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Molecular characterization of EG-VEGF-mediated angiogenesis: differential effects on microvascular and macrovascular endothelial cells.

Brouillet S, Hoffmann P, Benharouga M, Salomon A, Schaal JP, Feige JJ, Alfaidy N - Mol. Biol. Cell (2010)

Bottom Line: Here we characterized its angiogenic effect using different experimental procedures.More importantly, we demonstrated that PROKR1 mediates EG-VEGF angiogenic effects, whereas PROKR2 mediates cellular permeability.Altogether, these data characterized angiogenic processes mediated by EG-VEGF, depicted a new angiogenic factor in the placenta, and suggest a novel view of the regulation of angiogenesis in placental pathologies.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, Unité 878, Grenoble, France.

ABSTRACT
Endocrine gland derived vascular endothelial growth factor (EG-VEGF) also called prokineticin (PK1), has been identified and linked to several biological processes including angiogenesis. EG-VEGF is abundantly expressed in the highest vascularized organ, the human placenta. Here we characterized its angiogenic effect using different experimental procedures. Immunohistochemistry was used to localize EG-VEGF receptors (PROKR1 and PROKR2) in placental and umbilical cord tissue. Primary microvascular placental endothelial cell (HPEC) and umbilical vein-derived macrovascular EC (HUVEC) were used to assess its effects on proliferation, migration, cell survival, pseudovascular organization, spheroid sprouting, permeability and paracellular transport. siRNA and neutralizing antibody strategies were used to differentiate PROKR1- from PROKR2-mediated effects. Our results show that 1) HPEC and HUVEC express both types of receptors 2) EG-VEGF stimulates HPEC's proliferation, migration and survival, but increases only survival in HUVECs. and 3) EG-VEGF was more potent than VEGF in stimulating HPEC sprout formation, pseudovascular organization, and it significantly increases HPEC permeability and paracellular transport. More importantly, we demonstrated that PROKR1 mediates EG-VEGF angiogenic effects, whereas PROKR2 mediates cellular permeability. Altogether, these data characterized angiogenic processes mediated by EG-VEGF, depicted a new angiogenic factor in the placenta, and suggest a novel view of the regulation of angiogenesis in placental pathologies.

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EG-VEGF is a survival factor for HPEC and HUVEC cells. (A) Representative Western blots of MAP kinase and AKT phosphorylations after treatment with EG-VEGF in HPEC and HUVEC cells. Standardization of the protein signals was done with antibodies against dephospho-MAP kinases and -AKT. (B) shows the effect of EG-VEGF on caspase 3 expression in HPEC and HUVEC cells after serum starvation and challenging with EG-VEGF (25 ng/ml). (C) The percentage of caspase 3–positive cells. Three randomly selected microscopic fields were observed, and ≥200 cells/field were evaluated. (**p < 0.01, *p < 0.05). Bar, 20 μm.
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Figure 3: EG-VEGF is a survival factor for HPEC and HUVEC cells. (A) Representative Western blots of MAP kinase and AKT phosphorylations after treatment with EG-VEGF in HPEC and HUVEC cells. Standardization of the protein signals was done with antibodies against dephospho-MAP kinases and -AKT. (B) shows the effect of EG-VEGF on caspase 3 expression in HPEC and HUVEC cells after serum starvation and challenging with EG-VEGF (25 ng/ml). (C) The percentage of caspase 3–positive cells. Three randomly selected microscopic fields were observed, and ≥200 cells/field were evaluated. (**p < 0.01, *p < 0.05). Bar, 20 μm.

Mentions: It is well documented that mitogenic and survival effects of EG-VEGF on endothelial cells involve the induction of MAPK and PI3K phosphorylation (Kisliouk et al., 2003). We examined whether EG-VEGF activated these signaling pathways in HPECs and HUVECs. Cells were serum-starved for 24 h and then examined for the phosphorylation of p42/44 MAPKs and AKT in response to EG-VEGF treatment. Figure 3A shows the Western blot analysis of phospho-MAPKs and phospho-AKT after EG-VEGF treatment. Strong phosphorylation of p44/42 MAPKs and AKT proteins in response to EG-VEGF were observed after only 5 min in HPECs. In HUVECs, there was a slight increase in MAPK and AKT phosphorylation. EG-VEGF effect on AKT phosphorylation suggested to us that this factor might be a survival factor for both types of cells. This was confirmed by the measurement of HPEC and HUVEC survival after 24 h of serum starvation and a 24-h challenge with EG-VEGF (10, 25, 50, and 100 ng/ml). Caspase 3 staining was used to detect apoptotic cells. Figure 3B shows that serum starvation of HPEC and HUVEC resulted in 9.6 ± 1.23 and 11.7 ± 1.5% of cells becoming apoptotic in both cells types, respectively, and that the presence of EG-VEGF significantly decreased this proportion to 3.25 ± 0.23 and 7.6 ± 1.03%, respectively.


Molecular characterization of EG-VEGF-mediated angiogenesis: differential effects on microvascular and macrovascular endothelial cells.

Brouillet S, Hoffmann P, Benharouga M, Salomon A, Schaal JP, Feige JJ, Alfaidy N - Mol. Biol. Cell (2010)

EG-VEGF is a survival factor for HPEC and HUVEC cells. (A) Representative Western blots of MAP kinase and AKT phosphorylations after treatment with EG-VEGF in HPEC and HUVEC cells. Standardization of the protein signals was done with antibodies against dephospho-MAP kinases and -AKT. (B) shows the effect of EG-VEGF on caspase 3 expression in HPEC and HUVEC cells after serum starvation and challenging with EG-VEGF (25 ng/ml). (C) The percentage of caspase 3–positive cells. Three randomly selected microscopic fields were observed, and ≥200 cells/field were evaluated. (**p < 0.01, *p < 0.05). Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2921113&req=5

Figure 3: EG-VEGF is a survival factor for HPEC and HUVEC cells. (A) Representative Western blots of MAP kinase and AKT phosphorylations after treatment with EG-VEGF in HPEC and HUVEC cells. Standardization of the protein signals was done with antibodies against dephospho-MAP kinases and -AKT. (B) shows the effect of EG-VEGF on caspase 3 expression in HPEC and HUVEC cells after serum starvation and challenging with EG-VEGF (25 ng/ml). (C) The percentage of caspase 3–positive cells. Three randomly selected microscopic fields were observed, and ≥200 cells/field were evaluated. (**p < 0.01, *p < 0.05). Bar, 20 μm.
Mentions: It is well documented that mitogenic and survival effects of EG-VEGF on endothelial cells involve the induction of MAPK and PI3K phosphorylation (Kisliouk et al., 2003). We examined whether EG-VEGF activated these signaling pathways in HPECs and HUVECs. Cells were serum-starved for 24 h and then examined for the phosphorylation of p42/44 MAPKs and AKT in response to EG-VEGF treatment. Figure 3A shows the Western blot analysis of phospho-MAPKs and phospho-AKT after EG-VEGF treatment. Strong phosphorylation of p44/42 MAPKs and AKT proteins in response to EG-VEGF were observed after only 5 min in HPECs. In HUVECs, there was a slight increase in MAPK and AKT phosphorylation. EG-VEGF effect on AKT phosphorylation suggested to us that this factor might be a survival factor for both types of cells. This was confirmed by the measurement of HPEC and HUVEC survival after 24 h of serum starvation and a 24-h challenge with EG-VEGF (10, 25, 50, and 100 ng/ml). Caspase 3 staining was used to detect apoptotic cells. Figure 3B shows that serum starvation of HPEC and HUVEC resulted in 9.6 ± 1.23 and 11.7 ± 1.5% of cells becoming apoptotic in both cells types, respectively, and that the presence of EG-VEGF significantly decreased this proportion to 3.25 ± 0.23 and 7.6 ± 1.03%, respectively.

Bottom Line: Here we characterized its angiogenic effect using different experimental procedures.More importantly, we demonstrated that PROKR1 mediates EG-VEGF angiogenic effects, whereas PROKR2 mediates cellular permeability.Altogether, these data characterized angiogenic processes mediated by EG-VEGF, depicted a new angiogenic factor in the placenta, and suggest a novel view of the regulation of angiogenesis in placental pathologies.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, Unité 878, Grenoble, France.

ABSTRACT
Endocrine gland derived vascular endothelial growth factor (EG-VEGF) also called prokineticin (PK1), has been identified and linked to several biological processes including angiogenesis. EG-VEGF is abundantly expressed in the highest vascularized organ, the human placenta. Here we characterized its angiogenic effect using different experimental procedures. Immunohistochemistry was used to localize EG-VEGF receptors (PROKR1 and PROKR2) in placental and umbilical cord tissue. Primary microvascular placental endothelial cell (HPEC) and umbilical vein-derived macrovascular EC (HUVEC) were used to assess its effects on proliferation, migration, cell survival, pseudovascular organization, spheroid sprouting, permeability and paracellular transport. siRNA and neutralizing antibody strategies were used to differentiate PROKR1- from PROKR2-mediated effects. Our results show that 1) HPEC and HUVEC express both types of receptors 2) EG-VEGF stimulates HPEC's proliferation, migration and survival, but increases only survival in HUVECs. and 3) EG-VEGF was more potent than VEGF in stimulating HPEC sprout formation, pseudovascular organization, and it significantly increases HPEC permeability and paracellular transport. More importantly, we demonstrated that PROKR1 mediates EG-VEGF angiogenic effects, whereas PROKR2 mediates cellular permeability. Altogether, these data characterized angiogenic processes mediated by EG-VEGF, depicted a new angiogenic factor in the placenta, and suggest a novel view of the regulation of angiogenesis in placental pathologies.

Show MeSH
Related in: MedlinePlus