Attenuation of Hyperoxic Lung Injury in Newborn Thioredoxin-1-Overexpressing Mice through the Suppression of Proinflammatory Cytokine mRNA Expression.

The role of thioredoxin-1 (TRX), a small redox-active protein with antioxidant effects, during hyperoxic lung injury in newborns remains undetermined. We investigated TRX impact on hyperoxic lung injury in newborn TRX transgenic (TRX-Tg) and wildtype (WT) mice exposed to 21% or 95% O2 for four days, after which some mice were allowed to recover in room air for up to 14 days. Lung morphology was assessed by hematoxylin/eosin and elastin staining, as well as immunostaining for macrophages. The gene expression levels of proinflammatory cytokines were evaluated using quantitative real-time polymerase chain reaction. During recovery from hyperoxia, TRX-Tg mice exhibited an improved mean linear intercept length and increased number of secondary septa in lungs compared with the WT mice. Neonatal hyperoxia enhanced the mRNA expression levels of proinflammatory cytokines in the lungs of both TRX-Tg and WT mice. However, interleukin-6, monocyte chemoattractant protein-1, and chemokine (C-X-C motif) ligand 2 mRNA expression levels were reduced in the lungs of TRX-Tg mice compared with the WT mice during recovery from hyperoxia. Furthermore, TRX-Tg mice exhibited reduced macrophage infiltration in lungs during recovery. These results suggest that in newborn mice TRX ameliorates hyperoxic lung injury during recovery likely through the suppression of proinflammatory cytokines.

Increased expression of heme oxygenase-1 suppresses airway branching morphogenesis in fetal mouse lungs exposed to inflammation.

BACKGROUND: Intrauterine inflammation affects fetal lung development. BTB and CNC homology 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1) and interleukin-6 (IL-6) genes. We investigated the role of Bach1 in the development of fetal mouse lungs exposed to lipopolysaccharide (LPS) using a whole fetal lung tissue culture system. METHODS: We isolated and cultured embryonic day 12.5 fetal mouse lungs from pregnant Bach1 knockout (-/-) and wild-type (WT) mice. Airway branching morphogenesis was assessed by microscopically counting peripheral lung buds after incubation with/without LPS. Expression levels of genes related to inflammation and oxidative stress were evaluated using quantitative PCR. Zinc protoporphyrin, HO-1-specific inhibitor, was used. RESULTS: Branching morphogenesis was observed in Bach1-/- and WT fetal mice lungs without LPS exposure; after exposure to LPS, the number of peripheral lung buds was suppressed in Bach1-/- group only. Basal messenger RNA (mRNA) and protein expression of HO-1 was significantly higher in Bach1-/- group than in WT group; IL-6 and monocyte chemoattractant protein-1 mRNA expression was significantly increased after LPS exposure in both groups. Zinc protoporphyrin mitigated the LPS-induced suppression of branching morphogenesis in Bach1-/- mice. CONCLUSION: The ablation of Bach1 suppresses airway branching morphogenesis after LPS exposure by increased basal expression levels of HO-1.

Associations between the levels of soluble (pro)renin receptor in maternal and umbilical cord blood and hypertensive disorder of pregnancy.

INTRODUCTION: The prorenin (PR) receptor [(P)RR] contributes to the regulation of the tissue renin-angiotensin system (RAS) and Wnt signaling, which is involved in embryogenesis and the pathological progression of malignant tumors and diabetes mellitus. Placental (P)RR is significantly upregulated in placental tissues from preeclamptic women. However, because it cannot be examined during pregnancy, the chronological relationship between the acceleration of tissue RAS and the disease state of hypertensive disorder of pregnancy (HDP) has not been reported. In this study, we examined whether chronological changes in placental tissue RAS can be assessed by measuring soluble (P)RR [s(P)RR]. METHODS: We obtained maternal and umbilical cord blood samples from 517 pregnant women (441 singleton and 76 twin pregnancies). The concentrations of s(P)RR and prorenin (PR) were measured using enzyme-linked immunosorbent assays. RESULTS: Multivariate analysis showed that maternal serum s(P)RR levels were significantly higher in patients with HDP or fetal growth restriction (FGR) and were positively correlated with serum PR levels. Furthermore, the maternal s(P)RR level was significantly higher in HDP with severe hypertension and after the onset of HDP. However, maternal s(P)RR levels were not affected by the severity of proteinuria. Serum s(P)RR levels in umbilical cord blood of singleton pregnancies were significantly correlated with gestational week at delivery and PR level. DISCUSSION: Maternal serum s(P)RR concentrations may reflect acceleration of tissue RAS in the placenta and blood pressure severity; however, the umbilical serum s(P)RR concentration was not affected by maternal HDP.

Placental (pro)renin receptor expression and plasma soluble (pro)renin receptor levels in preeclampsia.

INTRODUCTION: The prorenin receptor ((P)RR) contributes to the regulation of the tissue renin-angiotensin system (RAS) and the function of V-ATPase, which are essential for Wnt signaling. Thus, (P)RRs may be involved in the control both of feto-placental and maternal circulation during pregnancy. This study was conducted to clarify how placental (P)RR expression and plasma soluble (P)RR [s(P)RR] levels are associated with blood pressure elevations and renal function during pregnancy. METHODS: We conducted a cross-sectional study, conducted at Saitama medical center in 2010-2013. Preeclamptic women (n = 16) diagnosed according to the criteria of Japan Society of Obstetrics and Gynecology and normotensive pregnant women (n = 15) participated in the study. We measured the expression of (P)RR in the placenta, plasma s(P)RR levels, systolic blood pressure (SBP), and estimated glomerular filtration rate (eGFR). RESULTS: Placental expression of (P)RR was significantly higher in preeclamptic women than in normotensive pregnant women. The plasma s(P)RR levels were significantly higher in preeclamptic women than in normotensive pregnant women. Systolic blood pressure (SBP) was positively correlated with placental (P)RR levels (P = 0.0001) and plasma s(P)RR levels (P = 0.005) in all pregnant women. In preeclamptic women, SBP was positively correlated with placental (P)RR levels (P = 0.004), but not with plasma s(P)RR levels (P = 0.15). The eGFR was negatively correlated with placental (P)RR levels (P = 0.02) and plasma s(P)RR levels (P = 0.0002) in all pregnant women. In preeclamptic women, eGFR was negatively correlated with plasma s(P)RR levels (P = 0.006), but not with placental (P)RR levels (P = 0.93). DISCUSSION: Placental (P)RR can be involved in blood pressure regulation via the tissue RAS. On the other hand, plasma s(P)RR may be involved in the pathogenesis of decreased renal function in preeclampsia.

The effect of progesterone on genes involved in preterm labor.

The decidua is known to be a major source of intrauterine PGF2α during late gestation and labor, and inflammatory cytokines, including IL-1ß, IL-6, and IL-8, are elevated in spontaneous preterm deliveries. In the present study, to elucidate how progesterone blocks the pathways associated with preterm birth, we determined the effects of P4 on the expression of PTGS-2 and PTGFR mRNA in human decidua fibroblast cells, as well as the genes, using microarray analysis. Senescence was induced in primary cultured human decidual cells treated with IL-1ß. The IL-1ß treatment implicated by microarray analysis increased gene expression levels of PTGS-2, PTGFR, NFκ-B p65, IL-17, and IL-8. In contrast, P4+IL-1ß decreased the expression levels of all of these genes in comparison to treatment with IL-1ß alone (p<0.05). IL-1ß also increased the proportion of SA-ß-gal-positive cells. Treatment with IL-1ß also increased the p21 protein level in comparison to cells treated either with the vehicle or P4. Neither the p21 protein level nor the number of SA-ß-gal-positive cells was increased in normal endometrial glandular cells by IL-1ß (p<0.05). Our studies demonstrated that P4 changes the level of gene expression in a manner that favors an anti-inflammatory milieu. Because IL-8 appears to be the cytokine whose expression is most significantly modulated by P4, further studies evaluating IL-8 as a therapeutic target are needed.

Examination of the expression of cyclooxygenase-2 in placenta villi from sufferers of pregnancy induced hypertension.

OBJECTIVES: The purpose of this paper is to elucidate the roles of phospholipase A(2) (PLA(2)), cyclooxygenase-2 (COX-2), and prostaglandin I(2) (PGI(2)) synthase in pregnancy induced hypertension (PIH). METHODS: In placentas from normal pregnant women and pregnant women with severe PIH, the enzyme expression of PLA(2), COX-2, and PGI(2) synthase was measured using real time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression of each enzyme was compared between normal (n=12) and PIH (n=12) groups. The expression levels of COX-2 and PGI(2) synthase during PIH pregnancy were significantly decreased to about 51% and 68%, respectively, of their values in normal pregnancy. However, the expression of PLA(2) was hardly changed by PIH. CONCLUSIONS: The decreases in COX-2 and PGI(2) synthase expression in severe PIH placentas may be causal factors in the disruption of the PGI(2)-thromboxane A(2) (TXA(2)) balance in favor of TXA(2). The decrease in COX-2 was more marked than that of PGI(2) synthase.

[Hormone excretion and peroxisomes of human immortalized extravillous trophoblast cells (TCL-2) derived from first-trimester placenta].

We studied the hormone excretion of human immortalized extravillous trophoblast cells (TCL-2, first-trimester cells) and determined whether peroxisomes are present in TCL-2. The results of TCL-2 were compared with those of TCL-1 (third-trimester cells). Morphologically, TCL-2 cells were fibroblast-like, and the growth rate of TCL-2 was slower than that of TCL-1 during 3 days culture. Progesterone was detected in the medium of TCL-2, and its concentration was approximately one-tenth of that in TCL-1. The activity of the peroxisomal marker enzyme catalase was detected in the TCL-2 homogenate, and it was about one-third the level of that in TCL-1. Fatty acyl-CoA oxidase activity was detected in TCL-2, and it was about one-seventh the level of that in TCL-1. On the other hand, human chorionic gonadotropin (hCG) was detected in the medium of TCL-2, and its concentration after 3 days of culture was about 2-fold that in TCL-1. Using the diaminobenzidine (DAB) method, peroxisomes were found in TCL-2, but only a very small amount of catalase was detected. These results indicate that human immortalized extravillous trophoblast cells (TCL-2) synthesize, secrete hCG and progesterone, and may contain peroxisomes. Because extravillous trophoblast cells are difficult to obtain from the first-trimester placenta, TCL-2 cells are useful for the study of the physiologic functions (including peroxisomal function) of first-trimester cells.

Influence of alpha-tumor necrosis factor and beta-interleukin-1 on production of angiogenetic factors and thymidine phosphorylase activity in immortalized human decidual fibroblasts in vitro.

AIM: The aim of the present study was to investigate regulatory mechanisms of angiogenesis in the decidua using immortalized human decidual fibroblasts. METHODS: A sample of decidual fibroblasts was taken from a woman in early pregnancy. A cell line, DE-1, was established by infecting the decidual fibroblasts with the simian virus 40 large T antigen. Using this cell line, the ability to produce vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), beta-transforming growth factor (TGF-beta), and thymidine phosphorylase (TP) activity was investigated using immunohistochemistry, and the influences of beta-interleukin-1 (IL-1beta) and alpha-tumor necrosis factor (TNF-alpha) on these angiogenetic factors was investigated using enzyme-linked immunosorbent assays. Furthermore, the effects of TNF-alpha on proliferative capacity and apoptosis induction in DE-1 were studied. RESULTS: It was demonstrated that DE-1 produced all of these angiogenetic factors. The production of VEGF, bFGF and TGF-beta respectively was enhanced by both IL-1beta and TNF-alpha. TP activity was increased by TNF-alpha, but no increase was observed as a result of IL-1beta. It was shown that TNF-alpha suppressed the proliferation of DE-1 cells and significantly increased the percentage of apoptotic cells. CONCLUSION: It is suggested that IL-1beta and TNF-alpha stimulate decidual fibroblasts to up-regulate angiogenesis in the human decidua.

PPARalpha agonists clofibrate and gemfibrozil inhibit cell growth, down-regulate hCG and up-regulate progesterone secretions in immortalized human trophoblast cells.

We studied effects of PPARalpha agonists clofibric acid and gemfibrozil on cell growth and functions of immortalized human extravillous trophoblast cells. Levels of DNA and protein gradually increased during incubation for 4 days. Gemfibrozil (>0.25mM) and clofibric acid (2.5mM) suppressed the rate of increase in DNA and protein. Specific activities of fatty acyl-CoA oxidase and catalase were increased to about 1.2-2.0 times the control value by 0.05mM gemfibrozil and 1.0 and 2.5mM clofibric acid after incubation for 3 days. Acid phosphatase activity showed a small increase in response to both agents, but esterase activity changed little. The secretion of progesterone from the cells into the medium was increased by 0.25mM gemfibrozil and 1.0 and 2.5mM clofibric acid after incubation for 3 days, but that of human chorionic gonadotropin (hCG) was decreased by 0.35mM gemfibrozil and 2.5mM clofibric acid. The specific activity of lactate dehydrogenase in the cells was hardly changed at all after incubation for 3 days. These results suggest that gemfibrozil and clofibric acid inhibit the proliferation of trophoblast cells. Cell metabolism is probably affected by both agents. The two agents may down-regulate hCG and up-regulate progesterone secretions.