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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 angiogenic effects are mediated by PROKR1 and not PROKR2. (A and B) EG-VEGF (25 ng/ml) effect on spheroid sprouting 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. (C and D) Quantifications of the number of sprouts in three independent experiments for both strategies. In the two sets of experiments, EG-VEGF significantly increased the number of sprouts. Both siRNA to PROKR1 and its blocking antibody inhibited EG-VEGF effect. However, nor siRNA to PROKR2, neither its blocking antibody did affect the spheroid sprouting. Data represent the mean ± SEM (***p < 0.001, **p < 0.01, ns, not significant). Bar, 100 μm.
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Figure 6: EG-VEGF angiogenic effects are mediated by PROKR1 and not PROKR2. (A and B) EG-VEGF (25 ng/ml) effect on spheroid sprouting 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. (C and D) Quantifications of the number of sprouts in three independent experiments for both strategies. In the two sets of experiments, EG-VEGF significantly increased the number of sprouts. Both siRNA to PROKR1 and its blocking antibody inhibited EG-VEGF effect. However, nor siRNA to PROKR2, neither its blocking antibody did affect the spheroid sprouting. Data represent the mean ± SEM (***p < 0.001, **p < 0.01, ns, not significant). Bar, 100 μm.

Mentions: In the aforementioned experiments, we have shown that HPEC express both type 1 and type 2 receptors for EG-VEGF (PROKR1 and PROKR2). To determine which type of receptor was involved in EG-VEGF effect on HPEC sprouting, we examined the effect of EG-VEGF on HPECs in which PROKR1 or PROKR2 mRNA expression was silenced by specific siRNAs, (see Figure S3 for siRNA strategy). In addition, the strategy of receptors blockade by specific neutralizing antibodies was also used (see Figure S3 for antibody strategy). Our results show that treatment with PROKR2 siRNA (Figure 6A) or antibody (Figure 6B) did not affect EG-VEGF stimulation of sprouting. However, PROKR1 siRNA or antibody reversed its effect. To measure the effect of EG-VEGF on HPEC sprouting, we quantified the number of sprouts formed under all conditions. The graphs in Figure 6, C and D, shows that EG-VEGF significantly increased the number of sprouts and that EG-VEGF effect was specifically reversed by the PROKR1 siRNA treatment and by PROKR1 antibody blockade.


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 angiogenic effects are mediated by PROKR1 and not PROKR2. (A and B) EG-VEGF (25 ng/ml) effect on spheroid sprouting 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. (C and D) Quantifications of the number of sprouts in three independent experiments for both strategies. In the two sets of experiments, EG-VEGF significantly increased the number of sprouts. Both siRNA to PROKR1 and its blocking antibody inhibited EG-VEGF effect. However, nor siRNA to PROKR2, neither its blocking antibody did affect the spheroid sprouting. Data represent the mean ± SEM (***p < 0.001, **p < 0.01, ns, not significant). Bar, 100 μm.
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Related In: Results  -  Collection

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Figure 6: EG-VEGF angiogenic effects are mediated by PROKR1 and not PROKR2. (A and B) EG-VEGF (25 ng/ml) effect on spheroid sprouting 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. (C and D) Quantifications of the number of sprouts in three independent experiments for both strategies. In the two sets of experiments, EG-VEGF significantly increased the number of sprouts. Both siRNA to PROKR1 and its blocking antibody inhibited EG-VEGF effect. However, nor siRNA to PROKR2, neither its blocking antibody did affect the spheroid sprouting. Data represent the mean ± SEM (***p < 0.001, **p < 0.01, ns, not significant). Bar, 100 μm.
Mentions: In the aforementioned experiments, we have shown that HPEC express both type 1 and type 2 receptors for EG-VEGF (PROKR1 and PROKR2). To determine which type of receptor was involved in EG-VEGF effect on HPEC sprouting, we examined the effect of EG-VEGF on HPECs in which PROKR1 or PROKR2 mRNA expression was silenced by specific siRNAs, (see Figure S3 for siRNA strategy). In addition, the strategy of receptors blockade by specific neutralizing antibodies was also used (see Figure S3 for antibody strategy). Our results show that treatment with PROKR2 siRNA (Figure 6A) or antibody (Figure 6B) did not affect EG-VEGF stimulation of sprouting. However, PROKR1 siRNA or antibody reversed its effect. To measure the effect of EG-VEGF on HPEC sprouting, we quantified the number of sprouts formed under all conditions. The graphs in Figure 6, C and D, shows that EG-VEGF significantly increased the number of sprouts and that EG-VEGF effect was specifically reversed by the PROKR1 siRNA treatment and by PROKR1 antibody blockade.

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