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Updated: Dec 30, 2025

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis
Published on: November 8, 2024
Calcium deficient placental growth restriction is mediated by an increase in non-invasive integrin α5 and β4
Bei Xu1, Angela Makris2, Chia-Chi Liu1
1Vascular Immunology Research Laboratory, The Heart Research Institute, University of Sydney, Australia.
Objectives:
Integrins are cell adhesion receptors that participated in endovascular invasion by cytotrophoblasts in preeclampsia. This study aimed to investigate the effect of calcium on cellular pathways influencing the trophoblast integration into endothelial cellular networks in vitro.
Study Design:
Red fluorescent-labelled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on Matrigel. Green fluorescent-labelled HTR-8/SVneo trophoblast cells were then co-cultured with endothelial cells in different concentrations of calcium for 24 h.
Main Outcome Measures:
The calcium effects on HTR-8/SVneo cell integration were quantified by Image J. Quantitative PCR was performed to measure mRNA expression of integrins α1, α5, α6, β1 and β4. The concentrations of interleukin IL-6, matrix metalloproteinase-2 (MMP-2), MMP-9, PlGF and sFlt-1 in the conditioned medium were measured by ELISA while levels of cytokines IL-1β, IL-8, IL-10, TNF-α and INF-γ were assessed by magnetic Luminex assays™.
Results:
Both calcium depletion (0.4 mM) and low calcium (1.8 mM) groups demonstrated inhibited integration of trophoblast cells into endothelial cellular networks, compared with the normal calcium group (2.4 mM). The IL-6 production was reduced from conditioned media in both calcium depletion and low calcium groups. In calcium depletion group, mRNA expression of integrin α5 and β4 in trophoblasts was increased while integrin α1 was decreased.
Conclusions:
The in vitro trophoblast cell integration into endothelial cellular networks could be modified by altering media calcium through integrin switch away from integrins α5 and β4 and towards integrin α1 which may be required for healthy early trophoblast integration.
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