Biology subjects

Gorham, I. K.

Publications and source records attributed to Gorham, I. K..

4 recordsLinked to original sources

bioRxiv2026

Fetal sex shapes placental inflammatory responses to extracellular mitochondrial DNA

Aberrant changes in circulating cell-free mitochondrial DNA (ccf-mtDNA) across gestation are associated with adverse pregnancy outcomes. Given the inflammatory properties of ccf-mtDNA via pattern recognition receptors such as Toll-like receptor 9 (TLR9), we hypothesized that extracellular mtDNA induces placental inflammation via TLR9 signaling and that this response differs by fetal sex. Pregnant Sprague-Dawley rats were treated intravenously with purified mtDNA (300 g/kg), nuclear DNA (nDNA), saline, and/or the TLR9 antagonist ODN2088 across five studies. Placental responses were evaluated 4 h (Studies 1-3) and 24 h (Study 4) post-treatment; pregnancy and neonatal outcomes were assessed at delivery (Study 5). Exposure to mtDNA, but not nDNA, increased placental il1{beta}, tnf, and il10 mRNA (p < 0.05), establishing response specificity. mtDNA-induced placental inflammation was fetal sex-dependent: mtDNA increased il6 and il1{beta} mRNA in male placentas (p [≤] 0.0004) but not female placentas, whereas ifn{gamma} was selectively induced in female placentas (p = 0.0004). TLR9 and MyD88 abundance increased in female but not male placentas, and TLR9 antagonism modified selected inflammatory responses with sex-specific patterns. The 4 h inflammatory transcriptional signature resolved by 24 h, whereas mtDNA exposure was associated with a sex-specific shift in antioxidant enzyme expression persisting to 24 h. Despite no effects on gestational length or neonatal biometrics, mtDNA exposure was associated with a higher estimated stillbirth count per litter (IRR = 4.23, 95% CI [0.89, 20.1], p = 0.069). These findings establish extracellular mtDNA as an acute, sex-differentiated placental inflammatory stimulus with partial TLR9 dependence and a potential impact on fetal viability. New & NoteworthyThis study demonstrates that acute exposure to extracellular mtDNA induces placental inflammatory responses in vivo. This response is specific to mtDNA, fetal-sex dependent, and partially mediated by TLR9, with male and female placentas engaging distinct inflammatory signals within hours of exposure. The biological effects extend beyond the initial inflammatory window, with mtDNA exposure producing lasting, sex-specific changes in antioxidant enzyme expression. mtDNA-exposed dams had higher expected stillbirth counts, suggesting extracellular mtDNA may affect fetal viability.

physiology↗

bioRxiv2026

Acute exposure to cell-free mitochondrial DNA induces pregnancy-specific aortic endothelial dysfunction and organ-selective inflammation in rats

Pregnancy complications such as preeclampsia are associated with circulating cell-free mitochondrial DNA (mtDNA), a damage-associated molecular pattern capable of activating Toll-like receptor 9 (TLR9). We hypothesized that acute mtDNA exposure induces maternal inflammation and endothelial dysfunction during pregnancy via TLR9 activation. Non-pregnant and pregnant rats (gestational days 14-15) were treated intravenously with saline or purified mtDNA and euthanized 4 h after treatment. mtDNA increased cytokine mRNA expression in lung and liver of non-pregnant and pregnant rats, with magnitude varying by pregnancy status and organ. Aortas from pregnant, but not non-pregnant, rats exhibited reduced acetylcholine (ACh)-induced relaxation following mtDNA treatment (Emax, saline: 90.1 {+/-} 3.9 % vs. mtDNA: 62.1 {+/-} 20.7 % KClmax, p<0.05), while uterine artery function was preserved, indicating vascular bed-specific effects. Ex vivo incubation of aortic rings with mtDNA {+/-} white blood cells did not replicate in vivo findings, implicating systemic rather than direct vascular mechanisms. Nuclear DNA did not affect ACh-induced relaxation (p>0.05), confirming that the vascular effects were mtDNA-specific. Pharmacological antagonism of TLR9 with ODN2088 partially attenuated mtDNA-induced maternal endothelial dysfunction. Although overt vascular ROS increases were not detected, aortas from pregnant rats had reduced sod-1 expression (p<0.05) and increased eNOS protein abundance (p<0.05). Acute mtDNA exposure during pregnancy induces maternal organ inflammation and impairs endothelium-dependent vasodilation, with partial TLR9 involvement. In conclusion, aortic transcriptional changes in antioxidant pathways and increased eNOS abundance were also observed, though their functional significance remains to be determined. New & NoteworthyTo our knowledge, this is the first study to demonstrate that acute exposure to circulating mtDNA induces pregnancy-specific maternal endothelial dysfunction and organ-selective inflammatory responses. Our findings reveal pregnancy- and vascular-bed specific responses of the maternal vasculature to mitochondrial danger signals, with partial TLR9 involvement. Aortic transcriptional changes in antioxidant pathways and increased nitric oxide synthase abundance were identified as molecular correlates of this dysfunction.

physiology↗

bioRxiv2025

Gestational Chronic Intermittent Hypoxia Triggers Maternal Inflammation and Disrupts Placental Stress Responses

Gestational hypoxia is associated with placental cellular responses, including oxidative stress and inflammation. Circulating cell-free mitochondrial DNA (ccf-mtDNA) is a marker of cell stress, that can be transported within extracellular vehicles (EVs), eliciting proinflammatory responses. We hypothesized that systemic exposure to chronic intermittent hypoxia (CIH) during late pregnancy would increase maternal inflammation, alter circulating EV characteristics, and disrupt placental stress responses. Pregnant rats were exposed to CIH (n=8) or normoxia (n=9) during gestational days 15-20 (term 22-23 days). On GD20, ccf-mtDNA and EV-associated mtDNA (EV-mtDNA) were quantified with qRT-qPCR, while maternal circulating cytokines were quantified using a MILLIPLEX(R) cytokine array. Systemic oxidative stress was measured by plasma advanced oxidation protein products (AOPP). Placental stress responses were evaluated by examining the balance between proinflammatory and antioxidant gene expression and the activation of proteins involved in apoptotic and autophagic processes. CIH exposure increased placental weights (p=0.015) and reduced placental efficiency (p=0.0006) without affecting fetal biometrics (p>0.05). Absolute ccf-mtDNA and EV-mtDNA content were unchanged (p>0.05), but EV concentrations were reduced (p=0.011) in response to CIH, suggesting an increase in EV-mtDNA per EV. Maternal interleukin-18 (IL-18) concentrations increased in the CIH group (p=0.047). Placental mRNA expression of catalase (p=0.048) and sod2 (p=0.038) were upregulated, while autophagy-related proteins Beclin-1 (p=0.006) and p62 (p=0.023) were also increased in response to CIH, with no changes in LC3A/B expression (p>0.05). Gestational CIH disrupts maternal EV and inflammatory profiles, reduces placental efficiency, and modulates placental antioxidant and autophagic mechanisms, without impairing fetal growth in rats.

physiology↗

bioRxiv2024

Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an indicator of cell death, inflammation, and oxidative stress. ccf-mtDNA differs in pregnancies with placental dysfunction from healthy pregnancies and the direction of this difference depends on gestational age and method of mtDNA quantification. Reactive oxygen species (ROS) trigger release of mtDNA from non-placental cells; yet it is unknown whether trophoblast cells release mtDNA in response to oxidative stress, a common feature of pregnancies with placental pathology. We hypothesized that oxidative stress would induce cell death and release of mtDNA from trophoblast cells. BeWo cells were treated with antimycin A (10-320 M) or rotenone (0.2-50 M) to induce oxidative stress. A multiplex real-time quantitative PCR (qPCR) assay was used to quantify mtDNA and nuclear DNA in membrane bound, non-membrane bound, and vesicular-bound forms in cell culture supernatants and cell lysates. Treatment with antimycin A increased ROS (p<0.0001), induced cell necrosis (p=0.0004) but not apoptosis (p=0.6471) and was positively associated with release of membrane-bound and non-membrane bound mtDNA (p<0.0001). Antimycin A increased mtDNA content in exosome-like extracellular vesicles (vesicular-bound form; p=0.0019) and reduced autophagy marker expression (LC3A/B, p=0.0002; p62, p<0.001). Rotenone treatment did not influence mtDNA release or cell death (p>0.05). Oxidative stress induces release of mtDNA into the extracellular space and causes non-apoptotic cell death and a reduction in autophagy markers in BeWo cells, an established in vitro model of human trophoblast cells. Intersection between autophagy and necrosis may mediate the release of mtDNA from the placenta in pregnancies exposed to oxidative stress. NEW & NOTEWORTHYThis is the first study to test whether trophoblast cells release mitochondrial DNA in response to oxidative stress and to identify mechanisms of release and biological forms of mtDNA from this cellular type. This research identifies potential cellular mechanisms that can be used in future investigations to establish the source and biomarker potential of circulating mitochondrial DNA in preclinical experimental models and humans.

cell biology↗