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Perivascular adipose tissue (PVAT) is distinct from other adipose depots, as it has differential gene and protein profiles and vasoactive functions. We have shown that pregnancy affects the morphology of PVAT surrounding the uterine arteries (utPVAT) differentially than the morphology of nonperivascular reproductive adipose depots (i.e., periovarian adipose tissue, OVAT). Here, we hypothesized that pregnancy modifies the profile (size and molecular mass) of exosome-like extracellular vesicles released by utPVAT (Exo-utPVAT) compared with exosome-like extracellular vesicles released by OVAT (Exo-OVAT) and that primary uterine vascular smooth muscle cells (utVSMCs) can internalize Exo-utPVAT. Our findings indicate that utPVAT from pregnant and nonpregnant rats secrete exosome-like vesicles. Exo-utPVAT from pregnant rats were smaller (i.e., molecular size) and heavier (i.e., molecular mass) than those from nonpregnant rats, whereas pregnancy did not affect the size of Exo-OVAT. Immunocytochemistry and confocal microscopy showed that primary utVSMCs internalized Exo-utPVAT (both tissues from the same pregnant rat) labeled by the lipophilic tracer DiO. Treatment of isolated uterine arteries with Exo-utPVAT did not affect relaxation responses to acetylcholine in pregnant or nonpregnant rats. Collectively, these findings demonstrate a novel type of intercellular communication between Exo-utPVAT and utVSMCs and indicate pregnancy modulates the morphology and cargo of Exo-utPVAT.

Bacterial infections and impaired mitochondrial DNA dynamics are associated with adverse pregnancy outcomes. Unmethylated cytosine-guanine dinucleotide (CpG) motifs are common in bacterial and mitochondrial DNA and act as potent immunostimulators. Here, we tested the hypothesis that exposure to CpG oligonucleotides (ODN) during pregnancy would disrupt blood pressure circadian rhythms and the placental molecular clock machinery, mediating aberrant fetoplacental growth dynamics. Rats were repeatedly treated with CpG ODN in the 3rdtrimester (gestational day, GD, 14, 16, 18) and euthanized on GD20 (near term) or with a single dose of CpG ODN and euthanized 4 hours after treatment on GD14. Hemodynamic circadian rhythms were analyzed via Lomb-Scargle periodogram analysis on 24-h raw data collected continuously via radiotelemetry. Ap-value ≥ 0.05 indicates the absence of a circadian rhythm. Following the first treatment with CpG ODN, maternal systolic and diastolic blood pressure circadian rhythms were lost (p≥ 0.05). Blood pressure circadian rhythm was restored by GD16 and remained unaffected after the second treatment with CpG ODN (p< 0.0001). Diastolic blood pressure circadian rhythm was again lost after the last treatment on GD18 (p≥ 0.05). CpG ODN increased placental expression ofPer2andPer3andTnfα(p≤ 0.05) and affected fetoplacental growth dynamics, such as reduced fetal and placental weights were disproportionately associated with increases in the number of resorptions in ODN-treated dams compared to controls. In conclusion, gestational exposure to unmethylated CpG DNA dysregulates placental molecular clock network and fetoplacental growth dynamics and disrupts blood pressure circadian rhythms.

Uterine perivascular adipose tissue (PVAT) contributes to uterine blood flow regulation in pregnancy, at least in part, due to its effects on uterine artery reactivity. Here, we investigated the effects of uterine PVAT on endothelium-dependent pathways involved in relaxation of main uterine arteries. We hypothesized that uterine PVAT modulates the balance between the contribution of nitric oxide synthase (NOS)- and cyclooxygenase (COX)-dependent pathways to acetylcholine (ACh)-induced relaxation in isolated uterine arteries. Concentration-response curves to ACh (1 nM – 30 µM) were performed on main uterine arteries from pregnant and non-pregnant rats. Arteries were exposed to Krebs-Henseleit solution (control) or PVAT-conditioned media (PVATmedia) in the presence of the following inhibitors: L-NAME (100 µM), indomethacin (COX inhibitor, 10 µM), SC560 (selective COX-1 inhibitor, 1 µM), NS398 (selective COX-2 inhibitor, 1 µM), SQ 29,548 (selective thromboxane receptor (TP) inhibitor, 1 µM). Indomethacin suppressed ACh-induced relaxation in control uterine arteries from pregnant rats (p1.0). In arteries incubated with PVATmedia, the presence of indomethacin increased ACh-induced relaxation, reversing the anti-dilatory effect of PVATmedia. NOS inhibition reduced ACh-induced relaxation in uterine arteries from pregnant rats, and exposure to PVATmedia did not change this effect. Selective inhibition of COX-1 but not COX-2 suppressed relaxation responses to ACh in control arteries. The presence of PVATmedia abolished the effect COX-1 inhibition. Incubation of uterine arteries from pregnant rats with PVATmedia increased production of thromboxane B2 (TxB2, p=0.01). TP inhibition did not have any effect on the anti-dilatory properties of PVATmedia. In conclusion, uterine PVAT releases transferable factors that reduce relaxation responses to ACh via a COX-dependent mechanism in isolated uterine arteries from pregnant rats.

Introduction: Uterine perivascular adipose tissue (PVAT) contributes to uterine blood flow regulation in pregnancy, at least in part, due to its effects on uterine artery tone. We hypothesized that the anti-dilatory effects of uterine PVAT are mediated by vascular nitric oxide synthase (NOS)- and cyclooxygenase (COX)-dependent mechanisms. Methods: Main uterine arteries from pregnant and non-pregnant rats were mounted onto a wire myograph. Concentration-response curves to acetylcholine (ACh, 10 -9 - 3x10 -5 M) were performed on arteries exposed to physiological salt solution or PVAT-conditioned media (PVAT media ) in the presence of the following inhibitors: a) L-NAME (NOS inhibitor, 100 μM), b) indomethacin (COX inhibitor, 10 μM), c) SC560 (COX-1 inhibitor, 1 μM), d) NS398 (COX-2 inhibitor, 1 μM)]. Results: NOS inhibition abolished ACh-induced relaxation in uterine arteries from pregnant rats and exposure to PVAT media did not change this effect [AUC, (-)PVAT media : 244.6 ± 18.1 vs. (-)PVAT media /(+)L-NAME: 52.64 ± 7.4, p < 0.0001; (+)PVAT media : 202.4 ± 15.5 vs. (+)PVAT media /(+)L-NAME: 56.17 ± 11.3, p < 0.0001]. Indomethacin suppressed ACh-induced relaxation in uterine arteries from pregnant rats [AUC, (-)PVAT media : 243.6 ± 6.6 vs. (-)PVAT media /(+)Indomethacin: 123.6 ± 12.3, p < 0.0001] but not in non-pregnant rats (p>1.0). In arteries incubated with PVAT media , the presence of indomethacin increased ACh-induced relaxation [AUC, (+)PVAT media : 125.2 ± 11.4 vs. (+)PVAT media /(+)Indomethacin: 179.1 ± 14.7, p = 0.01]. COX-1 but not COX-2 inhibition suppressed relaxation responses to ACh [AUC, COX-1 inhibition, (-)PVAT media : 244.6 ± 12.0 vs. (-)PVAT media /(+)SC560: 142.4 ± 15.4, p = 0.02; COX-2 inhibition, (-)PVAT media vs. (-)PVAT media /(+)NS398, p=0.1). The anti-dilatory effects of PVAT were not observed in the presence of SC560 or NS398 (p>0.05). Exposure to PVAT media increased the protein content of COX-1 (p=0.05) and COX-2 (p=0.03) and the production of thromboxane B 2 (p=0.01) in uterine arteries from pregnant rats. Conclusion: The anti-dilatory effects of PVAT-derived factors on uterine arteries are mediated in part by COX-derived products and this mechanism is specific to pregnancy.

Introduction: In utero insults have been proposed to lead to the onset of neurodegenerative diseases later in life, such as Parkinson’s disease (PD). In utero hypoxia is associated with a multitude of conditions, such as maternal sleep apnea, preeclampsia, gestational diabetes, and maternal hypertension. Exposure to in utero hypoxia may impact male progeny more than female progeny, which may underlie the male biased sex differences in PD. It is currently unknown whether late gestational hypoxic stress has a long-term effect on brain regions associated with PD, such as the nigrostriatal pathway. We hypothesized that exposure to late gestational hypoxia will result in nigrostriatal impairment in adult male progeny compared to adult female progeny. Methods: Timed pregnant female Long-Evans rats were exposed to five days (gestational days: 15-20) of chronic intermittent hypoxia (CIH) or room air (normoxia - 21% O2) for 8 hours during their sleep phase. Each CIH cycle was 6 min of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2) for a total of 10 CIH cycles/hour. Gestational age at delivery was recorded and neonate’s body weights were measured within 12-16 hours from birth. At weaning (postnatal day, PND 28), progeny was pair-housed with a conspecific of the same sex and similar weight. To examine PD, we focused on PD associated characteristics of oxidative stress in the nigrostriatal pathway and behavioral impairments of motor (open field activity and ultrasonic vocalizations) and cognitive (spatial memory) function during puberty (PND 40-45) and young adulthood (PND 60-65). Results: Gestational CIH had no effect on the duration of gestation, litter size, and neonatal weight at birth. Gestational CIH did not impact circulating oxidative stress, regardless of sex or age of progeny. Offspring gross motor function (open field activity) and cognitive (Morris Water maze) function were unaffected by gestational CIH. In contrast, gestational CIH impaired ultrasonic vocalizations in adult male progeny. Gestational CIH increased the latency to vocalize and decreased the loudness of the vocalizations in adult male progeny. Conclusion: Exposure to CIH during gestation resulted in nigrostriatal impairment in adult male progeny, as evidenced by impaired ultrasonic vocalizations that require a functional nigrostriatal pathway. In utero hypoxia during late gestation may increase the risk for PD in males.

Daily intake of low-dose aspirin after 12weeks of gestation is currently recommended as a preventative intervention in pregnancies in high risk of developing preeclampsia. This recommendation is based on epidemiological evidence, whereas experimental studies investigating the exact mechanisms of aspirin action during pregnancy are lacking. We previously showed that treating pregnant rats with a synthetic mimetic of unmethylated CpG DNA (bacterial DNA) caused preeclampsia-like characteristics such as maternal hypertension and increased cyclooxygenase (COX) expression and activity. In this study, we tested the hypothesis that daily maternal treatment with low-dose aspirin would prevent the development of maternal hypertension, reduce COX activity and thromboxane A2 (TxA2) production, and improve maternal vascular function in pregnant rats exposed to CpG ODN during gestation. Pregnant rats were treated with ODN2395 (synthetic CpG DNA) or saline (vehicle) on gestational days (GD) 14, 16, 18. Daily low-dose aspirin treatment (1.5mg/kgBW) started on GD10 and continued throughout gestation. Pregnant rats treated with ODN2395 had greater systolic blood pressure compared to controls (120±4mmHg vs. 100±5mmHg, p=0.03) and aspirin did not prevent this increase (p=0.86). Aspirin prevented ODN2395-induced increases of TxB2 (TxA2 metabolite) in serum and mesenteric arteries. ODN2395 increased expression of COX-1 and COX-2 in mesenteric and uterine arteries and aspirin abolished these effects. Aspirin reduced contractile responses to phenylephrine and U46619 (TxA2 mimetic) in mesenteric arteries from control rats but not from ODN2395-treated rats. In conclusion, treatment with low-dose aspirin reduced systemic and vascular COX expression and activity but did not prevent the development of maternal hypertension induced by exposure to unmethylated CpG DNA (bacterial DNA).

The main objective of these studies was to characterize metabolic, body composition, and cardiovascular responses to a free-choice high-fat, high-sucrose diet in female cycling and pregnant rats. In the nonpregnant state, female Sprague-Dawley rats offered a 3-wk free-choice high-fat, high-sucrose diet had greater energy intake, adiposity, serum leptin, and triglyceride concentrations compared with rats fed with standard chow and developed glucose intolerance. In addition, choice-diet-fed rats had larger cardiac ventricular weights, smaller kidney and pancreas weights, and higher blood pressure than chow-fed rats, but they did not exhibit resistance artery endothelial dysfunction. When the free-choice diet continued throughout pregnancy, rats remained hyperphagic, hyperleptinemic, and obese. Choice pregnant rats exhibited uterine artery endothelial dysfunction and had smaller fetuses compared with chow pregnant rats. Pregnancy normalized mean arterial blood pressure and pancreas weights in choice rats. These studies are the first to provide a comprehensive evaluation of free-choice high-fat, high-sucrose diet on metabolic and cardiovascular functions in female rats, extending the previous studies in males to female cycling and pregnant rodents. Free-choice diet may provide a new model of preconceptual maternal obesity to study the role of increased energy intake, individual food components, and preexisting maternal obesity on maternal and offspring physiological responses during pregnancy and after birth.

Key points In vivo, uterine perivascular adipose tissue (PVAT) potentiates uterine artery blood flow in pregnant rats, although not in non-pregnant rats. In isolated preparations, uterine PVAT has pro-contractile and anti-dilatory effects on uterine arteries. Pregnancy induces changes in uterine arteries that makes them responsive to uterine PVAT signalling. Abstract An increase in uterine artery blood flow (UtBF) is a common and necessary feature of a healthy pregnancy. In the present study, we tested the hypothesis that adipose tissue surrounding uterine arteries (uterine perivascular adipose tissue; PVAT) is a novel local mediator of UtBF and uterine artery tone during pregnancy. In vivo experiments in anaesthetized Sprague-Dawley rats showed that pregnant animals (gestational day 16, term = 22--23 days) had a three-fold higher UtBF compared to non-pregnant animals. Surgical removal of uterine PVAT reduced UtBF only in pregnant rats. In a series of ex vivo bioassays, we demonstrated that uterine PVAT had pro-contractile and anti-dilatory effects on rat uterine arteries. In the presence of PVAT-conditioned media, isolated uterine arteries from both pregnant and non-pregnant rats had reduced vasodilatory responses. In non-pregnant rats, these responses were mediated at the level of uterine vascular smooth muscle, whereas, in pregnant rats, PVAT-media reduced endothelium-dependent relaxation. Pregnancy increased adipocyte size in ovarian adipose tissue but had no effect on uterine PVAT adipocyte morphology. In addition, pregnancy down-regulated the gene expression of metabolic adipokines in uterine but not in aortic PVAT. In conclusion, this is the first study to demonstrate that uterine PVAT plays a regulatory role in UtBF, at least in part, as a result of its actions on uterine artery tone. We propose that the interaction between the uterine vascular wall and its adjacent adipose tissue may provide new insights for interventions in pregnancies with adipose tissue dysfunction and abnormal UtBF.

Epidemiological studies demonstrate disparities between men and women in cardiovascular disease prevalence, clinical symptoms, treatments, and outcomes. Enrollment of women in clinical trials is lower than men, and experimental studies investigating molecular mechanisms and efficacy of certain therapeutics in cardiovascular disease have been primarily conducted in male animals. These practices bias data interpretation and limit the implication of research findings in female clinical populations. This review will focus on the biological origins of sex differences in cardiovascular physiology, health, and disease, with an emphasis on the sex hormones, estrogen and testosterone. First, we will briefly discuss epidemiological evidence of sex disparities in cardiovascular disease prevalence and clinical manifestation. Second, we will describe studies suggesting sexual dimorphism in normal cardiovascular function from fetal life to older age. Third, we will summarize and critically discuss the current literature regarding the molecular mechanisms underlying the effects of estrogens and androgens on cardiac and vascular physiology and the contribution of these hormones to sex differences in cardiovascular disease. Fourth, we will present cardiovascular disease risk factors that are positively associated with the female sex, and thus, contributing to increased cardiovascular risk in women. We conclude that inclusion of both men and women in the investigation of the role of estrogens and androgens in cardiovascular physiology will advance our understanding of the mechanisms underlying sex differences in cardiovascular disease. In addition, investigating the role of sex-specific factors in the development of cardiovascular disease will reduce sex and gender disparities in the treatment and diagnosis of cardiovascular disease. © 2019 American Physiological Society. Compr Physiol 9:375-411, 2019.

Introduction: In utero insults have been linked with increased fear and anxiety in progeny. In utero hypoxic stress is associated with a multitude of gestational complications such as pregnancy-associated hypertensive disorders and intrauterine growth restriction. Maternal hypertension during pregnancy is also associated with increased mood and anxiety disorders in progeny. However, it is unknown if these associations are due to in utero hypoxic stress. We hypothesized that exposure to late gestational hypoxia will have a long-term impact on anxiety in progeny. Methods: Timed pregnant female Long-Evans rats were exposed to five days (gestational days: 15-20) of chronic intermittent hypoxia (CIH) or room air (normoxia - 21% O2) for 8 hours during their sleep phase. Each CIH cycle was 6 min of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2) for a total of 10 CIH cycles/hour. At weaning (PND 28), progeny was pair-housed with a conspecific of same sex and similar weight. To examine mood and anxiety disorders, we quantified anxiety-related behaviors (time spent in the center of open field arena, marble burying test, social and anti-social behaviors with conspecifics) along with quantifying food intake and circulating sex hormone levels during puberty (postnatal day, PND 40-45) and young adulthood (PND 60-65) in male and female progeny. Results: Gestational CIH did not impact circulating sex hormones or food intake, regardless of sex or age of progeny. However, gestational CIH increased anxiety related behaviors in pubertal females. These effects of gestational CIH on anxiety in pubertal females were not maintained, as these behaviors resolved in young adulthood. Gestational CIH did not impact male progeny, regardless of age. Conclusion: Exposure to CIH during gestation resulted in increased anxiety related behaviors in pubertal female progeny. In utero hypoxia during late gestation may temporarily increase the risk for mood and anxiety disorders in pubertal females.