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Impaired vascular endothelial growth factor expression and secretion during in vitro differentiation of human primary term cytotrophoblasts

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Abstract

Vascular endothelial growth factor A (VEGF-A) is one of the main growth factors involved in placental vasculogenesis and angiogenesis, but its placental expression is still ambiguous. During in vitro cultures of primary term cytotrophoblasts, VEGF could not be detected in the supernatants by enzyme-linked immunosorbent assays (ELISA). One hypothesis is that VEGF is immediately and completely bound to its soluble receptor after secretion, and cannot be recognized by the antibodies used in the commercial ELISA kits. We decided to verify this hypothesis by measuring VEGF-A expression during in vitro cultures of primary term cytotrophoblasts. Term cytotrophoblasts were cultured under 21% and 2.5% O2 for 4 days. VEGF-A transcripts were quantified by real-time polymerase chain reaction. The proteins from cell lysates and concentrated media were separated by polyacrylamide gel electrophoresis (PAGE) under denaturing and reducing conditions, and VEGF-A immunodetected by western blotting. VEGF mRNA expression did not increase during in vitro cell differentiation under 21% O2, but slightly increased under 2.5% O2 only at 24 h. VEGF-A monomer was not detected in the cell lysates and in the concentrated supernatants, while a ~ 42 KDa band corresponding to the precursor L-VEGF was detected in all the cellular extracts. Isolated term villous cytotrophoblasts produce the L-VEGF precursor but they do not secrete VEGF-A even under low-oxygen tension. The question remains about the origin of VEGF in pregnancy but also about the biological role of L-VEGF, which can represent a form of storage for rapid VEGF secretion when needed.

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Acknowledgements

The authors would like to acknowledge the financial support of “Fetus for Life” charity, Belgium. The authors are also grateful to Séverine Gonze for her help in trophoblastic cell cultures, real-time PCR, and western blotting experiments.

Funding

The work was supported by the FETUS FOR LIFE Charity in Brussels, Belgium.

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Authors and Affiliations

  1. Department of Obstetrics, Institut de Recherche Clinique Et Experimentale (IREC), Université Catholique de Louvain, Avenue Mounier 52, 5th floor, Woluwe-Saint-Lambert, 1200, Bruxelles, Belgium

    Christophe Louis Depoix, Arthur Colson, Corinne Hubinont & Frederic Debieve

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  1. Christophe Louis Depoix
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  2. Arthur Colson
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  3. Corinne Hubinont
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  4. Frederic Debieve
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Correspondence to Christophe Louis Depoix.

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Conflict of interest

Christophe Louis Depoix declares that he has no conflict of interest. Arthur Colson declares that he has no confict of interest. Corinne Hubinont declares that she has no conflict of interest. Frédéric Debiève declares that he has no conflict of interest.

Ethical approval

All procedures performed in studies involving our participants were in accordance with the ethical standards of the CUSL and Research Ethics Committee (approval # 2018-23OCT-397) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Informed consent was obtained from all individual participants included in the study.

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10456_2019_9702_MOESM1_ESM.tif

Supplemental data, s1 The anti-VEGFA antibody specifically recognized L-VEGF in cell lysates. Cell lysates from undifferentiated cytotrophoblasts (0 h), differentiated trophoblasts cultured for 4 days under 21% O2 and 2.5% O2, and SiHa cells cultured under 2.5% O2 for 24 hours were separated under denaturing/reducing conditions. The specificity of the immunodetection was verified by incubating the membranes with the anti-VEGFA antibody alone or in presence of a blocking peptide used for the immunization (left panel). GAPDH was used as a loading control and to verify the presence of proteins on both membranes (right panel). (Cyto, cytotrophoblasts; kD, kiloDalton). (TIF 5164 kb)

10456_2019_9702_MOESM2_ESM.tif

Supplemental data, s2 the absence of secreted VEGF monomers in cytotrophoblast-conditioned medium was verified using two anti-VEGF antibodies with different clonality. Unconditioned complete medium, supernatants from cytotrophoblasts cultured for 96 hours under 21% O2 and 2.5% O2, and from SiHa cells cultured 24 hours under 2.5% O2 were concentrated ~30 times. Forty micrograms of proteins were separated by PAGE under denaturing/reducing conditions and immunodetected with rabbit polyclonal or mouse monoclonal anti-VEGF antibodies (Ab). Rec.hVEGF (10 ng) was used as a positive control. Both antibodies recognized rec.hVEGF and VEGF monomer in SiHa-conditioned medium while there was no VEGF monomers detected in cytotrophoblast-conditioned media. A 50–57 kDa band was also detected by both antibodies. This band was also detected in the unconditioned medium and in the rechVEGF sample indicating that it was unspecific. (kD, kiloDalton). (TIF 5569 kb)

10456_2019_9702_MOESM3_ESM.tif

Supplemental data, s3 The 50–57 kDa band is nonspecific. The 50–57 kDa band in the concentrated media was nonspecific since it was also detected in pure fetal bovine serum (FBS) and in the concentrated complete medium that was not conditioned by cells, and it was not detected in medium without FBS. (TIF 3763 kb)

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Depoix, C.L., Colson, A., Hubinont, C. et al. Impaired vascular endothelial growth factor expression and secretion during in vitro differentiation of human primary term cytotrophoblasts. Angiogenesis 23, 221–230 (2020). https://doi.org/10.1007/s10456-019-09702-z

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