Browsing by Author "Goulopoulou, Styliani"
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Item A Comparative Analysis of Recruitment Methods used in Randomized Controlled Clinical Drug Trials(2019-05) Garud, Ashwini A.; Mathew, Stephen O.; Goulopoulou, Styliani; Anderson, Jessica; Maynard, BrianClinical trials are a crucial part of any drug development process. The reliability and validity of clinical trials depend on the successful recruitment of subjects. The overall goal of this project was to evaluate the effectiveness of subject recruitment methods used in Randomized Controlled Trials (RCTs) focusing on Major Depressive Disorder (MDD) and Postpartum Depression (PPD) studies. Recruitment methods (on-site and off-site) utilized in these trials were examined for their cost-effectiveness, recruitment return, including the number of subjects enrolled and the number of subjects randomized in the trial and subject demographics. Regarding cost effectiveness, on-site methods were found to be more effective in terms of recruitment return and less costly than off-site for both studies. There was a significant difference in race and gender when subjects were recruited from on-site versus off-site recruitment. For MDD study, a comparatively large number of enrolled and randomized participants were recruited from on-site recruitment methods as compared to off-site, this may be due to the number of physician referrals, indicating that physicians play a major role in subject recruitment. For the Postpartum Depression study, the number of enrolled and randomized subjects from off-site recruitment method was higher than those of on-site methods. Monitoring recruitment strategies implemented in the study and assessing their effectiveness would be helpful in employing strategies for future trials.Item Absolute Quantification of Mitochondrial DNA in Peripheral Blood from Women with Preeclampsia(2020) Silzer, Talisa; Phillips, Nicole; Goulopoulou, Styliani; Reid, Danielle; Sun, Jie; Scroggins, Sabrina; Santillan, Mark; Santillan, Donna; Cushen, SpencerIntroduction Mitochondrial DNA (mtDNA) in maternal blood has been proposed as a potential predictor of preeclampsia (PE). The objective of this study was to use an absolute PCR (abPCR) quantification protocol to determine concentrations of mtDNA in maternal plasma and peripheral blood mononuclear cells (PBMC) from normal and PE pregnancies. Methods Blood samples were collected from pregnant women with uncomplicated pregnancies and pregnancies with PE (University of Iowa IRB#200910784). abPCR quantification of mtDNA and nDNA was performed on DNA extracts from plasma (in the presence or absence of lysis buffer) and PBMCs using TaqMan(TM) probes and chemistry. Results When plasma DNA was extracted using lysis buffer, mtDNA concentrations were lower in women with PE than in controls (Control: 4.83 ± 1.09 vs. PE: 1.72 ± 0.38 pg/uL, n=19, P=0.017), while concentrations of nDNA did not differ (P=0.39). Without lysis buffer, plasma mtDNA remained lower in women with PE compared to controls (Control: 0.0106 ± 0.0019 vs PE: 0.0019 ± 0.0003 pg/uL, n=16-20, P< 0.0001). There were no group differences in PBMC mtDNA (P=0.66) and nDNA (P=0.13) concentrations. Conclusion mtDNA concentrations were lower in plasma of pregnant women with PE compared to controls. A significant amount of mtDNA was membrane bound as indicated by a 480-fold greater concentration in DNA isolated from plasma with lysis buffer vs. without. Use of this improved method of quantification of mtDNA in multiple blood fractions may allow for its development as a biomarker to detect PE prior to the onset of organ damage.Item CELL-FREE MEMBRANE-BOUND AND MEMBRANE-UNBOUND MITOCHONDRIAL DNA IN MATERNAL CIRCULATION IN PREECLAMPSIA(2021) Cushen, Spencer; Ricci, Contessa; Bradshaw, Jessica L.; Silzer, Talisa; Blessing, Alexandra M.; Sun, Jie; Scroggins, Sabrina; Santillan, Mark; Santillan, Donna; Phillips, Nicole; Goulopoulou, StylianiPURPOSE: Cell-free circulating mitochondrial DNA (CFCmtDNA) is a damage-associated molecular pattern (DAMP) that activates Toll-like receptor-9 (TLR-9). Previous studies suggested that CFCmtDNA may be a potential pathogenic trigger or a contributor to the maintenance of preeclampsia. The main objectives of this study were 1) to determine absolute concentrations of CFCmtDNA, in membrane-bound and -unbound states, independent of nuclear DNA (nDNA) changes, in cases with preeclampsia and healthy controls and 2) to implement a penalized regression analysis to establish the contribution of CFCmtDNA to preeclampsia diagnosis and its interaction with commonly collected patient characteristics. METHODS: Plasma CFCmtDNA (MT-ND5 gene) concentrations were quantified using an absolute quantification protocol. DNase I concentrations in maternal plasma were measured using an enzyme-linked immunosorbent assay and TLR-9 activity was monitored using SEAP reporter 293 cells expressing the human TLR-9 gene. RESULTS: Concentrations of CFCmtDNA were reduced in preeclampsia compared to healthy controls both in lysis buffer-treated samples (P=0.02) and in samples not treated with lysis buffer (P< 0.0001). Even though CFCmtDNA concentrations were reduced, plasma from women with preeclampsia induced greater TLR-9 activation than plasma from gestational age matched controls (P< 0.01). Multivariate analysis showed that high concentrations of nDNA and DNase I, a prior history of preeclampsia, and a lower concentration of CFCmtDNA are predictors of preeclampsia diagnosis. CONCLUSIONS: In conclusion, our data demonstrate an increased immunostimulatory potential of CFCmtDNA and upregulation of DNA degradation mechanisms in women with preeclampsia at the third trimester.Item Chronic Intermittent Hypoxia Increases Oxidative Stress and Impairs Spatial Memory in Male and Female Rats(2023) Gardner, Jennifer J.; Mabry, Steve; Bradshaw, Jessica L.; Wilson, E. Nicole; Little, Joel; Goulopoulou, Styliani; Cunningham, Rebecca L.Obstructive sleep apnea (OSA) is characterized by complex phenotypes and increased long-term risk of neurodegenerative disease. The impact of OSA in women is unknown due to sex differences in clinical presentation contributing to underdiagnosis. Using chronic intermittent hypoxia (CIH) to model OSA in rodents, our previous studies have shown CIH exposure increases oxidative stress and inflammation in male rats. However, the impact of CIH in female rats remains unclear. The objective of this study was to assess sex differences in CIH-mediated oxidative stress and rodent behaviors associated with neurodegenerative disease. Young adult male and female Long Evans and Sprague Dawley rats were exposed to CIH or normoxia for 14-15 days. Spatial memory and fine and gross motor skills were assessed. Plasma oxidative stress was measured and neuronal expression in the dorsal hippocampus was quantified. Female rats exhibited better spatial memory than males with increased neuronal expression in the CA1 region of the hippocampus. In both males and females, CIH impaired spatial memory and increased circulating oxidative stress. Yet, CIH increased CA1 neuronal expression in female rats only. CIH did not impact gross or fine motor skills, regardless of sex. Our preliminary findings indicate CIH increases oxidative stress and impairs spatial memory in males and females, but the impact of CIH on hippocampal neurons and region-specific contributions to spatial memory may be sexually dimorphic.Item Circulating Cell-free Mitochondrial DNA In Normal Human Pregnancy and In Experimental Preeclampsia(2017-03-14) Phillips, Nicole; Sprouse, Marc; Jarvis, Sara; Okada, Yoshiyuki; Morton, Jude; Davidge, Sandra; Fu, Qi; Goulopoulou, Styliani; Chaudhari, SarikaBackground: Mitochondrial DNA (mtDNA) is a damage-associated molecular pattern (DAMP) with potent immunogenic and inflammatory properties. Circulating cell-free mtDNA is increased in various inflammatory conditions associated with intense cell apoptotic processes. Pregnancy is characterized by systemic inflammation and placental apoptosis, which increase with advancing gestational age. The temporal changes of cell-free mtDNA during healthy pregnancy and in pregnancies with preeclampsia are unknown. Hypothesis: Circulating cell free mtDNA increases with gestational age in pregnant women and these changes positively correlate with maternal cardiovascular responses and neonatal biometrics. In a rat model of preeclampsia, circulating mtDNA is increased compared to normotensive control rats. Methods: Normal Human Pregnancy: Maternal blood samples were collected at early pregnancy (≤ 8 weeks of gestation), late pregnancy (32-36 weeks), and postpartum (6-10 weeks after delivery) in healthy, normotensive, pregnant women (n=21). Experimental Model of Preeclampsia: Reduced uterine perfusion pressure (RUPP) was surgically induced in pregnant rats on gestational day (GD) 14. Maternal blood samples were collected from RUPP rats (n=11) and control rats (Sham, n=11) on GD20. Absolute real-time PCR quantification of mtDNA was performed on whole genomic extracts from maternal human and rat sera using TaqMan® probes and chemistry. Results: Normal Human Pregnancy: Circulating mtDNA in late pregnancy were greater compared to early pregnancy (0.02 ± 1.2 pg/μL vs. 0.04 ± 1.2 pg/μL, p=0.04) and remained elevated post-partum (0.03 ± 1.2 pg/μL). Both blood pressure and heart rate increased from early to late pregnancy and decreased postpartum (pExperimental Model of Preeclampsia: RUPP rats had increased circulating mtDNA as compared to the sham group (0.30 ± 0.04 copy number/µL vs. 0.18 ± 0.04 copy number/µL, p=0.03). Conclusions: In normal pregnant women, circulating mtDNA change with advancing gestational age and may reflect rates of placental cell apoptosis. In a rat model of preeclampsia associated with placental ischemia, circulating cell free mtDNA is elevated in late pregnancy. The temporal changes in mtDNA in preeclampsia and their functional role in normal and preeclamptic pregnancies need to be further evaluated.Item Dysfunctional neuroimmune pathways promote the development and maintenance of lupus hypertension(2020-05) Pham, Grace S.; Mathis, Keisa W.; Rickards, Caroline A.; Goulopoulou, Styliani; Cunningham, J. Thomas; Ma, Rong; Mathew, Stephen O.Hypertension afflicts nearly half of the adults in the United States and the majority of cases have no known cause. Chronic inflammation has been implicated in the development and maintenance of hypertension, and autoimmunity may comprise one of its sources. Hypertension is highly prevalent in the autoimmune disease systemic lupus erythematosus (SLE), in which chronic aberrant inflammation may be a causative factor. Endogenous neuroimmune pathways, such as the hypothalamic-pituitary-adrenal (HPA) axis and the cholinergic anti-inflammatory pathway, likely contribute to this phenomenon. The HPA axis is a classical neuroimmune mechanism that senses peripheral inflammation via afferent vagal fibers, culminating in the release of the anti-inflammatory hormone cortisol. Previous studies have characterized HPA axis dysfunction in SLE, but less is known about how this dysregulation specifically impacts the hypertension that occurs in the setting of SLE. A second neuroimmune interaction, the cholinergic anti-inflammatory pathway, is an efferent vagus nerve-to-spleen mechanism that relies on T cell-produced acetylcholine to quell inflammation in acute settings and may be hypoactive in chronic inflammatory diseases like SLE. Notably, both of these neuroimmune mechanisms depend on vagus nerve function, identifying the vagus as a potential target for neuromodulation. Furthermore, the relationship between chronic inflammation and hypertension validates the investigation of neuroimmune pathway dysfunction towards novel mechanisms of hypertension. Herewithin, the HPA axis and cholinergic anti-inflammatory pathway are investigated using the well-established NZBWF1 mouse model of lupus hypertension. Our findings are that (1) administration of an inflammatory stimulus that activates vagal afferents elicits comparable neuronal activation in the paraventricular nucleus of the hypothalamus, compared to control mice, despite heightened peripheral inflammation; (2) amplification of efferent vagus nerve activity reduces blood pressure and renal inflammation; and (3) chronic unilateral vagotomy paradoxically results in decreased blood pressure and renal inflammation. Taken together, these findings identify dysfunction in two neuroimmune pathways while demonstrating that interventions targeting these pathways may have therapeutic benefits in lupus hypertension. In terms of future impact, these results may promote continuing inquiry in a more recently discovered neuroimmune pathway (i.e., cholinergic anti-inflammatory pathway), as well as reinstate curiosity in an older, abandoned area of research (i.e., HPA).Item Effects of the thromboxane receptor antagonist S18886 in the porcine coronary circulation(2023) Tucker, Selina M.; Warne, Cooper; Essajee, Salman; Goulopoulou, Styliani; Dick, Gregory; Tune, JohnathanThromboxane A2 (TxA2) is a potent coronary vasoconstrictor that has been implicated in promoting decreases in myocardial perfusion in a variety of (patho)-physiologic conditions. S18886 is a promising orally-active TxA2 receptor antagonist currently approved for investigational clinical use. However, the coronary vascular effects of S18886 are unknown and its specificity and affinity for the thromboxane receptor in the coronary circulation remain unclear. We tested the hypothesis that administration of S18886 dose-dependently attenuates coronary vasoconstriction to the TxA2 mimetic U46619 without influencing coronary responses to prostaglandin F2α, acetylcholine, or smooth muscle depolarization (K+). Experiments to test this hypothesis were performed in male (n = 5) and female (n = 6) domestic swine. Hearts were excised and the left circumflex coronary artery isolated, cleaned of periadventitial fat, and cut into 3 mm rings. Isometric tension of coronary artery rings was measured in response to log order increments of U46619 (1 nM to 1 µM) with and without S18886 (0.1-100 nM). Similar isometric studies were conducted with prostaglandin F2α (10 nM-10 µM), acetylcholine (0.1-10 µM), and KCl (5-90 mM). U46619 induced concentration dependent increases in tension development of isolated coronary artery rings (average EC50 of 42 ± 19 nM). Incubation of coronary arteries with S18886 (1 nM) significantly attenuated coronary vasoconstriction to U46619 resulting in a rightward shift of the EC50 to 187 ± 38 nM (P < 0.02). Vehicle had no effect on U46619-induced contractions. Higher concentrations of S18886 dose-dependently reduced U46619-induced contractions. S18886 (1 nM) antagonized coronary vasoconstriction of prostaglandin F2α (10 µM) by 68% ± 5 (P < 0.0001) but had no effect on either acetylcholine or KCl-induced contraction. Data from this investigation indicate that S18886 is an effective antagonist of U46619-induced vasoconstriction in the porcine coronary circulation. While S18886 does not influence coronary smooth muscle response to either acetylcholine or activation of L-type Ca2+ channels, attenuation of prostaglandin F2α suggests antagonists specificity may extend beyond TxA2 receptor signaling.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 Exposure to Synthetic CpG DNA During Pregnancy Increases Expression and Activity of Cyclooxygenase in Maternal Arteries(2017-03-14) Osikoya, Deborah; Jaini, Paresh; Nguyen, An; Valdes, Melissa; Goulopoulou, Styliani; Dimos, BradfordObjective: Preeclampsia is a pregnancy-associated disorder that is characterized by elevated maternal blood pressure, end organ damage, and/or proteinuria. This pregnancy syndrome affects 2-8% of pregnancies worldwide and has no treatment other than fetal delivery. The mechanisms behind the development of this condition remain unknown and is one of the barriers preventing the development of effective treatment. One of the suggested etiologies behind preeclampsia is activation of the innate immune system. We have previously determined that maternal exposure to CpG DNA (a ligand of Toll-like Receptor-9, TLR-9) causes maternal hypertension and excess vasoconstriction. In cancer cells, CpG DNA/TLR-9 activation leads to upregulation of cyclooxygenase (COX), which is the rate-limiting step in the conversion of arachidonic acid to prostaglandins and thromboxanes. It is unknown whether treatment with CpG DNA during pregnancy affects COX in maternal and fetal tissues. Hypothesis: Treatment of pregnant rats with CpG DNA induces maternal hypertension and increases COX function in maternal and fetal tissues. Methods: Pregnant Sprague-Dawley rats were treated with ODN 2395 (synthetic CpG DNA, 100μg/rat/ i.p. injection) and saline (control group) on gestational days (GD): 14, 16, 18 (term: 21-22 days). Tail cuff blood pressure was measured on GD19. On GD20, rats were euthanatized and maternal liver and arteries as well as fetal tissues were frozen for western blot analysis. Thromboxane B2 (TxB2) was measured in serum and media from maternal arteries by ELISA. Results: ODN 2395 increased systolic BP (Control: 100±4 mmHg vs. ODN2395 119±4 mmHg, p=0.006). Serum TxB2 was greater in the ODN 2395 group compared to control rats (Control: 65.2±8.9 ng/mL vs. ODN2395: 123.6±19.7 ng/mL, p=0.03). ODN2395 also increased release of TxB2 from mesenteric (Control: 28.2±4.3 pg/mg tissue vs ODN 54.5±6.2 pg/mg tissue, p=0.008) but not from uterine arteries (p>0.05). Western Blot analysis revealed greater expression of COX-2 in mesenteric (p=0.002) and uterine arteries (p=0.006) in ODN2395-treated animals. ODN2395 increased COX-1 expression in mesenteric arteries (p=0.006) and showed a moderate effect on uterine arteries (p=0.07). No differences were observed between treatment groups for COX-1 and COX-2 in maternal (p>0.07) and fetal liver (p>0.20). Conclusions: Exposure to CpG DNA during pregnancy induced maternal hypertension and augmented function of COX in maternal arteries.Item Free access to lard, sucrose, and chow results in expansion of rat periuterine adipose tissue.(2018-03-14) Goulopoulou, Styliani; Ahmed, HijabPurpose: Adipose tissue expansion, a common feature of obesity, is associated with metabolic dysfunction, endocrine dysregulation, and adipokine imbalance. Adipocyte hypertrophy and hyperplasia contribute to adipose tissue expansion in a depot-specific manner. Our objective was to determine whether a high-fat, high-carbohydrate diet induces expansion of periuterine adipose tissue (PUT: adipose tissue surrounding the uterus). We hypothesized that free access to lard, sucrose, and chow results in hypertrophy and hyperplasia of adipocytes from rat PUT. Methods: Sixteen Sprague-Dawley female rats were divided into 2 weight-matched groups (n=8 rats/group) after 5 days of baseline measurements of food intake and body weight. One group was offered free access to chow, 30% sucrose solution, and lard (choice group) and the other group remained on chow (chow group) for 3 weeks. Energy intake and body weight were recorded daily. After euthanization, PUT was collected, weighed, and fixed in 4% paraformaldehyde. Samples were embedded in paraffin, sliced into 5 μm sections, and stained with hematoxylin and eosin. To determine hypertrophy, hyperplasia, and cell morphology, we used NIS Elements software to measure cross-sectional area/cell, number of cells/unit area, and adipocyte size distribution, respectively. Results: Total energy intake was greater in choice rats than chow rats (1590 ± 40.60 kcal/21 days vs. 1036 ± 19.00 kcal/21 days, p2/cell vs. 492.0 ± 27.76 μm2/cell, p=0.0001). The number of cells/unit area was smaller in PUT from choice rats compared to chow rats (13.9 x 10-5 ± 0.880 x 10-5 cells/μm2 vs. 20.4 x 10-5 ±0.910 x 10-5 cells/μm2, p=0.0001). PUT from choice rats had large adipocytes in greater frequency compared to PUT from chow rats. Conclusion: Obesity induced by free access to chow, lard, and sucrose resulted in hyperplasia and hypertrophy of adipocytes, and in cell size distribution changes in PUT of female rats of breeding age. Future studies will investigate if obesity-induced PUT expansion influences reproductive capacity and gestational outcomes.Item Hemodynamic Oscillations: Physiological Consequences and Therapeutic Potential(2022-05) Anderson, Garen K.; Rickards, Caroline A.; Goulopoulou, Styliani; Romero, Steven A.; Cunningham, J. ThomasHemorrhage, or massive blood loss, continues to be a leading cause of preventable death. Therapeutic approaches that protect vital organ function are needed to improve outcomes from hemorrhage. In this dissertation, I explored the use of hemodynamic oscillations below the respiratory frequency (i.e., oscillations in arterial pressure and cerebral blood flow) as a novel technique for protecting tissue oxygenation during hemorrhage. In the first study of this dissertation, I hypothesized that hemodynamic oscillations would contribute to improved tolerance to central hypovolemia simulating hemorrhage. In further assessing the role of arterial blood gases on the physiological responses to forcing hemodynamic oscillations during a simulated hemorrhage, I hypothesized that forcing hemodynamic oscillations during simulated hemorrhage would protect tissue oxygenation during conditions of hypoxia and isocapnia, and improve cerebral blood flow. I also hypothesized that this protection would occur equally for both females and males. To address these hypotheses, I conducted five independent studies using lower body negative pressure as a method of simulating hemorrhage in healthy, conscious humans: in one study I utilized a maximal step-wise LBNP protocol to assess endogenous hemodynamic oscillations and tolerance to simulated hemorrhage, and in the remaining 4 studies, I utilized oscillatory and non-oscillatory LBNP to assess the potential therapeutic utility of forcing hemodynamic oscillations during simulated hemorrhage. The major findings from these investigations were: 1) greater amplitude of low frequency oscillations in arterial pressure are associated with greater LBNP tolerance, but the relative time to peak oscillatory power was not dependent on tolerance; 2) forced hemodynamic oscillations protect cerebral tissue oxygenation without protecting cerebral blood flow during the combined stress of simulated hemorrhage and hypobaric hypoxia; 3) isocapnia with simulated hemorrhage prevents the reduction in cerebral blood flow and tissue oxygenation, and forced hemodynamic oscillations during this stress protects stroke volume and arterial pressure; 4) females exhibit protected muscle tissue oxygenation to simulated hemorrhage, and the reduction in muscle tissue oxygenation in males can be attenuated with forced hemodynamic oscillations; and 5) forced hemodynamic oscillations at high altitude are greater in amplitude and result in similar protection of cerebral tissue oxygenation as low altitude conditions. These findings contribute to the growing body of literature highlighting the potential utility of oscillatory hemodynamics for therapeutic application.Item Hypoxia and oxidative stress reduce placental efficiency and impair the balance between autophagy and cell death mechanisms in trophoblasts(2024-03-21) Gardner, Jennifer; Bradshaw, Jessica L.; de Nazare Oliveria da, Renee; Hula, Nataliia; Mabry, Steve; Wilson, E. Nicole; Cunningham, Rebecca L.; Goulopoulou, StylianiIntroduction: Hypoxia and oxidative stress can activate autophagy, a lysosomal degradation pathway that maintains cellular homeostasis. Impairments in autophagy mechanisms have been observed in placentas from obstetric complications associated with placental hypoxia and oxidative stress, such as preeclampsia and intrauterine growth restriction. Purpose: The objective of this study was to investigate the effects of hypoxia and oxidative stress on placental autophagy. We hypothesized that exposure to oxidative stress and hypoxia would alter the balance between cytotoxic and cytoprotective mechanisms in human trophoblast cells and rat placentas and would adversely affect placental efficiency. Methods: We used an in vitro model incorporating human trophoblast cells (BeWo cells) exposed to an oxidative stressor, antimycin A (10, 100, 320 μM) or vehicle for 4 hours. Trophoblast cell death and autophagy mechanisms were assessed via flow cytometry and western blotting. Additionally, we used a rodent model of gestational sleep apnea, a pregnancy complication associated with placental hypoxia. Long Evans timed-pregnant dams were exposed to chronic intermittent hypoxia (CIH; n=6-8) or normoxia (NX; n=8-9) during their sleep cycle from gestational day (GD) 15 to 20 (late pregnancy, term=21-23 days). Results: In trophoblast cells (n=5-9 independent experiments), antimycin A increased necrosis and LC3 A/B II/I ratio (autophagy marker) at 100 μM compared to vehicle (p<0.015). Necrosis remained elevated at 320 μM, while BAX (pro-apoptotic marker) and p62 (autophagosomal flux marker) were reduced compared to vehicle (p<0.0001). LC3 A/B II/I ratio returned to vehicle levels at 320 μM (p>0.05 vs. vehicle). Placental weights from CIH exposed dams were greater (NX: 0.51±0.02 g vs. CIH: 0.60±0.03 g, p=0.015) and fetal to placental weight ratios (marker of placental efficiency) were reduced compared to control pregnancies (NX: 5.25±0.13 vs. CIH: 4.43±0.14, p=0.0006) on GD20. Gestational CIH did not affect (p>0.05) fetal weights (NX: 2.76±0.06 g vs. CIH: 2.61±0.06 g), crown to rump length (NX: 3.32±0.03 cm vs. CIH :3.18±0.12 cm), abdominal girth (NX: 3.22±0.06 cm vs. CIH: 3.32±0.12 cm), or litter size (NX: 11.9±0.90 vs. CIH: 10.5±0.82). Conclusion: Oxidative stress alters the balance between cytotoxic and cytoprotective mechanisms in trophoblast cells, promoting cell necrosis. Although assessment of autophagy machinery and cell death in placentas from hypoxic pregnancies is ongoing, our results indicate that maternal CIH during pregnancy adversely affects placental efficiency.Item Inhibition of Mitochondrial Respiratory Chain Complex I Induces Vascular Endothelial Cell Apoptosis and Release of Mitochondrial DNA(2019-03-05) Cushen, Spencer; Phillips, Nicole; Goulopoulou, Styliani; Riaz, MaryamPurpose: Vascular endothelial oxidative stress is a common feature of preeclampsia, a pregnancy specific hypertensive syndrome with high incidence of maternal and fetal mortality and morbidity. Cellular oxidative stress can lead to cell death, which promotes the release of cellular constituents (e.g. mitochondrial fragments) into the extracellular space. Circulating cell-free mitochondrial DNA (mtDNA) concentrations are increased in pregnant women with preeclampsia. The main objective of this study was to determine the mechanisms by which vascular endothelial cells may contribute to this increase in cell-free mtDNA. The hypothesis was that mitochondrial complex I inhibition results in extrusion of mtDNA from vascular endothelial cells via cell death-dependent mechanisms. Methods: Human umbilical vein endothelial cells (HUVEC, Lonza) were grown to 80-90% confluency before treatment with a mitochondrial complex I inhibitor (Rotenone: 5, 10, 25 μM - 4 h). Immunocytochemistry was used to confirm that HUVEC maintained endothelial cell characteristics. Cell death (apoptotic and non-apoptotic) was quantified using flow cytometry (staining for Annexin V and propidium iodide). mtDNA was measured on total nucleic acid extracts from cell culture supernatants using absolute real-time PCR techniques. Results: Treatment of HUVECs with rotenone increased early apoptosis and late apoptosis/necrosis [5μM (n=7), Veh: 11.16 ± 1.96% vs Rotenone: 14.74 ± 1.96% p=0.0159; 10μM (n=7), Veh: 10.54 ± 1.93% vs Rotenone: 14.83 ± 2.60% p=0.0033; 25μM (n=7), Veh: 10.34 ± 1.85% vs Rotenone: 15.87 ± 3.023% p=0.0002; 1-way ANOVA followed by Sidak’s post-hoc test]. Concentrations of mtDNA in HUVEC supernatant were increased in HUVECs treated with 5 μM of Rotenone [Veh (n=5): 2.45 ± 0.05 pg/mL vs. Rotenone (n=6): 3.65 ± 0.39 pg/mL, p=0.0700; Sidak’s post-hoc test]. Higher concentrations of Rotenone had no effect on concentrations of extracellular mtDNA (p [greater than] 0.84). Conclusions: Mitochondrial oxidative stress due to inhibition of mitochondrial respiratory chain complex I induces vascular endothelial cell death. Extrusion of mtDNA from apoptotic and necrotic endothelial cells may contribute to increased circulating mtDNA concentrations in preeclamptic pregnancies. Future studies will test this hypothesis using integrative pharmacological and physiological approaches.Item Innate immune system stimulation during pregnancy induces upregulation of thromboxane synthesis in rat maternal heart(2022) Tucker, Selina; Cushen, Spencer; Bradshaw, Jessica L.; Gardner, Jennifer; Ricci, Contessa; Dick, Gregory; Tune, Johnathan; Goulopoulou, StylianiPurpose: Infections during pregnancy are associated with adverse clinical outcomes. We previously showed that exposure to immunostimulatory ODN2395 (synthetic Toll-like receptor 9 agonist) during pregnancy induces maternal vascular inflammation and enhances vascular tone in pregnant rats. These outcomes were mediated in part by activation of the cyclooxygenase/thromboxane A2 (COX/TxA2) pathway. The objective of this study was to investigate the impact of ODN2395-induced immune system stimulation on maternal hearts during pregnancy. We hypothesize that exposure to TLR9-mediated immune system activation during pregnancy upregulates the COX/TxA2 signaling pathway in maternal cardiac tissues in rats. Methods: Rats were treated with a synthetic CpG DNA (ODN2395, 1 mg/kg, intraperitoneal injection) or vehicle (saline) in late pregnancy. Fetoplacental biometrics were recorded after euthanasia on gestational day 20 and maternal hearts were collected to assess COX-1 and COX-2 expression and 6-keto PGF1α (PGI2 metabolic byproduct) and TxB2 (TxA2 metabolic byproduct) production. Results: Left ventricular tissues from dams treated with ODN2395 released higher concentrations of TxB2 compared to tissues from vehicle-treated dams (ODN2395: 0.56 ± 0.06 ng/mg protein vs. Vehicle: 0.31 ± 0.04 ng/mg protein, n5, p=0.0041) but there were no differences in cardiac 6-keto PGF1α release between groups (p=0.16). COX-2 expression was lower in left ventricles from ODN2395-treated rats compared to vehicle-treated rats (p=0.009). There were no differences in cardiac COX-1 expression between groups (p=0.27). Exposure to ODN2395 during pregnancy increased fetal-placental weight ratio (ODN2395: 5.3 ± 0.22 vs. Vehicle: 4.7 ± 0.15, p = 0.04). COX-2 expression was greater in placental tissues from ODN2395-treated rats (p=0.004) but there were no differences in placental 6-keto PGF1α (p=0.51) and TxB2 release (p=0.32). Conclusion: TLR9 activation during pregnancy induces upregulation of TxB2 synthesis in maternal cardiac tissues coupled with a reduction in COX-2 expression. Maternal heart may have enhanced sensitivity to bacterial infections during pregnancy.Item Investigating the Use of Resistance Breathing for the Detection of Acute Hypovolemia(2021-05) Rusy, Ryan; Rickards, Caroline A.; Goulopoulou, Styliani; Mallet, Robert T.; Olivencia-Yurvati, Albert H.Introduction: Standard vital signs (e.g., heart rate and blood pressure) lack sensitivity and specificity to detect blood volume status following hemorrhage. Inspiratory resistance breathing has therapeutic potential to increase blood pressure and cardiac output following blood loss. We investigated the potential utility of resistance breathing as a novel method to detect volume loss. We hypothesized that resistance breathing would elicit greater increases in absolute and breath-to-breath amplitude of stroke volume and arterial pressure under hypovolemic vs. normovolemic conditions. Methods: Data were retrospectively analyzed from 23 healthy human subjects aged 23-40 years. Subjects underwent lower body negative pressure (LBNP) protocols to simulate hemorrhage with and without resistance breathing (via an impedance threshold device, ITD). Continuous arterial pressure and stroke volume were measured via finger photoplethysmography. Comparisons of absolute and changes in the breath-to-breath amplitude of arterial pressure and stroke volume were made under 4 conditions: 1) normovolemia; 2) normovolemia + resistance breathing; 3) hypovolemia, and; 4) hypovolemia + resistance breathing. The sensitivity and specificity of breath-to-breath arterial pressure and stroke volume amplitude responses in distinguishing between normovolemia and hypovolemia were assessed via area under the curve (AUC) of receiver operating characteristic (ROC) curves. Results: With resistance breathing the amplitude of systolic arterial pressure (P=0.007), diastolic arterial pressure (P<0.001), and mean arterial pressure (P<0.001) increased during hypovolemia vs. normovolemia, and the amplitude of stroke volume decreased (P=0.002). In distinguishing between normovolemia and hypovolemia, the ROC AUC were >0.86 for breath-by-breath mean, maximum and minimum stroke volume responses, and 0.77 for the amplitude response. The ROC AUC for mean arterial pressure amplitude was 0.88, and 0.64, 0.54, and 0.72 for the mean, maximum and minimum responses. Conclusions: The dynamic responses of arterial pressure and stroke volume with resistance breathing during hypovolemia show promise as a diagnostic tool for detection of hypovolemia in humans.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 Maternal and fetal mitochondrial gene dysregulation in hypertensive disorders of pregnancy(American Physiological Society, 2023-05-15) Ricci, Contessa A.; Reid, Danielle M.; Sun, Jie; Santillan, Donna A.; Santillan, Mark K.; Phillips, Nicole R.; Goulopoulou, StylianiMitochondrial dysfunction has been implicated in pregnancy-induced hypertension (PIH). The role of mitochondrial gene dysregulation in PIH, and consequences for maternal-fetal interactions, remain elusive. Here, we investigated mitochondrial gene expression and dysregulation in maternal and placental tissues from pregnancies with and without PIH; further, we measured circulating mitochondrial DNA (mtDNA) mutational load, an index of mtDNA integrity. Differential gene expression analysis followed by Time Course Gene Set Analysis (TcGSA) was conducted on publicly available high throughput sequencing transcriptomic data sets. Mutational load analysis was carried out on peripheral mononuclear blood cells from healthy pregnant individuals and individuals with preeclampsia. Thirty mitochondrial differentially expressed genes (mtDEGs) were detected in the maternal cell-free circulating transcriptome, whereas nine were detected in placental transcriptome from pregnancies with PIH. In PIH pregnancies, maternal mitochondrial dysregulation was associated with pathways involved in inflammation, cell death/survival, and placental development, whereas fetal mitochondrial dysregulation was associated with increased production of extracellular vesicles (EVs) at term. Mothers with preeclampsia did not exhibit a significantly different degree of mtDNA mutational load. Our findings support the involvement of maternal mitochondrial dysregulation in the pathophysiology of PIH and suggest that mitochondria may mediate maternal-fetal interactions during healthy pregnancy.NEW & NOTEWORTHY This study identifies aberrant maternal and fetal expression of mitochondrial genes in pregnancies with gestational hypertension and preeclampsia. Mitochondrial gene dysregulation may be a common etiological factor contributing to the development of de novo hypertension in pregnancy-associated hypertensive disorders.Item Mitochondrial oxidative stress and extrusion of mitochondrial DNA from endothelial cells: implications for maternal endothelial dysfunction in preeclampsia(2019-03-05) Cushen, Spencer; Phillips, Nicole; Goulopoulou, Styliani; Nguyen, RyanPurpose: Preeclampsia is one of the leading causes of maternal mortality during pregnancy and a risk factor of cardiovascular disease for the mother later in life. Hypertension and endothelial dysfunction are common characteristics of the maternal syndrome in preeclampsia, and oxidative stress is considered a pathogenic mediator of these maternal features. Mitochondria are the primary cellular producers of reactive oxygen species (ROS) and overproduction of mitochondrial ROS (mtROS) is detrimental to cellular processes, often leading to cell death. Mitochondrial DNA (mtDNA) has pro-inflammatory properties when released from dying cells into the extracellular space and its concentrations are increased in women with preeclampsia. The objective of this study was to determine the effects of mtROS in mtDNA release into the extracellular space. We hypothesize that inhibition of mitochondrial transport chain results in extrusion of mtDNA from vascular endothelial cells. Also, inhibition of Complex III causes a greater release of mtDNA compared to inhibition of Complex I. Methods: Human umbilical vein endothelial cells (HUVEC) were grown to 80-90% confluency before being treated with a mitochondrial complex I inhibitor (Rotenone: 5, 10, 25 mM – 4h) and mitochondrial complex III inhibitor (Antimycin A: 10, 50, 100 mM – 4h). After treatment, the cell media supernatant was collected and stored in -80 °C until further mtDNA quantification. mtDNA was isolated using the Mag-Bind Blood & Tissue DNA HDQ 96 Kit and quantified using the TaqMan chemistry-based method of absolute qPCR. Results: HUVEC cells treated with rotenone, regardless of dose, had no effect on concentrations of extracellular mtDNA (Figure A; One-way ANOVA followed by Sidak’s post-hoc test). Concentrations of mtDNA increased in HUVECs treated with 100 mM of Antimycin A (Figure B; One-way ANOVA followed by Sidak’s post-hoc test). Lower concentrations of Antimycin A had no effect on concentrations of extracellular mtDNA (Figure B). Conclusions: Inhibition of mitochondrial respiratory chain complex III, but not inhibition of complex I, results in extrusion of mtDNA. The increase in mtDNA released from dysfunctional cells may contribute to the increased circulating mtDNA concentrations seen in pregnancies with maternal endothelial dysfunction, such as pregnancies with preeclampsia.
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