Browsing by Author "Osikoya, Oluwatobiloba"
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Item Exosome-like vesicles facilitate intercellular communication between uterine artery smooth muscle cells and perivascular adipose tissue(2020) Cushen, Spencer; Raetz, Megan; Saranya Conjeevaram Nagarajan, Bhavani; Raut, Sangram; Goulopoulou, Styliani; Osikoya, OluwatobilobaIntroduction Perivascular adipose tissue (PVAT) regulates uterine artery tone during pregnancy. However, the mechanisms underlying its functional role in uterine arteries is unknown. Exosomes are cargo carrying membrane-bound extracellular vesicles used in intercellular communication. It is unknown whether PVAT secretes exosomes. We hypothesized that uterine PVAT sheds exosomes (Exo-PVAT) that are uptaken by neighboring uterine vascular smooth muscle cells (USMCs). Methods Exo-PVAT were isolated and purified with tissue culture and ultracentrifugation, and primary USMCs were isolated using enzymatic digestion from pregnant and non-pregnant rats. Exosome protein content, size and molecular weight were determined via western blot, Malvern Zetasizer and fast protein liquid chromatography (FPLC), respectively. To determine USMC uptake of Exo-PVAT, Exo-PVAT were labelled with a membrane-labeling dye and co-cultured with USMCs for 3 hours. Results Exo-PVAT expressed TSG101, Alix, and CD9. Pregnancy did not affect Exo-PVAT size [Median(IQR) (nm), Non-pregnant: 99.4 (80.5) vs. Pregnant: 46.7 (21.2), p=0.5]. Using FPLC, we identified exosomes of 40-200 kDa in samples from both pregnant and non-pregnant rats. PVAT from pregnant rats secreted a relatively high amount of exosomes of 2000 kDa compared to non-pregnat rats. Immunocytochemical assessments revealed that USMCs took up exosomes derived from their adjacent PVAT. Conclusion Uterine PVAT sheds exosome-like vesicles which are uptaken by adjacent USMCs. The interaction between Exo-PVAT and USMCs is a novel type of intercellular communication that may have important implications in uterine artery function and blood flow in pregnancy.Item L-sulforaphane Decreased Contractile Response in Mesenteric Arteries in A Rat Model of Gestational Hypertension(2017-03-14) Osikoya, Oluwatobiloba; Goulopoulou, Styliani PhD; Cushen, SpencerBackground: Maternal hypertension is a state of inflammation characterized by oxidative stress. Exposure of pregnant rats to the Toll-like Receptor 9 activator, ODN2395, induces hypertension and upregulates vascular oxidative stress. The transcription factor, Nuclear Factor Erythroid 2 Like 2 (Nrf2), is a regulator of antioxidant response, is overexpressed in placentas from patients with preeclampsia. However, the role of Nrf2 in maternal vascular dysfunction is unknown. Hypothesis: L-sulforaphane, an Nrf2 activator, will have anti-contractile effects on arteries from pregnant rats treated with CpG oligonucleotides (a model of gestational hypertension). Methods: Pregnant Sprague-Dawley rats were treated with synthetic unmethylated CpG oligonucleotides (ODN2395, 100µg/intraperitoneal injection) or saline (Control) on gestational day 14, 16, and 18 (term=21-22 days). Blood pressure was measured before pregnancy and on gestational day 19 using the tail cuff method. The contractile responses of mesenteric resistance arteries to a thromboxane A2 (TxA2) mimetic, U46619, in the presence or absence of Nrf2 activator, L-sulforaphane (L-S, 40 µM), were assessed by wire myography on gestational day 21. Results: Rats treated with ODN2395 had greater systolic blood pressure on gestational day 19 compared to control rats (Control, n=9: 100±4 mmHg vs. ODN2395, n=7: 119±4 mmHg, p=0.007). Three-hour but not one-hour incubation with L-sulforaphane reduced the contractile response to U46619 in mesenteric arteries from both ODN2395 and control rats (Peak contraction as %Max KCl (120mM), Control Veh: 120.5%±4.85; Control L-S: 39.1%±7.16; ODN2395 Veh: 111.1%±4.19; ODN2395 L-S: 42.6%±2.68). Conclusions: Pregnancy is a state of oxidative stress and this may explain the anti-contractile effects of L-sulforaphane in arteries from normal, healthy rats. In preeclampsia, levels of oxidative stress are greater compared to normotensive pregnancies and thus, systemic treatment with L-sulforaphane or other Nrf2 activators may improve poor cardiovascular outcomes in pregnancies with preeclampsia.Item Long-term effects of late gestational maternal hypoxic stress on mood disorders: Sex and age differences(2021) Mabry, Steve; Wilson, Elizabeth; Rybalchenko, Nataliya; Engelland, Rachel; Fadeyibi, Oluwadarasimi; Osikoya, Oluwatobiloba; Cushen, Spencer; Goulopoulou, Styliani; Cunningham, RebeccaPURPOSE: In utero insults have been linked with increased fear and anxiety in progeny. In utero hypoxic stress is also associated with multiple gestational complications. We hypothesized that exposure to maternal hypoxia during late gestation will have a long-term impact on anxiety in progeny. METHODS: 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 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 anxiety disorders, we quantified anxiety-related behaviors (time spent in 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: Maternal CIH did not impact circulating sex hormones or food intake, regardless of sex or age of progeny. However, maternal CIH increased anxiety related behaviors in pubertal females but were not observed in young adulthood. Maternal CIH did not impact male progeny, regardless of age. CONCLUSIONS: Maternal CIH during gestation resulted in increased anxiety related behaviors in pubertal female progeny. Maternal hypoxia during late gestation may temporarily increase the risk for anxiety disorders in pubertal females.Item Long-term effects of late gestational maternal hypoxic stress on neurodegeneration: Sex and age differences(2021) Wilson, Elizabeth; Mabry, Steve; Rybalchenko, Nataliya; Engelland, Rachel; Fadeyibi, Oluwadarasimi; Osikoya, Oluwatobiloba; Cushen, Spencer; Goulopoulou, Styliani; Cunningham, RebeccaIntroduction: In utero insults can lead to onset of neurodegenerative diseases, such as Parkinson's disease (PD). In utero hypoxic insults are associated with maternal sleep apnea or preeclampsia. It is unknown whether late gestational maternal hypoxic insults have long-term effects on brain regions associated with PD, such as the nigrostriatal pathway. We hypothesized that late gestational maternal hypoxia will result in sustained nigrostriatal impairment in male and female progeny. Methods: Timed pregnant 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 alternating 3 min hypoxia (10% O2) and normoxia (21% O2) totaling 10 CIH cycles/hour. Gestational age and biometrics were recorded 12-16 hours after birth. At postnatal day, PND 28, progeny were pair-housed with a conspecific of the same sex and similar weight. We focused on PD associated oxidative stress and behavioral impairments in the nigrostriatal pathway. Gross motor (open field), fine motor (ultrasonic vocalizations), and cognition (spatial memory) were examined during puberty and young adulthood. Results: Maternal CIH had no effect on gestational age, progeny biometrics, or progeny circulating oxidative stress. Gross motor and cognitive functions were unaffected by maternal CIH. However, a sustained fine motor impairment was observed in both male and female progeny. Conclusion: Maternal hypoxia during late gestation induced sustained nigrostriatal pathway impairment, which may increase the risk for neurodegeneration.Item Long-term effects of prenatal chronic intermittent hypoxia insult on the substantia nigra(2021) Engelland, Rachel; Fadeyibi, Oluwadarasimi; Rybalchenko, Nataliya; Wilson, Elizabeth; Mabry, Steve; Osikoya, Oluwatobiloba; Cushen, Spencer; Goulopoulou, Styliani; Cunningham, RebeccaPurpose: Prenatal chronic intermittent hypoxia (CIH) was employed to evaluate the effects of hypoxic insults on the substantia nigra (SN), which is impacted by Parkinson's disease (PD). SN loss during PD is linked with oxidative stress (OS) and apoptosis. We hypothesized that exposure to late gestational maternal hypoxia would result in an increase in increased OS, but not apoptosis, in the SN of adult male and female progeny. Methods: During gestational days 15-20, pregnant Long-Evans rats were exposed to CIH or room air (normoxia) for 8 hours. CIH consisted of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2). Animals were sacrificed at puberty (PND 44) or adulthood (PND 66). SN micropunches were obtained. OS was quantified by measuring calpain cleavage of spectrin. Results: OS (calpain cleavage of spectrin) was increased in the SN of adult male and female rats exposed to prenatal CIH compared to control (F1,17 = 3.606; p = 0.075). No effects on OS were observed in pubertal rats. Apoptosis (caspase-3 cleavage of spectrin) was not observed in any of the groups. Conclusions: These data suggest that prenatal CIH programming has a long-lasting impact on the SN of adult progeny, which may increase the susceptibility of SN to damage and PD risk. Although no sex differences were observed in this pilot study, we may see a sex effect upon increasing animal number, especially in male rats. This is consistent with the higher incidence of PD in men than in women.Item Low-dose Aspirin During Gestation Promotes Vascular Dysfunction and does not Ameliorate Maternal Hypertension in Rats Exposed to Innate Immune System Activation(2016-03-23) Nguyen, An; Valdes, Melissa; Osikoya, Oluwatobiloba; Goulopoulou, Styliani PhD; Jaini, PareshBackground: Daily low-dose aspirin after 12 weeks of gestation is recommended as a preventive intervention for women at high risk for preeclampsia, a hypertensive disorder of pregnancy with high rates of maternal and fetal mortality and morbidity. Activation of the innate immune system during pregnancy is implicated in the development of preeclampsia. Maternal exposure to synthetic CpG oligonucleotides (CpG ODN, specific ligand of the innate immune receptor Toll-like receptor 9) induces maternal hypertension, vascular dysfunction, and upregulation of cyclooxygenase enzymes in pregnant rats. Hypothesis: We hypothesized that maternal treatment with low-dose aspirin during gestation would ameliorate TLR9-induced hypertension and vascular dysfunction in pregnant rats. Methods: Pregnant Sprague-Dawley rats were treated with a synthetic CpG ODN (ODN2395) or vehicle on gestational day (GD) 14, 16, and 18. Aspirin treatment (or control) started on GD10 and continued throughout gestation for all groups [control (no treatment), ODN2395 (300 μg), aspirin (1.5 mg/kgBW), aspirin+ODN2395]. Blood pressure was measured on GD19 using the tail cuff method and mesenteric resistance artery (MES) function was assessed on GD21 using wire myography. Results: ODN2395-treated rats had higher blood pressure on GD19 compared to vehicle-treated dams and aspirin did not ameliorate ODN2395-induced hypertension (control: 97 ± 0.4 mmHg, ODN2395: 121 ± 7 mmHg, aspirin: 101 ± 5 mmHg, aspirin+ODN2395: 121 ± 7 mmHg, p Aspirin treatment increased MES sensitivity to PE (pEC50, ODN2395: 5.6 ± 0.1 vs. aspirin+ODN2395: 5.9 ± 0.1, ppEC50, ODN2395: 7.6 ± 0.1 vs. aspirin+ODN2395: 7.0 ± 0.1, pConclusion: Treatment with low-dose aspirin throughout gestation did not prevent the development of TLR9-induced maternal hypertension, augmented vascular sensitivity to α1-adrenergic receptor activation and attenuated endothelium-dependent dilation in rats exposed to innate immune system activation. The use of aspirin during gestation should be considered with caution in clinical cases associated with innate immune system-induced pregnancy complications.Item Placental Exposure to Hypoxia and Oxidative Stress Causes Mitochondrial DNA Release into the Extracellular Space(2019-03-05) Osikoya, Oluwatobiloba; Blessing, Alexandra; Phillips, Nicole; Goulopoulou, Styliani; Cushen, SpencerPurpose In preeclampsia, a severe hypertensive disorder of pregnancy, placentae experience reduced perfusion, increased cell death, and oxidative stress. Also, there is an increase in circulating cell-free mitochondrial DNA (mtDNA) in the maternal blood. The main objective of this study was to determine the role of hypoxia and oxidative stress in mtDNA release from placental cells, and to examine the effects of soluble factors from hypoxia-exposed placentae on vascular reactivity. To address this objective, the following hypotheses will be tested: a) Exposure to hypoxia and oxidative stress will result in mtDNA release via cell-death dependent mechanisms in human trophoblast cells. b) Soluble factors from hypoxia-exposed placentae will result in reduced vasodilation in rat maternal arteries. Methods To examine the effects of preeclampsia-related placental stressors on mtDNA release, we treated human trophoblast cells (BeWo cell line) with: 1) hypoxia (1% O2) vs. normoxia (21% O2) for 15 h, or 2) a mitochondrial complex I inhibitor (Rotenone, 10 μM) vs. vehicle for 4 h. mtDNA in cell culture supernatant was measured using absolute qPCR and cell death was quantified using flow cytometry. To test the effects of hypoxic placenta-derived factors on maternal vascular function, we used mesenteric arteries and placenta-conditioned media (PLmedia) from pregnant rats. Placentae were incubated in physiological salt solution (37oC) for 3 h in either 1% or 21% O2, while arteries were mounted on a wire myograph and underwent a baseline [(-) PLmedia] concentration-response curve (CRC) to acetylcholine (ACh, 10-9 – 3x10-5 M) followed by 30-min incubation with PLmedia, after which the CRC was repeated. Results Exposure of trophoblast cells to rotenone resulted in cell death (Vehicle: 28.17 ± 2.67% vs. Rotenone: 48.43 ± 1.22%, n = 3, P = 0.002) and mtDNA release (Vehicle: 1.69 ± 0.12 ng/uL vs. Rotenone: 2.39 ± 0.10 ng/uL, n = 5, P = 0.002). Hypoxia did not induce trophoblast cell death (Normoxia: 24.7 ± 0.50% vs. Hypoxia: 24.25 ± 0.45%, n = 2, P = 0.6), but increased release of mtDNA (Normoxia: 14.22 ± 1.20 pg/uL vs. Hypoxia: 20.64 ± 0.39 pg/uL, n = 3, P = 0.007). PLmedia from normoxic and hypoxic placentae reduced sensitivity to ACh (–logEC50, Normoxia: (–)PLmedia: 7.48 ± 0.03 vs. (+)PLmedia: 6.96 ± 0.10, n = 4, P = 0.02; Hypoxia: (–)PLmedia: 7.35 ± 0.35 vs. (+)PLmedia: 6.70 ± 0.29, n = 3, P = 0.08). Conclusion A placental cell model of mitochondrial stress results in cell death and release of mtDNA, while a hypoxic model of stress results in release of mtDNA without cell death. Placental factors decrease resistance artery sensitivity to vasodilators in both normoxic and hypoxic conditions, indicating that the placenta contributes to maternal vascular tone in healthy pregnancies and in pregnancies complicated with reduced perfusion. Ongoing studies investigate the vasoactive potential of placenta-derived cell-free mtDNA.Item Postpartum Maternal Vascular Function in a Rat Model of Gestational Obstructive Sleep Apnea(2020) Osikoya, Oluwatobiloba; Wilson, Elizabeth; Mabry, Steve; Cushen, Spencer; Cunningham, Rebecca; Goulopoulou, Styliani; Singhal, JuhiIntroduction: De novo obstructive sleep apnea (OSA) in pregnancy is associated with adverse gestational outcomes. In pregnant mice, exposure to chronic intermittent hypoxia (CIH), a model of OSA, induces endothelial dysfunction in maternal uterine arteries. It is currently unknown whether gestational OSA has a long-term effect on maternal vascular function. We hypothesized that exposure to gestational CIH during pregnancy will result in postpartum maternal vascular dysfunction. Methods: Pregnant rats were assigned to Normoxia and CIH groups. The CIH group was exposed to five days of intermittent hypoxia [6 min cycles of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2)]. Endothelial function was assessed in isolated maternal uterine arteries at weaning (postnatal day 28). Vascular reactivity to acetylcholine (ACh) was examined in the presence and absence of uterine perivascular adipose tissue (PVAT) using wire myography. Results: In the absence of PVAT, uterine arteries from dams exposed to gestational CIH had exaggerated responses to ACh compared to dams exposed to normoxia [(-)PVAT/pEC50, Normoxia: 6.77±0.08 vs. CIH: 7.13±0.09, p< 0.01], while PVAT normalized this difference [(+)PVAT/pEC50, Normoxia: 6.67±0.08 vs. CIH: 6.70±0.07, p=0.99]. The effects of PVAT were due to its anti-dilatory influences on uterine arteries from CIH-treated rats ([CIH (-PVAT) vs. CIH (+PVAT), p = 0.003), whereas it had no effect on arteries from normoxic rats (p = 0.77). Conclusion: Exposure to gestational CIH exaggerated postpartum uterine vascular smooth muscle relaxation responses. Gestational OSA may impair maternal vascular recovery after birth.Item Pregnancy Augments the Vasoactive, Metabolic, and Inflammatory Functions of Uterine Perivascular Adipose Tissue(2018-03-14) Goulopoulou, Styliani; Osikoya, OluwatobilobaIntroduction: Perivascular adipose tissue (PVAT) is functionally different from other adipose depots and has vasoactive effects that vary with anatomic location and disease state. Healthy pregnancy involves remodeling of the vessels that supply blood flow to the uteroplacental unit (i.e. uterine arteries) and increases adipose tissue metabolic and inflammatory functions. The main objective of this study was to examine whether pregnancy changes the function of PVAT surrounding the uterine arteries (utPVAT). Hypothesis: Healthy pregnancy augments the vasoactive, metabolic, and inflammatory functions of utPVAT. Methods: Pregnant (gestational day 16, term=22-23 days) and aged-matched non-pregnant rats were used. To evaluate the effects of utPVAT on endothelium-dependent dilation in uterine artery, we performed concentration-response curves to acetylcholine (ACh) in the presence or absence of utPVAT using wire myography. A proteome adipokine profiler and reverse transcription polymerase chain reaction (RT-PCR) were used to assess protein and gene expression of utPVAT adipocytokines, respectively. Results: Incubation of uterine arteries from pregnant rats with utPVAT reduced ACh-induced relaxation responses following constriction with 60 mM potassium chloride solution [pEC50, +PVAT (n=5): 6.25 ± 0.12 vs. –PVAT (n=5): 6.66 ± 0.18, p = 0.02] or 10-6 M phenylephrine [pEC50, +PVAT (n=9): 6.86 ± 0.10 vs. –PVAT (n=9): 7.61 ± 0.17, p=0.0004]. This effect was not seen in arteries from non-pregnant animals. Uterine PVAT from pregnant rats (n=3) had reduced mRNA expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) by 5.7 fold compared to utPVAT from non-pregnant rats (n=4). Leptin mRNA expression was reduced by 6.4 fold and protein expression was increased in utPVAT from pregnant rats compared to utPVAT from non-pregnant rats. Interleukin (IL)-10, IL-6, and monocyte chemoattractant protein (MCP)-1 mRNA expression in utPVAT did not differ between groups but protein expression of these adipocytokines was increased in utPVAT from pregnant rats. Conclusion: In pregnancy, utPVAT reduces endothelium-dependent relaxation in uterine arteries. In addition, pregnancy regulates metabolic and inflammatory adipocytokines in utPVAT at the level of protein translation. Future studies will determine the functional role of the vasoactive and molecular changes in utPVAT and their impact on uterine blood flow and fetal growth.Item Sex and age differences in social and cognitive function in offspring exposed to late gestational hypoxia(BioMed Central Ltd., 2023-11-12) Mabry, Steve; Wilson, E. Nicole; Bradshaw, Jessica L.; Gardner, Jennifer J.; Fadeyibi, Oluwadarasimi; Vera, Edward, Jr.; Osikoya, Oluwatobiloba; Cushen, Spencer C.; Karamichos, Dimitrios; Goulopoulou, Styliani; Cunningham, Rebecca L.BACKGROUND: Gestational sleep apnea is a hypoxic sleep disorder that affects 8-26% of pregnancies and increases the risk for central nervous system dysfunction in offspring. Specifically, there are sex differences in the sensitivity of the fetal hippocampus to hypoxic insults, and hippocampal impairments are associated with social dysfunction, repetitive behaviors, anxiety, and cognitive impairment. Yet, it is unclear whether gestational sleep apnea impacts these hippocampal-associated functions and if sex and age modify these effects. To examine the relationship between gestational sleep apnea and hippocampal-associated behaviors, we used chronic intermittent hypoxia (CIH) to model late gestational sleep apnea in pregnant rats. We hypothesized that late gestational CIH would produce sex- and age-specific social, anxiety-like, repetitive, and cognitive impairments in offspring. METHODS: Timed pregnant Long-Evans rats were exposed to CIH or room air normoxia from GD 15-19. Behavioral testing of offspring occurred during either puberty or young adulthood. To examine gestational hypoxia-induced behavioral phenotypes, we quantified hippocampal-associated behaviors (social function, repetitive behaviors, anxiety-like behaviors, and spatial memory and learning), hippocampal neuronal activity (glutamatergic NMDA receptors, dopamine transporter, monoamine oxidase-A, early growth response protein 1, and doublecortin), and circulating hormones in offspring. RESULTS: Late gestational CIH induced sex- and age-specific differences in social, repetitive, and memory functions in offspring. In female pubertal offspring, CIH impaired social function, increased repetitive behaviors, and elevated circulating corticosterone levels but did not impact memory. In contrast, CIH transiently induced spatial memory dysfunction in pubertal male offspring but did not impact social or repetitive functions. Long-term effects of gestational CIH on social behaviors were only observed in female offspring, wherein CIH induced social disengagement and suppression of circulating corticosterone levels in young adulthood. No effects of gestational CIH were observed in anxiety-like behaviors, hippocampal neuronal activity, or circulating testosterone and estradiol levels, regardless of sex or age of offspring. CONCLUSIONS: Our results indicate that hypoxia-associated pregnancy complications during late gestation can increase the risk for behavioral and physiological outcomes in offspring, such as social dysfunction, repetitive behaviors, and cognitive impairment, that are dependent on sex and age. Sleep apnea during late pregnancy is a common pregnancy complication that can impact the brain development of children born to mothers with sleep apnea. Children with impaired brain development may present with decreased social skills, memory issues, anxiety, and compulsivity. It is unclear if there is a cause and effect relationship between sleep apnea during late pregnancy and behavioral changes in offspring. Additionally, it is unknown whether male or female sex or age of the offspring affects these relationships. In this study, we exposed pregnant rats to a model of sleep apnea called chronic intermittent hypoxia (CIH) within late gestation and examined the behavior of the offspring and brain activity during puberty and young adulthood. We found that CIH during late pregnancy had long-term effects in the offspring that were different in males and females. Notably, female offspring displayed social impairments in response to late gestation CIH, whereas male offspring displayed cognitive dysfunction.Item Uterine Perivascular Adipose Tissue Potentiates Contractile Responses in Uterine Arteries from Pregnant rats(2016-03-23) Goulopoulou, Styliani PhD; Osikoya, OluwatobilobaBackground: Perivascular adipose tissue (PVAT) is the fourth and outer layer of the vascular wall. PVAT has vasoactive effects, mostly via paracrine actions. The effects of PVAT vary with anatomic location; PVAT has anti-contractile effects in peripheral vascular beds in animals and in humans but it potentiates contractions in coronary vascular smooth muscle. Pregnancy is characterized by adipose tissue expansion as well as structural and functional changes in the uterine vasculature. However, the effects of PVAT on uterine artery reactivity during pregnancy are not understood. Hypothesis: We hypothesized that uterine PVAT has a functional role in uterine artery contractile and dilatory responses and this role is modified by pregnancy. Methods: Pregnant Sprague-Dawley rats were sacrificed on gestational day 16 (term=21-22 days). Uterine arteries and their surrounding PVAT were harvested and cleaned for study. Concentration response curves (CRCs) to potassium chloride (KCl, 4.7 – 80 mM) and phenylephrine (PE, 10-9 - 3x10-5 M) were performed using wire myography. CRCs were performed in the presence and absence of the surrounding PVAT (0.1 g) or PVAT-conditioned media. Arteries were incubated with PVAT or PVAT-conditioned media for 30 minutes. To make the media, we incubated 0.4 g of PVAT in 15 ml physiological salt solution for 90 min (37oC - 5% CO2, 95% O2). Results: Uterine arteries incubated with PVAT (+PVAT) had greater contractile responses to KCl compared to control vessels [KCl (30 mM), control: 4.0 ± 0.81 mN vs. +PVAT: 14.7 ± 1.68 mN; KCl (40 mM), control: 14.4 ± 0.68 mN vs. +PVAT: 19.6 ± 0.88 mN, p50, control: 6.0 ± 0.08 vs. +PVAT: 6.3 ± 0.10, p50), control: 6.1 ± 0.08 vs. +PVAT-media: 6.4 ± 0.10, p=0.07]. Conclusions: Our data show that uterine PVAT has a pro-contractile effect on uterine arteries from pregnant rats. We propose that uterine PVAT provides signaling from the outer layer to the inner layers of the vascular wall that determines uteroplacental vascular adaptations to pregnancy.Item Uterine perivascular adipose tissue potentiates vasoconstriction in maternal arteries during rat pregnancy(2017-03-14) Goulopoulou, Styliani; Osikoya, OluwatobilobaBackground: Perivascular adipose tissue (PVAT) functions mostly to increase vasodilation in healthy conditions, but increases vasoconstriction in diseased states. During pregnancy, adipose tissue expands, and uterine arteries (UTA) undergo substantial remodeling. Previously, our laboratory showed that uterine PVAT potentiates contractile responses in UTA from pregnant but not in arteries from non-pregnant rats. It is unknown, however, if the effects of uterine PVAT are vascular bed specific and if they are mediated by pregnancy. Hypothesis: Uterine PVAT potentiates contractions in maternal arteries independently of vascular bed and this pro-contractile property is mediated by pregnancy specific factors. Methods: Sprague-Dawley pregnant rats were sacrificed on gestational day 16 (term=21-22) and aged-matched non-pregnant rats were used as controls. Uterine PVAT, and isolated segments of UTA and mesenteric arteries (MES) mounted onto a wire myograph. To determine whether uterine PVAT has pro-contractile effects independently of vascular bed, pregnant UTA and MES were incubated with uterine PVAT (0.1 g) for 30 minutes. To determine whether the pro-contractile effects of uterine PVAT are mediated by pregnancy specific changes, UTA from pregnant and non-pregnant rats were incubated for 30 minutes with non-pregnant PVAT (0.1 g) and pregnant PVAT (0.1 g), respectively. Concentration response curves to potassium chloride (KCl, 4.7 – 80 mM) were performed in all arteries. Force generated at each KCl concentration was expressed in mN and area under the curve (AUC) was used to quantify total contraction. Results: MES from pregnant rats had increased contractile responses to KCl when incubated with uterine PVAT (AUC, -PVAT: 366±46 vs. +PVAT: 529±53, P = 0.02). UTA from pregnant rats had increased contractile responses to KCl when incubated with uterine PVAT (AUC, -PVAT: 893±40 vs. +PVAT: 1092±59, p=0.004). Preliminary data showed that PVAT from non-pregnant rats increased contractile responses in UTA from pregnant rats (KCl 30 mM, -PVAT: 4.7±0.9 mN vs. +PVAT: 11.4±1.4 mN, p=0.047). Pregnant PVAT had no effect on UTA from non-pregnant rats (p [greater than] 0.05). Conclusions: Uterine perivascular adipose tissue potentiates vasoconstriction in both uterine and mesenteric arteries from pregnant rats. These effects may be due to vascular remodeling during pregnancy.Item Uterine perivascular adipose tissue: A novel regulator of uterine artery hemodynamics during normal pregnancy(2019-03-05) Ahmed, Hijab; Panahi, Sareh; Bourque, Stephane; Goulopoulou, Styliani; Osikoya, OluwatobilobaIntroduction During pregnancy, uterine artery (UtA) blood flow increases compared to non-pregnant state, in part due to reductions in uterine artery tone. The main objective of this study was to determine the role of adipose tissue surrounding UtA (perivascular adipose tissue, PVAT) in pregnancy-induced changes in UtA blood flow and vasodilatory capacity. We hypothesized that uterine PVAT augments UtA blood flow and potentiates UtA dilatory responses in pregnant rats. Also, we hypothesized that pregnancy induces distinct changes in uterine PVAT morphology and gene expression as compared to other adipose depots. Methods Blood flow and vascular reactivity were measured in UtA in pregnant and non-pregnant rats using transonic perivascular probes and wire myography techniques, respectively. Reactivity to acetylcholine (ACh: induces endothelium-dependent relaxation, 10-9 - 3x10-5 M) and sodium nitroprusside (SNP: induces endothelium-independent relaxation, 10-11 - 3x10-5 M) was measured in isolated UtA in the presence and absence of PVAT-conditioned media (PVATmedia, 30-min incubation). Adipocyte size was determined in hematoxylin and eosin-stained sections of uterine PVAT and ovarian adipose tissue. Gene expression was determined in uterine and periaortic PVAT using qRT-PCR. Results Maximum and minimum uterine artery blood flow (UBF) were increased in UtA with intact PVAT compared to PVAT-denuded UtA from pregnant rats (UBFmax (mL/min); denuded: 1.47 ± 0.3 vs. intact: 2.23 ± 0.2, p = 0.01; UBFmin (mL/min); denuded: 0.71 ± 0.1 vs. intact: 1.16 ± 0.1, p = 0.0002). Uterine PVAT had no effect on UBF in non-pregnant rats (p [greater than] 0.9). UtA from pregnant and non-pregnant rats incubated with PVATmedia had reduced sensitivity to ACh compared to UtA controls (Pregnant, pEC50; -PVATmedia: 7.14 ± 0.1 vs. +PVATmedia: 6.38 ± 0.2, p = 0.0006; Non-pregnant, pEC50; -PVATmedia: 7.01 ± 0.1 vs. +PVATmedia: 6.50 ± 0.1, p = 0.005). PVATmedia had no effect on UtA sensitivity to SNP in either pregnant (p = 0.48) or non-pregnant rats (p = 0.2). Adipocyte area was greater in ovarian adipose tissue from pregnant compared to non-pregnant rats [Area (μm2/unit cell); Non-pregnant: 563.6 ± 76.6 vs. Pregnant: 857.6 ± 31.0, p = 0.02] but there were no group differences in uterine PVAT morphology (p = 0.6). Expression of uncoupling protein-1 (UCP-1) was downregulated (p = 0.02) in aortic PVAT but was unchanged in uterine PVAT (p = 0.4). Expression of peroxisome proliferator-activated receptor gamma (PPAR-γ), adiponectin receptor (AdipoR1), and leptin were downregulated in uterine PVAT (p p [greater than] 0.6). Conclusions Uterine PVAT plays a regulatory role in uterine artery hemodynamics and reactivity during normal pregnancy and has a distinct and differential gene profile as compared to other perivascular depots. Ongoing studies investigate the effects of pregnancy on cross-talk between PVAT and maternal uterine arteries. Support or Funding Information University of North Texas Health Science Center Pilot Grant, CIHR (MOP142396) and the Women and Children's Health Research Institute at the University of Alberta