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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 increases HPEC but not HUVEC proliferation and migration. (A) [3H]Thymidine incorporation into HPEC and HUVEC cells, in the absence or presence of EG-VEGF. A significant increase of HPEC proliferation was observed with 25 and 50 ng/ml EG-VEGF (*p < 0.05). No significant effect was observed on HUVEC cells. (B and C) Photographs of wounded HPEC and HUVEC monolayers, respectively, at 0, and 12 h after wounding. The plots show percentages of wound closure after 12 h of treatment with EG-VEGF in the absence or presence of PD98059 and the LY294002, the inhibitors of MAP kinases and PI3K, respectively. Bars with different letters are significantly different from each other (p < 0.05).
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Figure 2: EG-VEGF increases HPEC but not HUVEC proliferation and migration. (A) [3H]Thymidine incorporation into HPEC and HUVEC cells, in the absence or presence of EG-VEGF. A significant increase of HPEC proliferation was observed with 25 and 50 ng/ml EG-VEGF (*p < 0.05). No significant effect was observed on HUVEC cells. (B and C) Photographs of wounded HPEC and HUVEC monolayers, respectively, at 0, and 12 h after wounding. The plots show percentages of wound closure after 12 h of treatment with EG-VEGF in the absence or presence of PD98059 and the LY294002, the inhibitors of MAP kinases and PI3K, respectively. Bars with different letters are significantly different from each other (p < 0.05).

Mentions: In a previous report from the group of Ferrara (LeCouter et al., 2001), it has been shown that EG-VEGF does not affect the proliferation of HUVECs. However, no data are available on its effect on HPEC proliferation. Here, we investigated the effect of EG-VEGF on the proliferation of both cell types. Proliferation was assessed using two different techniques: [3H]thymidine incorporation (Figure 2A) and Ki67 staining (Figure S2). Our results show that EG-VEGF significantly increased HPEC cell proliferation in a dose-dependent manner. However, no effect was observed on HUVEC proliferation. These results were confirmed by the observed increase in Ki67 staining in HPECs but not in HUVECs.


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 increases HPEC but not HUVEC proliferation and migration. (A) [3H]Thymidine incorporation into HPEC and HUVEC cells, in the absence or presence of EG-VEGF. A significant increase of HPEC proliferation was observed with 25 and 50 ng/ml EG-VEGF (*p < 0.05). No significant effect was observed on HUVEC cells. (B and C) Photographs of wounded HPEC and HUVEC monolayers, respectively, at 0, and 12 h after wounding. The plots show percentages of wound closure after 12 h of treatment with EG-VEGF in the absence or presence of PD98059 and the LY294002, the inhibitors of MAP kinases and PI3K, respectively. Bars with different letters are significantly different from each other (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: EG-VEGF increases HPEC but not HUVEC proliferation and migration. (A) [3H]Thymidine incorporation into HPEC and HUVEC cells, in the absence or presence of EG-VEGF. A significant increase of HPEC proliferation was observed with 25 and 50 ng/ml EG-VEGF (*p < 0.05). No significant effect was observed on HUVEC cells. (B and C) Photographs of wounded HPEC and HUVEC monolayers, respectively, at 0, and 12 h after wounding. The plots show percentages of wound closure after 12 h of treatment with EG-VEGF in the absence or presence of PD98059 and the LY294002, the inhibitors of MAP kinases and PI3K, respectively. Bars with different letters are significantly different from each other (p < 0.05).
Mentions: In a previous report from the group of Ferrara (LeCouter et al., 2001), it has been shown that EG-VEGF does not affect the proliferation of HUVECs. However, no data are available on its effect on HPEC proliferation. Here, we investigated the effect of EG-VEGF on the proliferation of both cell types. Proliferation was assessed using two different techniques: [3H]thymidine incorporation (Figure 2A) and Ki67 staining (Figure S2). Our results show that EG-VEGF significantly increased HPEC cell proliferation in a dose-dependent manner. However, no effect was observed on HUVEC proliferation. These results were confirmed by the observed increase in Ki67 staining in HPECs but not in HUVECs.

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