Nitric oxide (NO) reversed TNF-α inhibition of trophoblast interaction with endothelial cellular networks.
BACKGROUND
The interaction between trophoblast cells and maternal uterine endothelium is important for placental vascular modelling. Nitric oxide (NO) is a potent vasorelaxant that regulates systemic blood pressure and is reduced in preeclampsia. NO may affect placental cell interaction.


OBJECTIVE
This study was to examine whether NO plays a role in regulating TNF-α induced inhibition of trophoblast cell integration into endothelial cellular networks in-vitro.


METHODS
Red fluorescent-labelled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on Matrigel. After endothelial cellular networks formed, green fluorescent-labelled HTR-8/SVneo trophoblast cells were co-cultured with endothelial cells, together with/without TNF-α (0.5 ng/ml) and/or NO donor, sodium nitroprusside dihydrate (SNP) (100 μM). Images were captured after 24 h. The effects on HTR-8/SVneo cell integration were quantified by Image Analysis software. The cells were then recovered from Matrigel to extract mRNA. Quantitative PCR was performed to evaluate the expression of eNOS, VCAM-1 and E-selectin. The concentrations of sVCAM-1 and sE-selectin in the conditioned medium were measured by ELISA.


RESULTS
TNF-α inhibited HTR-8/SVneo trophoblast cell integration into endothelial cellular networks, as well as decreased eNOS mRNA expression. Increases in VCAM-1 and E-selectin in cellular mRNA and protein concentrations in the conditioned medium were also seen. The NO donor reversed the inhibitory effect of TNF-α on trophoblast integration and increased eNOS mRNA expression. SNP also reduced sE-selectin and sVCAM-1 expressions which were increased by TNF-α in the conditioned medium.


CONCLUSIONS
Our data suggest the inhibitory effect of TNF-α on trophoblast integration may be mediated by NO, via reducing endothelial cell activation.
School of Medicine, University of Western Sydney, Sydney, Australia; Vascular Immunology Research Laboratory, The Heart Research Institute, University of Sydney, Sydney, Australia. Electronic address: bei_xu2001@yahoo.com.
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B
Xu
Vascular Immunology Research Laboratory, The Heart Research Institute, University of Sydney, Sydney, Australia.
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Charlton
Renal Unit, Liverpool Hospital, Sydney, Australia; Vascular Immunology Research Laboratory, The Heart Research Institute, University of Sydney, Sydney, Australia.
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Makris
School of Medicine, University of Western Sydney, Sydney, Australia; Vascular Immunology Research Laboratory, The Heart Research Institute, University of Sydney, Sydney, Australia.
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Hennessy
D002478:Cells, Cultured; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000737:chemistry; D019040:E-Selectin / Q000032:analysis / Q000235:genetics; D042783:Endothelial Cells / Q000737:chemistry / Q000502:physiology*; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000032:analysis / Q000235:genetics; D009599:Nitroprusside / Q000494:pharmacology; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D014327:Trophoblasts / Q000737:chemistry / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014599:Uterus / Q000098:blood supply* / Q000166:cytology; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis / Q000235:genetics
D017077:Culture Media, Conditioned; D019040:E-Selectin; D005456:Fluorescent Dyes; D020030:Nitric Oxide Donors; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D009599:Nitroprusside; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III
E-selectin; Nitric oxide; TNF-α; Trophoblast–endothelial cell interaction; VCAM-1; eNOS