Browsing by Author "Gardner, Jennifer"
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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 Impact of sex and hypoxia on brain region-specific expression of androgen receptor AR45 and G protein Gαq in young adult rats(2024-03-21) Wilson, Elizabeth; Bradshaw, Jessica; Mabry, Steve; Shrestha, Pawan; Gardner, Jennifer; Cunningham, RebeccaPurpose: Sex differences in oxidative stress-associated cognitive decline are influenced by sex hormone levels. However, little is known regarding the expression of hormone receptors in brain regions associated with cognitive function. Notably, oxidative stress-associated neuronal cell death is exacerbated through testosterone signaling via membrane-associated androgen receptor AR45 and G protein Gαq. The objective of this study was to elucidate the expression of AR45 and Gαq in brain regions associated with cognitive function. Additionally, we investigated whether chronic intermittent hypoxia (CIH), an oxidative stressor with sex-specific effects, would modulate AR45 and Gαq expression. Methods: Adult male and female Sprague-Dawley rats were exposed to CIH or normoxia for 14 days. We quantified AR45 and Gαq protein expression in various cognition-associated brain regions [dorsal hippocampal CA1, CA3, DG, and entorhinal cortex (ETC)] via western blotting. For comparisons, AR45 and Gαq protein expression were also assessed in brain regions outside the hippocampal-ETC circuit [thalamus (TH) and striatum (STR)]. Results: The highest AR45 levels were expressed in the CA1 while the lowest expression was observed in the STR. The highest Gαq levels were expressed in the DG and ETC while the lowest expression was observed in the TH. We observed no effect of sex on AR45 or Gαq expression regardless of brain region assessed. Similarly, there was no effect of CIH on AR45 expression in any of the brain regions examined. However, CIH exposure increased Gαq expression only in the CA3 regardless of sex. Conclusions: Our findings reveal enrichment of AR45 and Gαq protein expression within the hippocampal-ETC circuit, which is vulnerable to oxidative stress and neurodegeneration during cognitive decline. Moreover, our data suggest the CA3 is the most vulnerable region to CIH-mediated oxidative stress. Overall, these findings were observed in both sexes, indicating that there are no observed sex differences in AR45 and Gαq expression or their modulation by CIH.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 Oxidative Stress and Release of Cell-free Mitochondrial DNA from Trophoblast Cells(2022) Gardner, Jennifer; Cushen, Spencer; Bradshaw, Jessica L.; Garlotte, Isabelle; Phillips, Nicole; Cunningham, Rebecca; Goulopoulou, StylianiCell free mitochondrial DNA (mtDNA) is an indicator of cellular stress and systemic inflammation. These properties are accentuated when mtDNA undergoes oxidative damage. In addition, toll-like receptor 9 (TLR9), a receptor of the innate immune system, is activated by mtDNA. Inflammation, oxidative stress, and cell death are characteristics of placental ischemia, a common feature of preeclampsia. Recent work from our lab has shown dysregulation of circulating cell-free mtDNA in pregnancies with preeclampsia and association of this dysregulation with preeclampsia diagnosis. However, mechanisms underlying the release of mtDNA remain unclear. We hypothesized that human trophoblast cells exposed to oxidative stress via antimycin A, an inhibitor of complex III of the electron transport chain, would induce release of mtDNA via cell death-dependent mechanisms, leading to increased TLR9 activation. BeWo cells (ATCC? CCL-98) were treated with increasing concentrations of antimycin A (10, 50, 100, 320 µM) and vehicle (ethanol, 0.16% v/v) for 4 hours. Supernatants were collected and snap frozen in liquid nitrogen. Absolute real-time qPCR quantification with TaqMan™ probes and chemistry was used to quantify cell-free mtDNA (amplification target: MT-ND5 gene) and nuclear DNA (nDNA). Flow cytometry was used to assess the activation of cell death mechanisms in response to oxidative stress. To determine TLR-9-associated immunostimulatory potency of cell culture supernatants, we used an engineered cell line of human embryonic kidney 293 cells transfected with a human TLR-9 gene (HEK-BlueTM hTLR9). Exposure of trophoblast cells to antimycin A did not induce the release of mtDNA (p>0.05) or nDNA (p>0.05). Similarly, there were no differences in TLR9 activation between groups (p>0.28). Antimycin A (320 µM) reduced cell viability (Vehicle: 64.44 ± 5.46% vs Antimycin A: 18.14 ± 5.78%, p< 0.05) and increased necrosis (Vehicle: 10.39 ± 3.11% vs Antimycin A (100, 320 µM): 30.51 ± 4.43%, 40.16 ± 5.08%, P< 0.05), while apoptosis levels remained unchanged (P>0.1). Activation of oxidative stress pathways, via inhibition of complex III of the electron transport chain, leads to cell death, but does not affect release of mtDNA. These data suggest other cellular mechanisms, such as mitophagy or activation of antioxidant pathways, may serve a cytoprotective role against oxidative stressors in trophoblast cells. This study extends our pre-clinical knowledge about the links between placental oxidative stress and immunogenic factors in trophoblast cells. These findings may contribute to development of novel therapeutic targets for treatment of maternal cardiovascular dysfunction in preeclampsia.Item Rats with Placental Ischemia and Preeclampsia-like Symptoms Have Increased Circulating Cell-free Mitochondrial DNA(2020) Goulopoulou, Styliani; Phillips, Nicole; Davidge, Sandra; Cushen, Spencer; Morton, Jude; Spaans, Floortje; Kirschenman, Raven; Gardner, JenniferPreeclampsia (PE) is a hypertensive disorder of pregnancy, which is characterized by placental mitochondrial dysfunction. Increased circulating cell-free mitochondrial DNA (mtDNA) has been also reported in PE. Animal models are commonly used to study the role of placental dysfunction in the maternal syndrome of PE. The objective of this study was to determine the concentrations of circulating mtDNA in rat models of placental ischemia. Placental ischemia was induced in rats on gestational day (GD) 14 by placing clips on a) the abdominal aorta and ovarian arteries (reduced uterine perfusion pressure (RUPP)) and b) ovarian arteries and uterine arteries (selective RUPP (sRUPP)). Sham rats had clips placed on intraabdominal fat. Different groups of rats were exposed to hypoxia (11% O2) or maintained at atmospheric conditions (21% O2) from GD6 to GD21. Blood samples were collected on GD21. Real time PCR quantification of mtDNA was performed on DNA extracts from serum using TaqMan™ probes and chemistry. mtDNA copy number (CN) was greater in RUPP and sRUPP rats compared to their respective controls (Sham (11) vs. RUPP (11): 0.18 ± 0.04 CN/μl vs. 0.30 ± 0.04 CN/μl, p-value: 0.04; Sham (8) vs. sRUPP (10): 24.84 ± 3.29 CN/μl vs. 54.38 ± 3.29 CN/μl, p-value: 0.016)). Hypoxia did not affect mtDNA CN (Control (7) vs. Hypoxia (9): 0.28 ± 0.05 CN/μl vs. 0.36 ± 0.04 CN/μl, p-value: 0.28). Rats with placental ischemia have increased circulating cell-free mtDNA similar to what is seen in pregnant women with PE.Item Rats with placental ischemia and preeclampsia-like symptoms have increased circulating cell-free mtDNA(2021) Gardner, Jennifer; Cushen, Spencer; Morton, Jude; Spaans, Floortje; Kirschenman, Raven; Davidge, Sandra; Phillips, Nicole; Goulopoulou, StylianiPreeclampsia (PE) is a hypertensive disorder of pregnancy, which is characterized by placental mitochondrial dysfunction. Increased circulating cell-free mitochondrial DNA (mtDNA) has been also reported in PE. Animal models are commonly used to study the role of placental dysfunction in the maternal syndrome of PE. The objective of this study was to determine the concentrations of circulating mtDNA in rat models of placental ischemia. Placental ischemia was induced in rats on gestational day (GD) 14 by placing clips on a) the abdominal aorta and ovarian arteries (reduced uterine perfusion pressure (RUPP)) and b) ovarian arteries and uterine arteries (selective RUPP (sRUPP)). Sham rats had clips placed on intraabdominal fat. Different groups of rats were exposed to hypoxia (11% O2) or maintained at atmospheric conditions (21% O2) from GD6 to GD21. Blood samples were collected on GD21. Real time PCR quantification of mtDNA was performed on DNA extracts from serum using TaqMan™ probes and chemistry. mtDNA copy number (CN) was greater in RUPP and sRUPP rats compared to their respective controls (Sham (11) vs. RUPP (11): 0.18 ± 0.04 CN/µl vs. 0.30 ± 0.04 CN/µl, p-value: 0.04; Sham (8) vs. sRUPP (10): 24.84 ± 3.29 CN/µl vs. 54.38 ± 3.29 CN/µl, p-value: 0.016)). Hypoxia did not affect mtDNA CN (Control (7) vs. Hypoxia (9): 0.28 ± 0.05 CN/µl vs. 0.36 ± 0.04 CN/µl, p-value: 0.28). Rats with placental ischemia have increased circulating cell-free mtDNA similar to what is seen in pregnant women with PE.Item Sex-dependent effects of chronic intermittent hypoxia: Implication for obstructive sleep apnea(2024-03-21) Mabry, Steve; Bradshaw, Jessica; Gardner, Jennifer; Wilson, Elizabeth; Cunningham, RebeccaBackground: Obstructive sleep apnea (OSA) is a highly prevalent sleeping disorder in the USA with known sex differences in prevalence and severity. Men have a higher incidence and experience greater severity of OSA than women. However, recent reports indicate the incidence of OSA in women, particularly mild cases of OSA, may be under-reported and left untreated. OSA is characterized by elevated oxidative stress and inflammation, mechanisms that involve mitochondrial function. This study addressed the role of 1) sex and 2) mitochondrial oxidative stress in OSA induced circulatory oxidative stress and inflammatory cytokines. Methods: Adult Sprague-Dawley male and female rats were implanted (s.c.) with an osmotic pump containing either MitoTEMPOL (mitochondrial oxidative stress inhibitor; MT) or saline vehicle and then exposed to a model of OSA, chronic intermittent hypoxia (CIH), or normoxic room-air for 14 days. The CIH protocol consisted of 10 CIH cycles/hour/8 hrs/day, in which each CIH cycle was composed of 3 minutes of normoxia at 21% O2 and 3 minutes of hypoxia at 10% O2. This protocol replicates an apnea-hypopnea index (AHI) of 10, which is consistent with mild OSA in humans. At the conclusion of the CIH protocol, rats were sacrificed and plasma was collected to quantify markers of oxidative stress (Advanced Oxidized Protein Products, AOPP) and inflammation (pro-inflammatory IL-6, anti-inflammatory IL-10, IL-6/IL-10 ratio). To determine statistical significance, ANOVA followed by Tukey’s post-hoc test was used. Significance level was set a p<0.05. Results: We found circulating oxidative stress was dependent on CIH and sex. Sex differences were observed in control normoxic rats, in which females had higher oxidative stress than males. Interestingly, the impact of CIH on oxidative stress was dependent on sex, wherein CIH decreased oxidative stress in females but increased oxidative stress in males. Inhibiting mitochondria-associated oxidative stress reduced oxidative stress in vehicle females, but only blocked the effect of CIH-induced oxidative stress in males. In contrast to oxidative stress, CIH increased the level of IL-6 only in females. Further, CIH overall induced a pro-inflammatory state as measured by an elevated IL6/IL10 ratio in females. The inflammatory effects of CIH in females were blocked by inhibiting mitochondrial-associated oxidative stress, despite no effect on circulating oxidative stress in CIH. Neither CIH nor MT impacted inflammatory markers in males. Discussion: These results indicate CIH-induced mechanisms underlying oxidative stress and inflammation are dependent on sex. Specifically, males experience a mitochondria-associated oxidative stress phenotype and females experience a mitochondria-associated inflammatory phenotype. These findings indicate that the OSA phenotype is sex-dependent, which may be related to the under-reported OSA incidence in women compared to men. Further, these data indicate that women may be at unique risk from OSA, particularly when AHIs are mild. Interestingly, inhibition of mitochondrial oxidative stress may be a potential drug target for both men and women with OSA.Item The impact of healthy pregnancy on maternal cognitive impairment in Sprague Dawley rats(2022) Wilson, E. Nicole; Bradshaw, Jessica L.; Tucker, Selina; Gardner, Jennifer; Goulopoulou, Styliani; Cunningham, RebeccaIntroduction/Background: There is clinical evidence of impaired attention, learning, and memory in pregnant women during pregnancy and in the postpartum period, suggesting an association between pregnancy and maternal cognitive dysfunction. Yet, the effects of pregnancy on memory impairment are unclear. We hypothesized that pregnancy would induce maternal cognitive dysfunction that would persist postpartum in a rat model of healthy pregnancy. Methods: To observe recollective memory, the novel object recognition test was performed using Sprague Dawley female rats with different reproductive histories [non-pregnant virgin, late gestation (gestational day 20, term = 22-23 days), postpartum (28 days after birth), and parous non-pregnant (60 days after birth); n = 7-8/group]. Each rat was placed into an empty arena without objects, to allow for adjustments to the open arena. Thirty-minutes after habituation, each rat was given a period of five minutes to explore the arena with two objects of identical size, color, and texture. Upon completion, one hour was given before the animal was placed back in the arena. To test short term recollective memory, each rat was given three minutes to explore two items: one familiar item and a novel item of different size, color, and texture. The latency to which the animal made the initial contact for each object was recorded, and the number of contacts made with the novel object were tallied and compared with overall contacts to each object. Results: Pregnant rats had increased latency to initial contact of the novel object (p < 0.05) compared to virgin females, postpartum dams, and parous non-pregnant dams. Additionally, parous, non-pregnant dams displayed significantly greater contacts with the novel object (p < 0.05) compared to pregnant rats and postpartum dams. Conclusion: Overall, healthy pregnancy results in decreased short term memory recognition that can be repaired over time. Future directions include evaluating the impact of healthy pregnancy on long term memory recognition, examining underlying mechanisms contributing to cerebral impairments during pregnancy, and determining the effects of pregnancy complications on memory impairment.