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Impaired vascular endothelial growth factor expression and secretion during in vitro differentiation of human primary
Christophe Louis Depoix1, Arthur Colson2, Corinne Hubinont2
1Department 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.depoix@uclouvain.be.
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.
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.
