. This was followed by assessing blood pressure, pulse wave velocity, echocardiography, and ex vivo passive vascular mechanics. RESULTS: Arterial stiffening but not blood pressure was more significant in castrated than testes-intact mice independent of sex chromosome complement. Castrated mice showed a leftward shift in stress-strain curves and carotid wall thinning. Sex chromosome complement (XX) in the absence of testosterone increased collagen deposition in the aorta and Kdm6a gene expression. CONCLUSION: Testosterone deprivation increases arterial stiffening and vascular wall remodeling. Castration increases Col1α1 in male mice with XX sex chromosome complement. Our study shows decreased aortic contractile genes in castrated mice with XX than XY sex chromosomes.

Cardiovascular disease is the leading cause of death worldwide. Cardiovascular disease presents differently in men and women. While men develop plaque buildup in large arteries, women develop buildup in the microvessels in the heart. Arterial stiffening, which is the hardening of arteries, increases with age in both men and women. Aging, coupled with the decline in sex hormones, exacerbates cardiovascular disease in women compared to men. Men with XY sex chromosomes have higher circulating testosterone, while women with XX sex chromosomes have increased circulating estradiol. The potential benefits of sex hormone replacement therapy are shown in men and women. Indeed, testosterone replacement deficiency is associated with adverse cardiovascular outcomes in men. Whether adverse events are dependent or independent of sex hormones' interaction with sex chromosomes is unknown. This study used the four core genotype mice comprising males with either XX or XY sex chromosome complement. We show castration increases arterial stiffening and collagen deposition on the arterial wall. We also identified the escapee and smooth muscle contractile genes that may play a role in arterial stiffening. Our data suggests that testosterone deprivation mediates arterial stiffening and remodeling.

Dihydrotestosterone Induces Arterial Stiffening in Female Mice.

Background: Testosterone is the predominant sex hormone in men and is increased in women with polycystic ovarian syndrome. These patients also experience an increased risk for cardiovascular diseases including hypertension and arterial stiffness. Since our previous work shows an important role for the G protein-coupled estrogen receptor (GPER) in arterial stiffness, we hypothesized that other hormones including androgens may impact arterial stiffness in female mice via regulation of GPER. Methods: The impact of the non-aromatizable androgen dihydrotestosterone (DHT), the glucocorticoid dexamethasone, and the progestin medroxyprogesterone acetate (all 100 nM for 24 h) on GPER and ERα expression was assessed in cultured vascular smooth muscle cells using droplet digital PCR (ddPCR). To assess the in vivo impact of the DHT-induced downregulation of GPER, female ovary-intact C57Bl/6 mice were treated with silastic capsules containing DHT for 4 weeks, one with a dosage expected to mimic human male DHT levels and another to double the expected human concentration (n=8-9/group). Results: GPER mRNA was only decreased by DHT (P=0.001), while ERα expression was significantly suppressed by all hormones (P<0.0001). While blood pressure was not different between groups (P= 0.59), there was a dose-dependent increase in body weight (control 22±2 g, single dose 24±2 g, double dose 26±2 g; P=0.0002). Intracarotid stiffness measured via pulse wave velocity showed a more than two-fold increase in both DHT-treated groups (control 1.9±0.3 m/s, single dose 4.3±0.8 m/s, double dose 4.8±1.0 m/s). Histological analysis of aortic sections using Masson's trichrome showed a significant decrease in collagen between the control group (24 ± 5%) and the double dose group (17 ± 3%, P=0.007), despite no changes in aortic wall thickness or smooth muscle content. Lastly, ddPCR showed that in vivo DHT treatment decreased aortic expression of both GPER (control 20±5, single dose 10.5 ± 5.6, double dose 10±4 copies/ng; P=0.001) and ERα (control 54±2, single dose 24±13, and double dose 23 ± 12 copies/ng; P=0.003). Conclusions: These findings indicate that testosterone promotes arterial stiffening and cardiovascular damage in female mice and is associated with decreased estrogen receptor expression. These data are important not only for polycystic ovarian syndrome patients but also women using testosterone for fitness, gender transitioning, or reduced libido.

Smooth muscle contribution to vaginal viscoelastic response.

Smooth muscle cells contribute to the mechanical function of various soft tissues, however, their contribution to the viscoelastic response when subjected to multiaxial loading remains unknown. The vagina is a fibromuscular viscoelastic organ that is exposed to prolonged and increased pressures with daily activities and physiologic processes such as vaginal birth. The vagina changes in geometry over time under prolonged pressure, known as creep. Vaginal smooth muscle cells may contribute to creep. This may be critical for the function of vaginal and other soft tissues that experience fluctuations in their biomechanical environment. Therefore, the objective of this study was to develop methods to evaluate the contribution of smooth muscle to vaginal creep under multiaxial loading using extension - inflation tests. The vaginas from wildtype mice (C57BL/6 × 129SvEv; 3-6 months; n = 10) were stimulated with various concentrations of potassium chloride then subjected to the measured in vivo pressure (7 mmHg) for 100 s. In a different cohort of mice (n = 5), the vagina was stimulated with a single concentration of potassium chloride then subjected to 5 and 15 mmHg. A laser micrometer measured vaginal outer diameter in real-time. Immunofluorescence evaluated the expression of alpha-smooth muscle actin and myosin heavy chain in the vaginal muscularis (n = 6). When smooth muscle contraction was activated, vaginal creep behavior increased compared to the relaxed state. However, increased pressure decreased the active creep response. This study demonstrated that extension - inflation protocols can be used to evaluate smooth muscle contribution to the viscoelastic response of tubular soft tissues.

Estrogen-mediated mechanisms in hypertension and other cardiovascular diseases.

Cardiovascular disease (CVD) is the leading cause of death globally for men and women. Premenopausal women have a lower incidence of hypertension and other cardiovascular events than men of the same age, but diminished sex differences after menopause implicates 17-beta-estradiol (E2) as a protective agent. The cardioprotective effects of E2 are mediated by nuclear estrogen receptors (ERα and ERß) and a G protein-coupled estrogen receptor (GPER). This review summarizes both established as well as emerging estrogen-mediated mechanisms that underlie sex differences in the vasculature during hypertension and CVD. In addition, remaining knowledge gaps inherent in the association of sex differences and E2 are identified, which may guide future clinical trials and experimental studies in this field.

Ovariectomy-Induced Arterial Stiffening Differs from Vascular Aging and is Reversed by GPER Activation.

Arterial stiffness is a cardiovascular risk factor and dramatically increases as women transition through menopause. The current study assessed whether a mouse model of menopause increases arterial stiffness in a similar manner to aging, and whether activation of the G protein-coupled estrogen receptor (GPER) could reverse stiffness. Female C57Bl/6J mice were ovariectomized (OVX) at 10 weeks of age or aged to 52 weeks, and some mice were treated with GPER agonists. OVX and aging increased pulse wave velocity to a similar extent independent of changes in blood pressure. Aging increased carotid wall thickness, while OVX increased material stiffness without altering vascular geometry. RNA-Seq analysis revealed that OVX downregulated smooth muscle contractile genes. The enantiomerically pure GPER agonist, LNS8801, reversed stiffness in OVX mice to a greater degree than the racemic agonist G-1. In summary, OVX and aging induced arterial stiffening via potentially different mechanisms. Aging was associated with inward remodeling while OVX induced material stiffness independent of geometry and a loss of the contractile phenotype. This study helps to further our understanding of the impact of menopause on vascular health and identifies LNS8801 as a potential therapy to counteract this detrimental process in women.

Estrogen Metabolite 2-Methoxyestradiol Attenuates Blood Pressure in Hypertensive Rats by Downregulating Angiotensin Type 1 Receptor.

The therapeutic potential of 2-Methoxyestradiol (2ME2) is evident in cardiovascular disease. Our laboratory has previously demonstrated the mechanism involved in the 2ME2 regulation of angiotensin type 1 receptor (AT1R) in vitro. However, 2ME2 regulation of angiotensin receptors and its effects on blood pressure (BP) and resting heart rate (RHR) are uncertain. In this study, male and female Wistar-Kyoto (WKY) rats infused with angiotensin II (65 ng/min) and male spontaneously hypertensive rats (SHR) were surgically implanted with telemetric probes to continuously assess arterial BP and RHR. In both male and female WKY rats, 2ME2 treatment (20 mg/kg/day for 2 weeks) resulted in a significant reduction of Ang II-induced systolic, diastolic, and mean arterial BP. Moreover, significant weight loss and RHR were indicated in all groups. In a separate set of experiments, prolonged 2ME2 exposure in male SHR (20 mg/kg/day for 5 weeks) displayed a significant reduction in diastolic and mean arterial BP along with RHR. We also found downregulation of angiotensin receptors and angiotensinogen (AGT) in the kidney and liver and a reduction of plasma Ang II levels. Collectively, we demonstrate that 2ME2 attenuated BP and RHR in hypertensive rats involves downregulation of angiotensin receptors and body weight loss.

Alterations in the estrogen receptor profile of cardiovascular tissues during aging.

Estrogen exerts protective effects on the cardiovascular system via three known estrogen receptors: alpha (ERα), beta (ERß), and the G protein-coupled estrogen receptor (GPER). Our laboratory has previously showed the importance of GPER in the beneficial cardiovascular effects of estrogen. Since clinical studies indicate that the protective effects of exogenous estrogen on cardiovascular function are attenuated or reversed 10 years post-menopause, the hypothesis was that GPER expression may be reduced during aging. Vascular reactivity and GPER protein expression were assessed in female mice of varying ages. Physiological parameters, blood pressure, and estrogen receptor transcripts via droplet digital PCR (ddPCR) were assessed in the heart, kidney, and aorta of adult, middle-aged, and aged male and female C57BL/6 mice. Vasodilation to estrogen (E2) and the GPER agonist G-1 were reduced in aging female mice and were accompanied by downregulation of GPER protein. However, ERα and GPER were the predominant receptors in all tissues, whereas ERß was detectable only in the kidney. Female sex was associated with higher mRNA for both ERα and GPER in both the aorta and the heart. Aging impacted receptor transcript in a tissue-dependent manner. ERα transcript decreased in the heart with aging, while GPER expression increased in the heart. These data indicate that aging impacts estrogen receptor expression in the cardiovascular system in a tissue- and sex-specific manner. Understanding the impact of aging on estrogen receptor expression is critical for developing selective hormone therapies that protect from cardiovascular damage.

Sex and the G Protein-Coupled Estrogen Receptor Impact Vascular Stiffness.

[Figure: see text].

Medroxyprogesterone opposes estradiol-induced renal damage in midlife ovariectomized Long Evans rats.

OBJECTIVE: Our laboratory previously published that long-term administration of estradiol (E2) was detrimental to the kidneys of midlife ovariectomized Long Evans rats, contrasting clinical studies in showing that menopausal hormone therapy is associated with decreased albuminuria. However, it is unknown whether this renal benefit was due to estrogen and/or the combination with progestogen. Therefore, the objective of the current study was to determine the impact of medroxyprogesterone (MPA) on E2-mediated renal damage using a rodent model. METHODS: Female Long Evans retired breeders underwent ovariectomy at 11 months of age and were treated for 40 days with subcutaneous E2, E2+MPA or vehicle at doses mimicking that of menopausal hormone therapy (N = 5-7 per group). Systolic blood pressure was measured along with indices of renal damage and function to investigate the impact of MPA on E2-mediated renal outcomes. Renal estrogen receptor alpha and G protein-coupled estrogen receptor transcript copy numbers were measured in all treatment groups through droplet digital PCR. RESULTS: Middle-aged female Long Evans rats displayed spontaneous hypertension with similar systolic blood pressures and heart weights between groups. Even though blood pressure was comparable, E2 reduced glomerular filtration rate and increased proteinuria indicating pressure-independent renal damage. Coadministration with MPA prevented E2-induced glomerular filtration rate impairment and proteinuria by promoting renal hypertrophy and preventing renal interstitial fibrosis. Both E2 and E2+MPA reduced renal estrogen receptor alpha (ERα) and increased renal G protein-coupled estrogen receptor mRNA, but neither ERα nor ERß protein was different between groups. CONCLUSION: MPA was protective against E2-induced renal damage and dysfunction in middle-aged female Long Evans rats. Assessing the impact of hormone therapy on renal outcomes may be an important clinical factor when considering treatment options for postmenopausal women.

Estrogen receptor profiles across tissues from male and female Rattus norvegicus.

BACKGROUND: Estrogen is formed by the enzyme aromatase (CYP19A1) and signals via three identified receptors ERα (ESR1), ERß (ESR2), and the G protein-coupled estrogen receptor (GPER). Understanding the relative contribution of each receptor to estrogenic signaling may elucidate the disparate effects of this sex hormone across tissues, and recent developments in PCR technology allow absolute quantification and direct comparison of multiple targets. We hypothesized that this approach would reveal tissue- and sex-specific differences in estrogen receptor mRNA. METHODS: ESR1, ESR2, GPER, and CYP19A1 were measured in four cardiovascular tissues (heart, aorta, kidney, and adrenal gland), three brain areas (somatosensory cortex, hippocampus, and prefrontal cortex), and reproductive tissues (ovaries, mammary gland, uterus, testes) from six male and six female adult Sprague-Dawley rats. RESULTS: GPER mRNA expression was relatively stable across all tissues in both sexes, ranging from 5.49 to 113 copies/ng RNA, a 21-fold difference. In contrast, ESR1/ESR2 were variable across tissues although similar within an organ system. ESR1 ranged from 4.46 to 614 copies/ng RNA (138-fold difference) while ESR2 ranged from 0.154 to 83.1 copies/ng RNA (540-fold). Significant sex differences were broadly absent except for renal ESR1 (female 206 vs. male 614 copies/ng RNA, P < 0.0001) and GPER (62.0 vs. 30.2 copies/ng RNA, P < 0.05) as well as gonadal GPER (5.49 vs. 47.5 copies/ng RNA, P < 0.01), ESR2 (83.1 vs. 0.299 copies/ng RNA, P < 0.01), and CYP19A1 (322 vs. 7.18 copies/ng RNA, P < 0.01). Cardiovascular tissues showed a predominance of ESR1, followed by GPER. In contrast, GPER was the predominant transcript in the brain with similarly low levels of ESR1 and ESR2. CYP19A1 was detected at very low levels except for reproductive tissues and the hippocampus. CONCLUSION: While the data indicates a lack of sex differences in most tissues, significant differences were found in the range of receptor gene expression across tissues as well as in the receptor profile between organ systems. The data provide a guide for future studies by establishing estrogen receptor expression across multiple tissues using absolute PCR quantification. This knowledge on tissue-specific estrogen receptor profiles will aid the development of hormonal therapies that elicit beneficial effects in specific tissues.

Evaluating residual strain throughout the murine female reproductive system.

Mounting evidence suggests that cells within soft tissues seek to maintain a preferred biomechanical state. Residual stress is defined as the stress that remains in a tissue when all external loads are removed and contributes to tissue mechanohomeostasis by decreasing the transmural gradient of wall stress. Current computational models of pelvic floor mechanics, however, often do not consider residual stress. Residual strain, a result of residual stress can be quantitatively measured through opening angle experiments. Therefore, the objective of this study is to quantify the regional variations in opening angles along the murine female reproductive system at estrus and diestrus, to quantify residual strain in the maintenance state of sexually mature females. Further, evidence suggests that hydrophilic glycosaminoglycan/proteoglycans are integral to cervical remodeling. Thus, variations in opening angles following hypo-osmotic loading are evaluated. Opening angle experiments were performed along the murine reproductive system in estrus (n = 8) and diestrus (n = 8) and placed in hypo-osmotic solution. Measurements of thickness and volume were also obtained for each group. Differences (p < 0.05) in opening angle were observed with respect to region and loading, however, differences with respect to estrous stage were not significant. Thickness values were significant (p < 0.05) with respect to region only. The effects of both estrous cycle and region resulted in significant differences (p < 0.05) in observed volume. The observed regional differences indicate variation in the stress-free state among the reproductive system which may have implications for future computational models to advance women's reproductive health.

G Protein-Coupled Estrogen Receptor Protects From Angiotensin II-Induced Increases in Pulse Pressure and Oxidative Stress.

Our previous work showed that the G protein-coupled estrogen receptor (GPER) is protective in the vasculature and kidneys during angiotensin (Ang) II-dependent hypertension by inhibiting oxidative stress. The goal of the current study was to assess the impact of GPER deletion on sex differences in Ang II-induced hypertension and oxidative stress. Male and female wildtype and GPER knockout mice were implanted with radiotelemetry probes for measurement of baseline blood pressure before infusion of Ang II (700 ng/kg/min) for 2 weeks. Mean arterial pressure was increased to the same extent in all groups, but female wildtype mice were protected from Ang II-induced increases in pulse pressure, aortic wall thickness, and Nox4 mRNA. In vitro studies using vascular smooth muscle cells found that pre-treatment with the GPER agonist G-1 inhibited Ang II-induced ROS and NADP/NADPH. Ang II increased while G-1 decreased Nox4 mRNA and protein. The effects of Ang II were blocked by losartan and Nox4 siRNA, while the effects of G-1 were inhibited by adenylyl cyclase inhibition and mimicked by phosphodiesterase inhibition. We conclude that during conditions of elevated Ang II, GPER via the cAMP pathway suppresses Nox4 transcription to limit ROS production and prevent arterial stiffening. Taken together with our previous work, this study provides insight into how acute estrogen signaling via GPER provides cardiovascular protection during Ang II hypertension and potentially other diseases characterized by increased oxidative stress.

Stable Density and Dynamics of Dendritic Spines of Cortical Neurons Across the Estrous Cycle While Expressing Differential Levels of Sensory-Evoked Plasticity.

Periodic oscillations of gonadal hormone levels during the estrous cycle exert effects on the female brain, impacting cognition and behavior. While previous research suggests that changes in hormone levels across the cycle affect dendritic spine dynamics in the hippocampus, little is known about the effects on cortical dendritic spines and previous studies showed contradictory results. In this in vivo imaging study, we investigated the impact of the estrous cycle on the density and dynamics of dendritic spines of pyramidal neurons in the primary somatosensory cortex of mice. We also examined if the induction of synaptic plasticity during proestrus, estrus, and metestrus/diestrus had differential effects on the degree of remodeling of synapses in this brain area. We used chronic two-photon excitation (2PE) microscopy during steady-state conditions and after evoking synaptic plasticity by whisker stimulation at the different stages of the cycle. We imaged apical dendritic tufts of layer 5 pyramidal neurons of naturally cycling virgin young female mice. Spine density, turnover rate (TOR), survival fraction, morphology, and volume of mushroom spines remained unaltered across the estrous cycle, and the values of these parameters were comparable with those of young male mice. However, while whisker stimulation of female mice during proestrus and estrus resulted in increases in the TOR of spines (74.2 ± 14.9% and 75.1 ± 12.7% vs. baseline, respectively), sensory-evoked plasticity was significantly lower during metestrus/diestrus (32.3 ± 12.8%). In males, whisker stimulation produced 46.5 ± 20% increase in TOR compared with baseline-not significantly different from female mice at any stage of the cycle. These results indicate that, while steady-state density and dynamics of dendritic spines of layer 5 pyramidal neurons in the primary somatosensory cortex of female mice are constant during the estrous cycle, the susceptibility of these neurons to sensory-evoked structural plasticity may be dependent on the stage of the cycle. Since dendritic spines are more plastic during proestrus and estrus than during metestrus/diestrus, certain stages of the cycle could be more suitable for forms of memory requiring de novo formation and elimination of spines and other stages for forms of memory where retention and/or repurposing of already existing synaptic connections is more pertinent.