Adhesion development and the expression of endothelial nitric oxide synthase.

OBJECTIVE: This study was conducted to determine whether nitric oxide (NO), a potent vasodilator and inhibitor of thrombus formation, is involved in the formation and maintenance of adhesions. METHODS: Skin, subcutaneous tissues, peritoneum and adhesions were collected from surgical patients and total RNA was isolated. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) was performed to quantitate endothelial nitric oxide synthase (eNOS) and beta-actin mRNA levels. RESULTS: eNOS mRNA levels for skin, subcutaneous tissue, peritoneum and adhesions were < or = 3.12 x 10(-4), < or = 3.12 x 10(-4), 6.24 x 10(-4) and 2.5 x 10(-3) attomoles/microl, respectively. Beta-actin mRNA levels for all tissues were between 1.25 x 10(-1) and 6.25 x 10(-2) attomoles/microl. CONCLUSION: eNOS mRNA can be identified in tissue adhesions, and may therefore play a role in adhesion formation and maintenance.

Expression and regulation of vascular endothelial growth factor in a first trimester trophoblast cell line.

Embryo implantation and development are critically dependent upon the spatial and temporal regulation of angiogenesis and localized vascular permeability. A key mediator of these effects is the endothelial cell mitogen vascular endothelial growth factor (VEGF). VEGF has been shown to promote endometrial vascular permeability, fetal vasculogenesis and placental, fetal and maternal angiogenesis. However, the mechanism through which this regulation occurs in the placenta is poorly understood. This study was conducted to determine if the pro-angiogenic cytokines, TNF-alpha and TGF-beta1, affect VEGF expression in human first trimester trophoblasts. Culture of a first trimester trophoblast cell line (HTR-8/SVneo), in the presence of either TNF-alpha or TGF-beta1, resulted in the expression of significant levels of VEGF in culture. The trophoblast cell line also showed a time-dependent and a dose-dependent increase in VEGF mRNA levels when cultured in the presence of either TNF-alpha or TGF-beta1. These results suggest that both TNF-alpha and TGF-beta1 may regulate the production of VEGF in early gestational trophoblasts and may therefore serve to modulate placental vascular permeability and angiogenesis that are necessary for embryo implantation and placentation.

Effect of imiquimod on cytokine induction in first trimester trophoblasts.

OBJECTIVES: Imiquimod (IQ) is used clinically for the topical treatment of external genital warts. IQ is an immune response modifier and induces the expression of interferon-alpha and other cytokines in human Peripheral Blood Monocytes (PBMC). Trophoblasts have been previously shown to express inflammatory cytokines upon lipopolysaccharide (LPS) stimulation. The objective of this study was to evaluate the ability of IQ to induce transcription of cytokines in trophoblasts. METHODS: A transformed human first trimester trophoblast cell line, HTR-8/SVneo, was cultured in DMEM containing IQ at concentrations of 0 to 5.0 microg/ml. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability assays were conducted to control for any drug-induced cell death. Total RNA was isolated from trophoblasts at 0, 8 and 24 hours of culture and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was conducted using specific amplimers for the inflammatory cytokines interleukin (IL)-1alpha, IL-1beta, IL-6 and IL-8. RT-PCR of beta-actin was performed to control for equal RNA loading. RESULTS: RT-PCR was unable detect an increase in either IL-1alpha, IL-1beta, IL-6 or IL-8 mRNA in first trimester trophoblasts cultured in the presence of 0 to 5.0 microg/mL of IQ for up to 24 hours. RT-PCR confirmed equal RNA loading and MTT viability assays did not show loss of cell viability at concentrations of IQ up to 5.0 microg/ml. CONCLUSIONS: IQ, at the concentrations tested, did not induce the transcriptional expression of inflammatory cytokines in human first trimester trophoblasts. These data suggest that IQ would not induce the expression of inflammatory cytokines in placental trophoblasts.

Ethanol-induced expression of cytokines in a first-trimester trophoblast cell line.

OBJECTIVES: Altered cytokine expression at the fetoplacental interface may be a potential mechanism for the development of fetal immune dysfunction in children with fetal alcohol syndrome. This study was conducted to determine whether first-trimester trophoblasts respond to ethanol exposure by the induction of specific cytokines. STUDY DESIGN: HTR-8/SVneo trophoblast cells were cultured in vitro in the presence of either ethanol (0.5% [vol/vol]), lipopolysaccharide (1 microg/mL), or ethanol and lipopolysaccharide. Expression of granulocyte colony-stimulating factor, regulated on activation normal T cell expressed and secreted, and interleukin-6 was examined by Northern analysis and enzyme-linked immunosorbent assay. RESULTS: Culture in the presence of ethanol, lipopolysaccharide, or lipopolysaccharide and ethanol resulted in the increased transcription and secretion of granulocyte colony-stimulating factor, regulated on activation normal T cell expressed and secreted, and interleukin-6 at significantly greater levels (P < .01) than control cultures. CONCLUSIONS: Human first-trimester trophoblasts express high levels of cytokines when cultured in the presence of ethanol. Trophoblasts may therefore be an important exogenous source of cytokines for the fetus, and altered cytokine levels during early gestation may have an adverse effect on the development of the fetal immune system.

Decreased gene expression of endothelial nitric oxide synthase in newborns with persistent pulmonary hypertension.

Previous studies in adults have shown that chronic pulmonary hypertension is associated with decreased endothelial nitric oxide synthase (eNOS) expression in pulmonary arteries. However, the role of decreased eNOS expression in persistent pulmonary hypertension of the newborn (PPHN) is unknown. We investigated the hypothesis that umbilical vein endothelial cells cultured from infants with PPHN will have decreased eNOS expression. Umbilical cords were collected from meconium-stained infants at birth, and endothelial cells were isolated if the infants developed PPHN. Endothelial cells were grown in primary culture, and total RNA was isolated. cDNA was reverse transcribed from mRNA and amplified by PCR. An expected product of approximately 550 bp was found in all control infants but only in two of the six infants with PPHN. Identity of the PCR product was confirmed by Southern hybridization to a separate internal eNOS-specific probe. Amplification of beta-actin cDNA, an internal control, was detected in all controls and in all infants with PPHN, including the four infants without the eNOS band. There was no difference in the course and outcome of patients with presence or absence of the eNOS band. However, there was an acidotic arterial blood pH (7.19-7.29) and intrapartum fetal heart rate decelerations in all four infants without eNOS expression. In conclusion, eNOS mRNA was detected in all normal term infants but was notably absent in the majority of infants with PPHN in this pilot study. The development of PPHN is multifactorial, and a decrease in eNOS gene expression may occur in some infants. Whether the decreased eNOS transcript is a cause of PPHN or a result of intrapartum stress remains to be determined.