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Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165.

Miao HQ, Soker S, Feiner L, Alonso JL, Raper JA, Klagsbrun M - J. Cell Biol. (1999)

Bottom Line: To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined.Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner.These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical Research, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Neuropilin-1 (NRP1) is a receptor for two unrelated ligands with disparate activities, vascular endothelial growth factor-165 (VEGF165), an angiogenesis factor, and semaphorin/collapsins, mediators of neuronal guidance. To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined. Collapsin-1 inhibited the motility of porcine aortic EC (PAEC) expressing NRP1 alone; coexpressing KDR and NRP1 (PAEC/KDR/NRP1), but not parental PAEC; or PAEC expressing KDR alone. The motility of PAEC expressing NRP1 was inhibited by 65-75% and this inhibition was abrogated by anti-NRP1 antibody. In contrast, VEGF165 stimulated the motility of PAEC/KDR/NRP1. When VEGF165 and collapsin-1 were added simultaneously to PAEC/KDR/NRP1, dorsal root ganglia (DRG), and COS-7/NRP1 cells, they competed with each other in EC motility, DRG collapse, and NRP1-binding assays, respectively, suggesting that the two ligands have overlapping NRP1 binding sites. Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner. In an in vitro angiogenesis assay, collapsin-1 inhibited the capillary sprouting of EC from rat aortic ring segments. These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.

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Collapsin-1 inhibits the motility of EC expressing NRP1. (A) PAEC, PAEC/NRP1, PAEC/KDR, and PAEC/KDR/NRP1 were seeded in the upper wells of a Boyden chamber. Collapsin-1 (150 ng/ml) was added (gray bars) or not added (white bars) to the lower wells. (B) PAEC/NRP1 and PAEC/KDR/NRP1 were seeded in the upper wells and increasing concentrations of collapsin-1 were added to the lower wells of the Boyden chamber. (C) PAEC/NRP1 and PAEC/KDR/NRP1 were cultured in complete medium and seeded in the upper wells of a Boyden chamber. For each cell type, either 0 (lanes 1 and 2) or 150 ng/ml (lanes 3 and 4) of collapsin-1 was added in the absence (hatched bars) or presence (solid bars) of 30 μg/ml anti-NRP1 antibodies. For each set of experiments, after a 4-h incubation, the numbers of cells that had migrated through the filter in each field were counted. Each data point represents the mean ± SD of four independent wells.
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Figure 2: Collapsin-1 inhibits the motility of EC expressing NRP1. (A) PAEC, PAEC/NRP1, PAEC/KDR, and PAEC/KDR/NRP1 were seeded in the upper wells of a Boyden chamber. Collapsin-1 (150 ng/ml) was added (gray bars) or not added (white bars) to the lower wells. (B) PAEC/NRP1 and PAEC/KDR/NRP1 were seeded in the upper wells and increasing concentrations of collapsin-1 were added to the lower wells of the Boyden chamber. (C) PAEC/NRP1 and PAEC/KDR/NRP1 were cultured in complete medium and seeded in the upper wells of a Boyden chamber. For each cell type, either 0 (lanes 1 and 2) or 150 ng/ml (lanes 3 and 4) of collapsin-1 was added in the absence (hatched bars) or presence (solid bars) of 30 μg/ml anti-NRP1 antibodies. For each set of experiments, after a 4-h incubation, the numbers of cells that had migrated through the filter in each field were counted. Each data point represents the mean ± SD of four independent wells.

Mentions: Collapsin/semaphorins are inhibitors of axonal motility acting via NRP1 (Kolodkin et al. 1997). Accordingly, the ability of collapsin-1 to affect the motility of EC expressing NRP1 was tested in a Boyden chamber assay (Fig. 2). Collapsin-1 (150 ng/ml) inhibited the motility of PAEC/NRP1 and PAEC/KDR/NRP1 by ∼65–70%, but did not inhibit the motility of PAEC or PAEC/KDR at all (Fig. 2 A). The specific inhibition of only those EC expressing NRP1 was consistent with the binding data. Collapsin-1 inhibition of PAEC/NRP1 and PAEC/KDR/NRP1 motility was dose-dependent with an ID50 (inhibitory dose) of ∼3 ng/ml (Fig. 2 B). A maximal inhibition of ∼65–75% occurred with ∼15 ng/ml. To test whether the collapsin-1–mediated inhibition of motility was due to repulsion, collapsin-1 was placed in both upper and lower chambers. The same results were found as with the presence of collapsin-1 in the lower chamber alone (not shown), suggesting that collapsin-1 is not repelling the cells in this particular motility assay.


Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165.

Miao HQ, Soker S, Feiner L, Alonso JL, Raper JA, Klagsbrun M - J. Cell Biol. (1999)

Collapsin-1 inhibits the motility of EC expressing NRP1. (A) PAEC, PAEC/NRP1, PAEC/KDR, and PAEC/KDR/NRP1 were seeded in the upper wells of a Boyden chamber. Collapsin-1 (150 ng/ml) was added (gray bars) or not added (white bars) to the lower wells. (B) PAEC/NRP1 and PAEC/KDR/NRP1 were seeded in the upper wells and increasing concentrations of collapsin-1 were added to the lower wells of the Boyden chamber. (C) PAEC/NRP1 and PAEC/KDR/NRP1 were cultured in complete medium and seeded in the upper wells of a Boyden chamber. For each cell type, either 0 (lanes 1 and 2) or 150 ng/ml (lanes 3 and 4) of collapsin-1 was added in the absence (hatched bars) or presence (solid bars) of 30 μg/ml anti-NRP1 antibodies. For each set of experiments, after a 4-h incubation, the numbers of cells that had migrated through the filter in each field were counted. Each data point represents the mean ± SD of four independent wells.
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Related In: Results  -  Collection

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Figure 2: Collapsin-1 inhibits the motility of EC expressing NRP1. (A) PAEC, PAEC/NRP1, PAEC/KDR, and PAEC/KDR/NRP1 were seeded in the upper wells of a Boyden chamber. Collapsin-1 (150 ng/ml) was added (gray bars) or not added (white bars) to the lower wells. (B) PAEC/NRP1 and PAEC/KDR/NRP1 were seeded in the upper wells and increasing concentrations of collapsin-1 were added to the lower wells of the Boyden chamber. (C) PAEC/NRP1 and PAEC/KDR/NRP1 were cultured in complete medium and seeded in the upper wells of a Boyden chamber. For each cell type, either 0 (lanes 1 and 2) or 150 ng/ml (lanes 3 and 4) of collapsin-1 was added in the absence (hatched bars) or presence (solid bars) of 30 μg/ml anti-NRP1 antibodies. For each set of experiments, after a 4-h incubation, the numbers of cells that had migrated through the filter in each field were counted. Each data point represents the mean ± SD of four independent wells.
Mentions: Collapsin/semaphorins are inhibitors of axonal motility acting via NRP1 (Kolodkin et al. 1997). Accordingly, the ability of collapsin-1 to affect the motility of EC expressing NRP1 was tested in a Boyden chamber assay (Fig. 2). Collapsin-1 (150 ng/ml) inhibited the motility of PAEC/NRP1 and PAEC/KDR/NRP1 by ∼65–70%, but did not inhibit the motility of PAEC or PAEC/KDR at all (Fig. 2 A). The specific inhibition of only those EC expressing NRP1 was consistent with the binding data. Collapsin-1 inhibition of PAEC/NRP1 and PAEC/KDR/NRP1 motility was dose-dependent with an ID50 (inhibitory dose) of ∼3 ng/ml (Fig. 2 B). A maximal inhibition of ∼65–75% occurred with ∼15 ng/ml. To test whether the collapsin-1–mediated inhibition of motility was due to repulsion, collapsin-1 was placed in both upper and lower chambers. The same results were found as with the presence of collapsin-1 in the lower chamber alone (not shown), suggesting that collapsin-1 is not repelling the cells in this particular motility assay.

Bottom Line: To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined.Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner.These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical Research, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Neuropilin-1 (NRP1) is a receptor for two unrelated ligands with disparate activities, vascular endothelial growth factor-165 (VEGF165), an angiogenesis factor, and semaphorin/collapsins, mediators of neuronal guidance. To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined. Collapsin-1 inhibited the motility of porcine aortic EC (PAEC) expressing NRP1 alone; coexpressing KDR and NRP1 (PAEC/KDR/NRP1), but not parental PAEC; or PAEC expressing KDR alone. The motility of PAEC expressing NRP1 was inhibited by 65-75% and this inhibition was abrogated by anti-NRP1 antibody. In contrast, VEGF165 stimulated the motility of PAEC/KDR/NRP1. When VEGF165 and collapsin-1 were added simultaneously to PAEC/KDR/NRP1, dorsal root ganglia (DRG), and COS-7/NRP1 cells, they competed with each other in EC motility, DRG collapse, and NRP1-binding assays, respectively, suggesting that the two ligands have overlapping NRP1 binding sites. Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner. In an in vitro angiogenesis assay, collapsin-1 inhibited the capillary sprouting of EC from rat aortic ring segments. These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.

Show MeSH
Related in: MedlinePlus