Exposure to unmethylated CpG oligonucleotides disrupts blood pressure circadian rhythms and placental clock gene network in pregnant rats.

Bacterial infections and impaired circulating mitochondrial DNA dynamics are associated with adverse pregnancy outcomes. Unmethylated cytosine-guanine dinucleotide (CpG) motifs are common in bacterial and mitochondrial DNA and act as potent immunostimulators. We tested the hypothesis that exposure to CpG oligonucleotides (ODN) during pregnancy would disrupt blood pressure circadian rhythms and placental molecular clock network, mediating aberrant fetoplacental growth dynamics. Rats were repeatedly treated with CpG ODN in the third trimester [gestational days (GD) 14, 16, and 18] and euthanized on GD20 (near term) or treated with a single dose of CpG ODN on GD14 and euthanized 4 h after treatment. Hemodynamic circadian rhythms were analyzed via Lomb-Scargle periodogram analysis on 24-h raw data collected continuously via radiotelemetry. A P value ≥ 0.05 indicates the absence of a circadian rhythm. Following the first treatment with CpG ODN, maternal systolic and diastolic blood pressure circadian rhythms were lost (P ≥ 0.05). Blood pressure circadian rhythm was restored by GD16 and remained unaffected after the second treatment with CpG ODN (P < 0.0001). Diastolic blood pressure circadian rhythm was again lost after the last treatment on GD18 (P ≥ 0.05). CpG ODN increased placental expression of Per2, Per3, and Tnfα (P ≤ 0.05) and affected fetoplacental growth dynamics. Reduced fetal and placental weights were disproportionately associated with increases in the number of resorptions in ODN-treated dams compared with controls. In conclusion, gestational exposure to unmethylated CpG ODN dysregulates the placental molecular clock network and fetoplacental growth dynamics and disrupts blood pressure circadian rhythms.NEW & NOTEWORTHY Gestational exposure to unmethylated CpG ODN dysregulates placental molecular clock network and fetoplacental growth dynamics and disrupts blood pressure circadian rhythms. These findings provide novel insights into the relationship between circadian rhythms and immune responses in pregnancy and propose new mechanisms by which maternal responses to immune triggers could dictate circadian rhythms of cardiovascular processes and placental clock machinery function to determine fetal growth trajectories.

Maternal and fetal mitochondrial gene dysregulation in hypertensive disorders of pregnancy.

Mitochondrial 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.

Gestational exposure to unmethylated CpG oligonucleotides dysregulates placental molecular clock network and fetoplacental growth dynamics, and disrupts maternal blood pressure circadian rhythms in rats.

Bacterial infections and impaired mitochondrial DNA dynamics are associated with adverse pregnancy outcomes. Unmethylated cytosine-guanine dinucleotide (CpG) motifs are common in bacterial and mitochondrial DNA and act as potent immunostimulators. Here, we tested the hypothesis that exposure to CpG oligonucleotides (ODN) during pregnancy would disrupt blood pressure circadian rhythms and the placental molecular clock machinery, mediating aberrant fetoplacental growth dynamics. Rats were repeatedly treated with CpG ODN in the 3 rd trimester (gestational day, GD, 14, 16, 18) and euthanized on GD20 (near term) or with a single dose of CpG ODN and euthanized 4 hours after treatment on GD14. Hemodynamic circadian rhythms were analyzed via Lomb-Scargle periodogram analysis on 24-h raw data collected continuously via radiotelemetry. A p -value ≥ 0.05 indicates the absence of a circadian rhythm. Following the first treatment with CpG ODN, maternal systolic and diastolic blood pressure circadian rhythms were lost ( p ≥ 0.05). Blood pressure circadian rhythm was restored by GD16 and remained unaffected after the second treatment with CpG ODN ( p < 0.0001). Diastolic blood pressure circadian rhythm was again lost after the last treatment on GD18 ( p ≥ 0.05). CpG ODN increased placental expression of Per2 and Per3 and Tnfα ( p ≤ 0.05) and affected fetoplacental growth dynamics, such as reduced fetal and placental weights were disproportionately associated with increases in the number of resorptions in ODN-treated dams compared to controls. In conclusion, gestational exposure to unmethylated CpG DNA dysregulates placental molecular clock network and fetoplacental growth dynamics and disrupts blood pressure circadian rhythms.

Reduced Maternal Circulating Cell-Free Mitochondrial DNA Is Associated With the Development of Preeclampsia.

Background Circulating cell-free mitochondrial DNA (ccf-mtDNA) is a damage-associated molecular pattern that reflects cell stress responses and tissue damage, but little is known about ccf-mtDNA in preeclampsia. The main objectives of this study were to determine (1) absolute concentrations of ccf-mtDNA in plasma and mitochondrial DNA content in peripheral blood mononuclear cells and (2) forms of ccf-mtDNA transport in blood from women with preeclampsia and healthy controls. In addition, we sought to establish the association between aberrance in circulating DNA-related metrics, including ccf-mtDNA and DNA clearance mechanisms, and the clinical diagnosis of preeclampsia using bootstrapped penalized logistic regression. Methods and Results Absolute concentrations of ccf-mtDNA were reduced in plasma from women with preeclampsia compared with healthy controls (P≤0.02), while mtDNA copy number in peripheral blood mononuclear cells did not differ between groups (P>0.05). While the pattern of reduced ccf-mtDNA in patients with preeclampsia remained, DNA isolation from plasma using membrane lysis buffer resulted in 1000-fold higher ccf-mtDNA concentrations in the preeclampsia group (P=0.0014) and 430-fold higher ccf-mtDNA concentrations in the control group (P<0.0001). Plasma from women with preeclampsia did not induce greater Toll-like receptor-9-induced nuclear factor kappa-light-chain enhancer of activated B cells-dependent responses in human embryonic kidney 293 cells overexpressing the human TLR-9 gene (P>0.05). Penalized regression analysis showed that women with preeclampsia were more likely to have lower concentrations of ccf-mtDNA as well as higher concentrations of nuclear DNA and DNase I compared with their matched controls. Conclusions Women with preeclampsia have aberrant circulating DNA dynamics, including reduced ccf-mtDNA concentrations and DNA clearance mechanisms, compared with gestational age-matched healthy pregnant women.

Cyclooxygenase-dependent mechanisms mediate in part the anti-dilatory effects of perivascular adipose tissue in uterine arteries from pregnant rats.

Uterine perivascular adipose tissue (PVAT) contributes to uterine blood flow regulation in pregnancy, at least in part, due to its effects on uterine artery reactivity. We tested the hypothesis that uterine PVAT modulates the balance between the contribution of nitric oxide synthase (NOS)- and cyclooxygenase (COX)-dependent pathways to acetylcholine (ACh)-induced relaxation in isolated uterine arteries. Concentration-response curves to ACh (1 nM - 30 µM) were performed on uterine arteries from pregnant and non-pregnant rats. Arteries were exposed to Krebs-Henseleit solution (control) or PVAT-conditioned media (PVATmedia) in the presence of the following inhibitors: L-NAME (NOS inhibitor), indomethacin (COX inhibitor), SC560 (COX-1 inhibitor), NS398 (COX-2 inhibitor), SQ 29,548 (thromboxane receptor (TP) inhibitor). In arteries incubated with PVATmedia, the presence of indomethacin increased ACh-induced relaxation, reversing the anti-dilatory effect of PVATmedia. NOS inhibition reduced ACh-induced relaxation in uterine arteries from pregnant rats, and exposure to PVATmedia did not change this effect. Selective inhibition of COX-1 but not COX-2 suppressed relaxation responses to ACh in control arteries. The presence of PVATmedia abolished the effect of COX-1 inhibition. Incubation of uterine arteries from pregnant rats with PVATmedia increased production of thromboxane B2 (TxB2, p = 0.01) but thromboxane receptor (TP) inhibition did not affect the anti-dilatory properties of PVATmedia. In conclusion, inhibition of COX signaling suppressed the anti-dilatory effects of PVATmedia, while PVATmedia had no effect on the contribution of the NOS/NO pathway to ACh-induced relaxation in uterine arteries from pregnant rats, indicating that the anti-dilatory effects of uterine PVAT are mediated in part by COX-dependent mechanisms.

Responding to Threats Both Foreign and Domestic: NOD-Like Receptors in Corals.

Historically mechanisms with which basal animals such as reef-building corals use to respond to changing and increasingly stressful environments have remained elusive. However, the increasing availability of genomic and transcriptomic data from these organisms has provided fundamental insights into the biology of these critically important ecosystem engineers. Notably, insights into cnidarians gained in the post-genomics age have revealed a surprisingly complex immune system which bears a surprising level of similarity with the vertebrate innate immune system. This system has been critically linked to how corals respond to the two most prominent threats on a global scale, emerging coral diseases and increasing water temperature, which are recognized cellularly as either foreign or domestic threats, respectively. These threats can arise from pathogenic microbes or internal cellular dysfunction, underscoring the need to further understand mechanisms corals use to sense and respond to threats to their cellular integrity. In this investigation and meta-analysis, we utilize resources only recently available in the post-genomic era to identify and characterize members of an underexplored class of molecules known as NOD-like receptors in the endangered Caribbean coral Orbicella faveolata. We then leverage these data to identify pathways possibly mediated by NLRs in both O. faveolata and the ecologically important branching coral Acropora digitifera. Overall, we find support that this class of proteins may provide a mechanistic link to how reef-building corals respond to threats both foreign and domestic.

Proteomic Investigation of a Diseased Gorgonian Coral Indicates Disruption of Essential Cell Function and Investment in Inflammatory and Other Immune Processes.

As scleractinian coral cover declines in the face of increased frequency in disease outbreaks, future reefs may become dominated by octocorals. Understanding octocoral disease responses and consequences is therefore necessary if we are to gain insight into the future of ecosystem services provided by coral reefs. In Florida, populations of the octocoral Eunicea calyculata infected with Eunicea black disease (EBD) were observed in the field in the fall of 2011. This disease was recognized by a stark, black pigmentation caused by heavy melanization. Histological preparations of E. calyculata infected with EBD demonstrated granular amoebocyte (GA) mobilization, melanin granules in much of the GA population, and the presence of fungal hyphae penetrating coral tissue. Previous transcriptomic analysis also identified immune trade-offs evidenced by increased immune investment at the expense of growth. Our investigation utilized proteogenomic techniques to reveal decreased investment in general cell signaling while increasing energy production for immune responses. Inflammation was also prominent in diseased E. calyculata and sheds light on factors driving the extreme phenotype observed with EBD. With disease outbreaks continuing to increase in frequency, our results highlight new targets within the cnidarian immune system and provide a framework for understanding transcriptomics in the context of an organismal disease phenotype and its protein expression.