Systemic manifestation of preeclampsia (PE) is associated with circulating factors, including inflammatory cytokines and damage-associated molecular patterns (DAMPs), or alarmins. However, it is unclear whether the placenta directly contributes to the increased levels of these inflammatory triggers. Here, we demonstrate that pyroptosis, a unique inflammatory cell death pathway, occurs in the placenta predominantly from early onset PE, as evidenced by elevated levels of active caspase-1 and its substrate or cleaved products, gasdermin D (GSDMD), IL-1β, and IL-18. Using cellular models mimicking pathophysiological conditions (e.g., autophagy deficiency, hypoxia, and endoplasmic reticulum (ER) stress), we observed that pyroptosis could be induced in autophagy-deficient human trophoblasts treated with sera from PE patients as well as in primary human trophoblasts exposed to hypoxia. Exposure to hypoxia elicits excessive unfolded protein response (UPR) and ER stress and activation of the NOD-like receptor pyrin-containing 3 (NLRP3) inflammasome in primary human trophoblasts. Thioredoxin-interacting protein (TXNIP), a marker for hyperactivated UPR and a crucial signaling molecule linked to NLRP3 inflammasome activation, is significantly increased in hypoxia-treated trophoblasts. No evidence was observed for necroptosis-associated events. Importantly, these molecular events in hypoxia-treated human trophoblasts are significantly observed in placental tissue from women with early onset PE. Taken together, we propose that placental pyroptosis is a key event that induces the release of factors into maternal circulation that possibly contribute to severe sterile inflammation and early onset PE pathology.

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Heme oxygenase (HO) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, this NADPH and cytochrome P450 reductase (CPR)-dependent oxidation of heme also releases free iron and carbon monoxide. Much of the recent research involving heme oxygenase is done using a 30-kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a GST-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30-kDa degradation product that could not be eliminated. Therefore, we attempted to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces lysine with arginine. This mutation allowed the expression and purification of a full length hHO-1 protein. Unlike wild-type HO-1, the K254R mutant could be purified to a single 32-kDa protein capable of degrading heme at the same rate as the wild-type enzyme. The K254R full-length form had a specific activity of ~200-225 nmol bilirubin hr −1 nmol −1 HO-1 as compared to ~140-150 nmol bilirubin hr −1 nmol −1 for the WT form, which contains the 30-kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30-kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other ER-resident enzymes.Heme oxygenase catalyzes the breakdown of heme to biliverdin, iron, and carbon monoxide (CO) (1). The physiological significance of these metabolites, most notably CO and biliverdin, is a reason for the escalating interest in HO research within the last 15 years (2). HO regulates iron homeostasis in mammals by recycling the iron released from the porphyrin ring. Biliverdin reductase, a soluble enzyme found in the cytosol, converts biliverdin to bilirubin. Bilirubin is a potent antioxidant that is conjugated to glucuronic acid in order to be excreted (3). Obstruction of bilirubin elimination has been implicated in common conditions such as neonatal jaundice, which can lead to neurological damage (4). CO, a third product of heme oxygenase, has been *Correspondence should be addressed to: Wayne L. Backes, Ph.D., Department of Pharmacology and the Stanley S. Scott Cancer Center, LSU Health Sciences Center, 533 Bolivar Street, New Orleans, La 70112, Voice 504-568-6557, FAX 504-568-6888, wbacke@lsuhsc.edu. shown to have signaling properties that parallel those of nitric oxide (5-7). CO is believed to elicit its effects via the guanylate cyclase pathway, common to NO. NIH Public AccessTwo disti...

BackgroundThis study sought to clarify the roles of Anti-müllerian hormone (AMH) and follicle stimulating hormone (FSH) in predicting live birth, especially in patients with discordant AMH and FSH. A large IVF data set provided by eIVF®, consisting of 13,964 cycles with AMH, FSH, age, BMI, and birth outcomes were evaluated. Patients were categorized into four groups: Good prognosis group (AMH ≥1 ng/ml; FSH < 10 mIU/ml), Poor prognosis group (AMH < 1 ng/ml; FSH ≥10 mIU/ml), Reassuring AMH group (AMH ≥1 ng/ml; FSH ≥10 mIU/ml), and Reassuring FSH group (AMH < 1 ng/ml; FSH < 10 mIU/ml). The interaction between AMH, FSH, and their impact on live birth rate among these four groups was evaluated using Generalized Additive Mixed Modeling (GAMM).ResultsAnalysis revealed a nonlinear relationship of AMH and FSH with live birth rate among all ages. Among the four groups, the good prognosis group had the highest live birth rate while the poor prognosis group had the lowest live birth rate (29.3% vs 13.1%, p < 0.005). In the discordant groups, the live birth rate of the reassuring AMH group was significantly higher than the reassuring FSH group (22.8% vs 15.6%, p < 0.005).ConclusionsAlthough both FSH and AMH are widely use to assess the ovarian reserve in women undergoing evaluation for infertility, AMH appears to be superior to FSH among all age groups. This is particularly important for patients with discordant AMH and FSH where reassuring AMH is a better clinical predictor of cycle success.Electronic supplementary materialThe online version of this article (10.1186/s13048-018-0430-z) contains supplementary material, which is available to authorized users.

ABSTRACT:Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the K m for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ ml) and hBVR (0.025-0.05 M), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment.

Heme oxygenase-1 (HO-1) catalyzes the oxidative degradation of heme to biliverdin, carbon monoxide, and free iron in a reaction requiring the interaction of HO-1 with NADPH-cytochrome P450 reductase (CPR). HO-1 is bound to the endoplasmic reticulum by 23 C-terminal amino acids; however, a soluble HO-1 (sHO-1) lacking this membrane spanning region has been extensively studied. The goal of this project was to characterize the effect of the C-terminal hydrophobic domain on formation of the HO-1/CPR complex. Full-length HO-1 was shown to exhibit higher reaction rates than sHO-1, particularly at subsaturating CPR; indicating that the C-terminal region influences HO-1 binding to CPR. The increased activity of HO-1 was attributable to a time dependent formation of a low Km HO-1/CPR complex that was not seen with sHO1. Gel filtration analysis confirmed the formation of multiple high molecular weight complexes in the presence and absence of the synthetic lipid dilauroylphosphatidylcholine (DLPC). However, the largest complex appeared following a two hour incubation of HO-1 and CPR in DLPC, suggesting that C-terminal region was required for the high affinity HO-1/CPR complex formation and membrane incorporation. These data demonstrate that the C-terminal region of HO-1 influenced complex formation and ultimately its affinity for CPR.

Hypertensive disorders of pregnancy remain among the leading causes of maternal morbidity and mortality. The onset of headaches in patients with hypertensive disorders of pregnancy has been considered as a premonitory symptom for eclampsia and other adverse maternal outcomes. Headaches are very common symptoms during pregnancy and the postpartum period with a reported incidence of 39%; however, headache is absent in 30-50% of women before the onset of eclampsia and is a poor predictor of eclampsia and adverse maternal outcomes. If included in the definition of cerebral or visual disturbances, headache may be considered a symptom of preeclampsia, a diagnostic feature of preeclampsia with severe features, a premonitory symptom of eclampsia, and an indication for delivery. Inclusion of this nonspecific symptom in the diagnosis and management of hypertensive disorders of pregnancy in the absence of an evidence basis may lead to unintended consequences including excessive testing, visits to outpatient offices or emergency departments, additional hospitalization, and iatrogenic preterm delivery without proven benefit. If a cerebral disturbance such as severe or persistent headache presents for the first time during pregnancy or postpartum, an evaluation should be performed that considers a broad differential diagnosis, including but not limited to hypertensive disorders of pregnancy, and the diagnostic evaluation is similar to that in nonpregnant adults. This commentary draws attention to the implications of considering the cerebral disturbance of headache as a symptom that portends adverse pregnancy outcome in the current recommendations for diagnosing and managing hypertensive disorders of pregnancy.

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