Dose-response effects of betamethasone on maturation of the fetal sheep lung.

OBJECTIVE: Glucocorticoid administration to women in preterm labor improves neonatal mortality and morbidity. Fetal exposure to glucocorticoid levels higher than those appropriate to the current gestational stage has multiple organ system effects. Some, eg, fetal hypertension, are maximal at lower than the clinical dose. We hypothesized that the clinical dose has supramaximal lung maturational effects. STUDY DESIGN: We evaluated the full, half, and quarter clinical betamethasone dose (12 mg/70 kg or 170 microg/kg intramuscularly twice 24 hours apart) on fetal sheep lung pressure volume curves (PVC) after 48 hours' exposure at 0.75 gestation. We measured key messenger RNAs and protein products that affect lung function and total lung dipalmitoyl phosphatidyl choline. RESULTS: Full and half doses had similar PVC and total lung dipalmitoyl phosphatidyl choline effects. Messenger RNA for surfactant proteins A, B, and D and elastin increased in a dose-dependent fashion. CONCLUSION: Half the clinical betamethasone dose produces maximal PVC improvement in fetal sheep at 0.75 gestation.

Expression and regulation of glucocorticoid receptor in human placental villous fibroblasts.

The human placenta is a glucocorticoid (GC)-responsive organ consisting of multiple cell types including smooth muscle cells, fibroblasts, and trophoblast that demonstrate changes in gene expression after hormone treatment. However, little is known about the relative expression or activity of the GC receptor (GR) among the various placental cell types. Normal term human placentas were examined by immunohistochemistry using either GR phosphorylation site-specific antibodies that are markers for various activation states of the GR or a GR antibody that recognizes the receptor independent of its phosphorylation state (total GR). We found strong total GR and phospho-GR immunoreactivity in stromal fibroblasts of terminal villi, as well as perivascular fibroblasts and vascular smooth muscle cells of the stem villi. Lower levels of both total GR and phospho-GR were found within cytotrophoblast cells relative to fibroblasts, whereas syncytiotrophoblast showed very little total GR or phospho-GR immunoreactivity. This pattern holds true for immunoblot analysis of extracts from cell fractions cultured ex vivo. In cultured placental fibroblasts, phosphorylation of GR increased upon short-term GC treatment, consistent with a role for GR phosphorylation in receptor transactivation. Total GR levels were reduced by nearly 90% after long-term hormone treatment; however, this down-regulation was independent of changes in GR mRNA levels. These findings demonstrate that GR levels in fibroblasts can be modulated by changes in hormone exposure. Such cell type-specific differences in GR protein expression and phosphorylation may provide the means of differentially regulating the GC response among the cells of the human placenta.

Differential effects of thrombin and hypoxia on endometrial stromal and glandular epithelial cell vascular endothelial growth factor expression.

Ovarian steroids and/or premenstrual endometrial hypoxia are thought to restore the endometrial vasculature shed during menstruation by elevating endometrial vascular endothelial growth factor (VEGF) levels. During the luteal phase, VEGF levels peak, progesterone induces estradiol (E(2))-primed human endometrial stromal cells (HESCs) to decidualize and express tissue factor (TF), and endometrial vascular permeability is enhanced. The latter would present circulating clotting factors to decidual cell-expressed TF to form local thrombin. HESCs were incubated in serum-supplemented medium containing vehicle (control) or 10(-8) M E(2) or 10(-7) M medroxyprogesterone acetate (MPA) or E(2) + MPA for 7 d to induce decidualization, while monolayers of human endometrial glandular epithelial cells (HEGECs) formed during 4-d incubation of glands. The medium was exchanged for a defined medium containing corresponding vehicle or steroids +/- thrombin under normoxia or hypoxia (0-1% O(2)). Hypoxia enhanced secreted immunoreactive VEGF levels by severalfold in HESCs and HEGECs, but the steroids did not affect VEGF output in either cell type under normoxia or hypoxia. In E(2) + MPA-decidualized HESCs, VEGF levels were elevated by 0.1 U/ml of thrombin, and 0.5-2.5 U/ml of thrombin elicited maximum effects. The addition of 0.5 U/ml of thrombin evoked a time-dependent enhancement of VEGF levels and about an 8-fold increase at 48 h (P < 0.02; n = 6). Northern blotting indicated that E(2) + MPA-decidualized HESCs expressed VEGF(121), VEGF(165), and VEGF(189) mRNA, which were enhanced severalfold during 5- to 20-h incubation with thrombin. Moreover, TRAP, a synthetic peptide activator of the constitutively expressed protease activated receptor-1 thrombin receptor in decidualized HESCs, also elevated secreted VEGF levels. By contrast, HEGECs were unresponsive to thrombin added alone or with ovarian steroids. These results suggest that thrombin formed by progestin-augmented TF levels acts as an autocrine enhancer of VEGF expression in decidualized HESCs. Because angiogenesis occurs in a matrix of decidualized HESCs, these in vitro results provide a novel mechanism to account for both the peak in VEGF and angiogenesis in luteal phase human endometrium.

Effects of thrombin, hypoxia, and steroids on interleukin-8 expression in decidualized human endometrial stromal cells: implications for long-term progestin-only contraceptive-induced bleeding.

Abnormal uterine bleeding is the major reason for discontinuing long-term progesterone-only contraceptives (LTPOCs). Prior studies demonstrated that endometria exposed to the LTPOC, Norplant, display aberrant angiogenesis, leukocyte infiltration, and hypoxia-associated impaired blood flow. Paradoxically, human endometrial stromal cells (HESCs) of these specimens exhibit elevated expression of tissue factor (TF), the primary initiator of hemostasis via thrombin generation. The current study demonstrates that TF levels are also elevated in HESCs that are decidualized after insertion of Mirena, an intrauterine system that releases levonorgestrel directly into the endometrial canal and produces elevated perivascular levels of the proinflammatory and angiognenic cytokine IL-8. Because bleeding, inflammation, and ischemia-associated increased vascular permeability enhance access of plasma factor VII to HESC-expressed TF to generate thrombin, we evaluated the effects of steroids, thrombin, and hypoxia on HESC expression of IL-8. Confluent HESCs were incubated in a serum-containing medium for 7 d with vehicle control or estradiol (E(2)) plus medroxyprogesterone acetate (MPA). The medium was then exchanged for corresponding defined medium with and without thrombin, and the cultures were incubated in parallel for up to 48 h in a standard incubator (normoxia) or a sealed chamber at 0-1% O(2) (hypoxia). Under normoxia, immunoreactive IL-8 levels in the conditioned medium were reduced to one-third of control levels during decidualization with E(2)+MPA (P < 0.05; n = 5). In E(2)+MPA-treated cultures, thrombin (0.1 U/ml to 2.5 U/m) elicited a dose-dependent reversal of this inhibition, elevating IL-8 up to 60-fold (P < 0.05; n = 5) for more than 24 h and steady-state IL-8 mRNA levels by 3-fold for 3 h. The specific inactivator, hirudin, blocked most of the effects of thrombin, whereas TRAP-14, an agonist of the protease-activated receptor for thrombin, enhanced IL-8 output. In the absence of thrombin, hypoxia elevated IL-8 output 5-fold in E(2)+MPA-treated HESCs (P < 0.02, n = 4), with thrombin exerting additive effects. In contrast to its effects in progestin-treated HESCs, hypoxia did not elevate IL-8 output in control cultures. This study suggests that inhibition of IL-8 expression in decidualized HESCs contributes to the antiinflammatory milieu of the luteal phase. However, LTPOC-induced hypoxia and excess thrombin generation enhance IL-8 expression in decidualized HESCs, thereby eliciting aberrant angiogenesis and inflammation that promote the onset of abnormal uterine bleeding.

Differential effects of lipopolysaccharide and thrombin on interleukin-8 expression in syncytiotrophoblasts and endothelial cells: implications for fetal survival.

Syncytiotrophoblasts (SCTs) are directly bathed by maternal blood and, as such, are in direct contact with proinflammatory stimuli present in the maternal circulation. The extent and nature of cytokine responses induced in SCTs play a central role in the maintenance of pregnancy. Thrombin is a critical mediator of tissue factor-initiated blood coagulation. Thrombin has been more recently demonstrated to induce cytokine expression and inflammation in several cell types. To dissect the patterns of regulation of cytokine production in the placental villus, we compared the effects of thrombin and lipopolysaccharide (LPS) treatments on cytokine expression in SCTs and endothelial cells. For studies, primary cultures of cytotrophoblasts from human term placentas were differentiated to SCTs. We observed that the presence of thrombin only modestly enhanced interleukin-8 (IL-8) levels in SCTs in a manner that was not dose-dependent. Conversely, SCTs were exquisitely sensitive to LPS, the presence of which induced approximately a 10-fold increase in IL-8 levels with an EC(50) approximately 1 ng/mL. Northern blotting and real-time PCR results indicated that LPS (but not thrombin) treatment induced a >4-fold increase in levels of IL-8 mRNA. The addition of the anti-inflammatory steroid, dexamethasone, significantly reduced the LPS-mediated increase in levels of IL-8 in SCTs. Conversely, in human umbilical vein endothelial cells, thrombin and LPS treatments induced 10- and 20-fold increases in IL-8 expression, respectively. These results indicate that LPS, but not thrombin, promotes proinflammatory processes in SCTs, with cell-type specificity. The inability of thrombin in the intervillous space to evoke inflammatory responses in SCTs may constitute an important aspect of fetal survival. Conversely, our results suggest that SCTs do play a key role in infection-associated changes in placental cytokine expression.

Glucocorticoid enhances transforming growth factor-beta effects on extracellular matrix protein expression in human placental mesenchymal cells.

Extracellular matrix (ECM) proteins synthesized by human placental mesenchymal cells (PMCs) provide structural support for the villus. Aberrant expression of ECM proteins by PMCs has been associated with intrauterine growth restriction (IUGR). To provide insight into the mechanisms of ECM protein regulation in the stroma of the placental villus, in the current study, we examined the interaction of glucocorticoid (GC) and transforming growth factor-beta (TGFbeta) in the modulation of ECM proteins in cultures of PMCs isolated from human term placentas. Initial results obtained by ELISA showed that combined treatment with dexamethasone (DEX) and TGFbeta enhanced oncofetal fibronectin (FFN) protein levels in serum-free culture medium severalfold in a dose-dependent manner. Northern blotting and real-time polymerase chain reaction (PCR) analyses revealed a similar enhancement in levels of FN mRNA in cells treated with TGFbeta and DEX. Real-time PCR results also revealed that DEX and TGFbeta enhanced collagen (Col) I and Col IV expression, but did not affect levels of Col III or laminin, indicative of selective stimulation of ECM proteins. Hypoxic treatment moderately enhanced FFN levels in control cells but not in those treated with DEX and TGFbeta. In contrast with the results obtained with PMCs, we noted that DEX treatment suppressed FFN levels in untreated and TGFbeta-treated cytotrophoblasts, suggesting that GC and TGFbeta modulate FFN expression in placenta in a cell-type-specific manner. We conclude that GC and TGFbeta are key regulators of ECM protein synthesis in PMCs, suggesting a role in modulating placental architecture in uncomplicated pregnancies and those associated with aberrant ECM protein expression.

Mechanisms of abruption-induced premature rupture of the fetal membranes: Thrombin enhanced decidual matrix metalloproteinase-3 (stromelysin-1) expression.

OBJECTIVE: The aim of the study was to evaluate thrombin and progestin effects on matrix metalloproteinase-3 expression in term decidual cells as a mechanism of abruption-related preterm delivery. STUDY DESIGN: Decidual cells were isolated by standard techniques, purified to homogeneity, grown to confluence, and passaged. Cultures were primed with 10 (-8) M estradiol or estradiol plus 10 (-7) progestin and then incubated in a defined medium with corresponding steroid(s) plus or minus thrombin or the protease-activated thrombin receptor-1 agonist for 24 hours. Secreted matrix metalloproteinase-3 levels were assessed by enzyme-linked immunosorbent assay, and immunoblotting and messenger RNA levels were measured by Northern blotting and quantitative reverse transcription-polymerase chain reaction. RESULTS: Immunoreactive matrix metalloproteinase-3 levels were inhibited 66% by estradiol plus progestin versus estradiol ( P < .05). Thrombin elicited a dose-dependent reversal in this progestin inhibition, producing a 2.5-fold increase at 2.5 U/mL ( P < .05) that attained 33% of matrix metalloproteinase-3 levels in parallel incubations with estradiol plus thrombin. Protease-activated thrombin receptor-1 agonist mimicked 60% of thrombin-enhanced matrix metalloproteinase-3 output. Immunoblotting validated the enzyme-linked immunosorbent assay results. Northern blotting and quantitative reverse transcription-polymerase chain reaction demonstrated corresponding effects on steady-state messenger RNA levels. CONCLUSION: Abruption-generated thrombin promotes preterm delivery by mediating fetal membrane extracellular matrix degradation via enhanced decidual cell matrix metalloproteinase-3 expression, whereas progesterone blunts this thrombin-induced effect.