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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 effects on HPEC permeability are mediated by PROKR2 and not PROKR1: Panels (A) and (B) show EG-VEGF (25 ng/ml) effect on the permeability of HPEC cells that had been silenced for PROKR1 and PROKR2 mRNA using siRNA (siRNA strategy), or treated with PROKR1 and R2 blocking antibodies (antibody strategy), respectively. In the two sets of experiments, EG-VEGF significantly increased HPEC permeability. Both siRNA to PROKR2 and its blocking antibody inhibited EG-VEGF effect. However, nor siRNA to PROKR1, neither its blocking antibody did affect the permeability. Data represent the mean ± SEM. Bars with different letters are significantly different from each other (p < 0.05).
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Figure 8: EG-VEGF effects on HPEC permeability are mediated by PROKR2 and not PROKR1: Panels (A) and (B) show EG-VEGF (25 ng/ml) effect on the permeability of HPEC cells that had been silenced for PROKR1 and PROKR2 mRNA using siRNA (siRNA strategy), or treated with PROKR1 and R2 blocking antibodies (antibody strategy), respectively. In the two sets of experiments, EG-VEGF significantly increased HPEC permeability. Both siRNA to PROKR2 and its blocking antibody inhibited EG-VEGF effect. However, nor siRNA to PROKR1, neither its blocking antibody did affect the permeability. Data represent the mean ± SEM. Bars with different letters are significantly different from each other (p < 0.05).

Mentions: As for the sprouting experiments, we also sought to determine the type of receptor that mediates EG-VEGF effects on permeability. Both, siRNA and antibody strategies were used to differentiate between the two receptor types. As expected, EG-VEGF significantly increased HPEC permeability in the absence of any other treatment. Scramble siRNA, siRNA PROKR1, or siRNA PROKR2 alone did not affect basal HPEC permeability. However, invalidation of PROKR2 significantly abolished the response to EG-VEGF. Under PROKR1 mRNA invalidation EG-VEGF effect was maintained (Figure 8A). These results were substantiated with the antibody strategy, showing that only PROKR2 blockade affects EG-VEGF effects on HPEC permeability. Altogether these results demonstrate that EG-VEGF mediates its effect on the permeability via the activation of PROKR2 and not PROKR1 receptor (Figure 8B).


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 effects on HPEC permeability are mediated by PROKR2 and not PROKR1: Panels (A) and (B) show EG-VEGF (25 ng/ml) effect on the permeability of HPEC cells that had been silenced for PROKR1 and PROKR2 mRNA using siRNA (siRNA strategy), or treated with PROKR1 and R2 blocking antibodies (antibody strategy), respectively. In the two sets of experiments, EG-VEGF significantly increased HPEC permeability. Both siRNA to PROKR2 and its blocking antibody inhibited EG-VEGF effect. However, nor siRNA to PROKR1, neither its blocking antibody did affect the permeability. Data represent the mean ± SEM. Bars with different letters are significantly different from each other (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2921113&req=5

Figure 8: EG-VEGF effects on HPEC permeability are mediated by PROKR2 and not PROKR1: Panels (A) and (B) show EG-VEGF (25 ng/ml) effect on the permeability of HPEC cells that had been silenced for PROKR1 and PROKR2 mRNA using siRNA (siRNA strategy), or treated with PROKR1 and R2 blocking antibodies (antibody strategy), respectively. In the two sets of experiments, EG-VEGF significantly increased HPEC permeability. Both siRNA to PROKR2 and its blocking antibody inhibited EG-VEGF effect. However, nor siRNA to PROKR1, neither its blocking antibody did affect the permeability. Data represent the mean ± SEM. Bars with different letters are significantly different from each other (p < 0.05).
Mentions: As for the sprouting experiments, we also sought to determine the type of receptor that mediates EG-VEGF effects on permeability. Both, siRNA and antibody strategies were used to differentiate between the two receptor types. As expected, EG-VEGF significantly increased HPEC permeability in the absence of any other treatment. Scramble siRNA, siRNA PROKR1, or siRNA PROKR2 alone did not affect basal HPEC permeability. However, invalidation of PROKR2 significantly abolished the response to EG-VEGF. Under PROKR1 mRNA invalidation EG-VEGF effect was maintained (Figure 8A). These results were substantiated with the antibody strategy, showing that only PROKR2 blockade affects EG-VEGF effects on HPEC permeability. Altogether these results demonstrate that EG-VEGF mediates its effect on the permeability via the activation of PROKR2 and not PROKR1 receptor (Figure 8B).

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