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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 alters RAEC morphology. RAEC were seeded in 8-well culture slides precoated with fibronectin. The next day, medium was replaced with fresh medium (A, D, and G), medium containing 300 ng/ml collapsin-1 (B, E, and H), and medium containing 300 ng/ml collapsin-1 preheated at 70°C for 30 min (C, F, and I). After 30 min of incubation with or without collapsin-1, cells were fixed. (A–C) Analysis by DIC optic microscopy. (D–F) Staining with phalloidin-FITC and analysis by fluorescence microscopy. (G–I) The cells in D–F were stained for DNA with DAPI. The cells in A–C are not the same as those in D–F, because different fixation methods were used.
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Figure 7: Collapsin-1 alters RAEC morphology. RAEC were seeded in 8-well culture slides precoated with fibronectin. The next day, medium was replaced with fresh medium (A, D, and G), medium containing 300 ng/ml collapsin-1 (B, E, and H), and medium containing 300 ng/ml collapsin-1 preheated at 70°C for 30 min (C, F, and I). After 30 min of incubation with or without collapsin-1, cells were fixed. (A–C) Analysis by DIC optic microscopy. (D–F) Staining with phalloidin-FITC and analysis by fluorescence microscopy. (G–I) The cells in D–F were stained for DNA with DAPI. The cells in A–C are not the same as those in D–F, because different fixation methods were used.

Mentions: Maintenance and physiological reorganization of the cytoskeleton play a crucial role in cell motility in response to mechanical and humoral stimuli (Hall 1998; Mackay and Hall 1998; Sheetz et al. 1998). RAEC sprouting from aortic rings were cultured. These cells formed a typical EC cobblestone monolayer at confluence (not shown), expressed NRP1 as demonstrated by Northern blot analysis (Fig. 6 A), and their motility was inhibited by collapsin-1 in a dose-dependent manner with an ID50 of ∼10 ng/ml and a maximal inhibition of 60–65% at 30 ng/ml (Fig. 6 B). However, in the presence of anti-NRP1 antibody, the inhibitory effects of 150 ng/ml collapsin-1 were reduced to ∼18% (not shown), consistent with the inhibitory effects of these antibodies on collapsin-1 inhibition of PAEC/NRP1 and PAEC/KDR/NRP1 motility that was shown in Fig. 2 C. To clarify possible mechanisms of collapsin-1 inhibition of EC, RAEC were seeded on fibronectin-coated glass chamber slides, grown for one day, treated with collapsin-1 (300 ng/ml) for 30 min, and analyzed by DIC optic microscopy (Fig. 7, A–C) and phalloidin-FITC staining (Fig. 7, D–F). RAEC typically exhibit numerous active lamellipodia, as characterized by membrane ruffling (Fig. 7A and Fig. D). However, in the presence of collapsin-1, there was a significant retraction of the lamellipodia, as shown in Fig. 7B and Fig. E, that occurred in 30–50% of the RAEC population. Time-lapse video microscopy showed that these alterations in lamellipodia structures began ∼10 min after exposure to collapsin-1 (not shown). Within a given responsive cell, almost of all the lamellipodia were retracted. The cell membranes became thinner, ruffling was undetectable, and cell surface blebs were observed. Phalloidin-FITC staining showed that collapsin-1 treatment resulted in the loss of polymerized actin fibers (Fig. 7E compared with D). In these cells, ∼70–80% of the F-actin was depolymerized. It appeared that depolymerized actin was clustered in the retracted lamellipodia. The collapsin-1 effects on RAEC morphology were abrogated when collapsin-1 was heat-inactivated by 70°C treatment for 30 min (Fig. 7C and Fig. F). DAPI staining of nuclei showed no DNA breakage, indicating that collapsin-1 did not induce apoptosis in the RAEC (Fig. 7, G–I).


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 alters RAEC morphology. RAEC were seeded in 8-well culture slides precoated with fibronectin. The next day, medium was replaced with fresh medium (A, D, and G), medium containing 300 ng/ml collapsin-1 (B, E, and H), and medium containing 300 ng/ml collapsin-1 preheated at 70°C for 30 min (C, F, and I). After 30 min of incubation with or without collapsin-1, cells were fixed. (A–C) Analysis by DIC optic microscopy. (D–F) Staining with phalloidin-FITC and analysis by fluorescence microscopy. (G–I) The cells in D–F were stained for DNA with DAPI. The cells in A–C are not the same as those in D–F, because different fixation methods were used.
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Related In: Results  -  Collection

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Figure 7: Collapsin-1 alters RAEC morphology. RAEC were seeded in 8-well culture slides precoated with fibronectin. The next day, medium was replaced with fresh medium (A, D, and G), medium containing 300 ng/ml collapsin-1 (B, E, and H), and medium containing 300 ng/ml collapsin-1 preheated at 70°C for 30 min (C, F, and I). After 30 min of incubation with or without collapsin-1, cells were fixed. (A–C) Analysis by DIC optic microscopy. (D–F) Staining with phalloidin-FITC and analysis by fluorescence microscopy. (G–I) The cells in D–F were stained for DNA with DAPI. The cells in A–C are not the same as those in D–F, because different fixation methods were used.
Mentions: Maintenance and physiological reorganization of the cytoskeleton play a crucial role in cell motility in response to mechanical and humoral stimuli (Hall 1998; Mackay and Hall 1998; Sheetz et al. 1998). RAEC sprouting from aortic rings were cultured. These cells formed a typical EC cobblestone monolayer at confluence (not shown), expressed NRP1 as demonstrated by Northern blot analysis (Fig. 6 A), and their motility was inhibited by collapsin-1 in a dose-dependent manner with an ID50 of ∼10 ng/ml and a maximal inhibition of 60–65% at 30 ng/ml (Fig. 6 B). However, in the presence of anti-NRP1 antibody, the inhibitory effects of 150 ng/ml collapsin-1 were reduced to ∼18% (not shown), consistent with the inhibitory effects of these antibodies on collapsin-1 inhibition of PAEC/NRP1 and PAEC/KDR/NRP1 motility that was shown in Fig. 2 C. To clarify possible mechanisms of collapsin-1 inhibition of EC, RAEC were seeded on fibronectin-coated glass chamber slides, grown for one day, treated with collapsin-1 (300 ng/ml) for 30 min, and analyzed by DIC optic microscopy (Fig. 7, A–C) and phalloidin-FITC staining (Fig. 7, D–F). RAEC typically exhibit numerous active lamellipodia, as characterized by membrane ruffling (Fig. 7A and Fig. D). However, in the presence of collapsin-1, there was a significant retraction of the lamellipodia, as shown in Fig. 7B and Fig. E, that occurred in 30–50% of the RAEC population. Time-lapse video microscopy showed that these alterations in lamellipodia structures began ∼10 min after exposure to collapsin-1 (not shown). Within a given responsive cell, almost of all the lamellipodia were retracted. The cell membranes became thinner, ruffling was undetectable, and cell surface blebs were observed. Phalloidin-FITC staining showed that collapsin-1 treatment resulted in the loss of polymerized actin fibers (Fig. 7E compared with D). In these cells, ∼70–80% of the F-actin was depolymerized. It appeared that depolymerized actin was clustered in the retracted lamellipodia. The collapsin-1 effects on RAEC morphology were abrogated when collapsin-1 was heat-inactivated by 70°C treatment for 30 min (Fig. 7C and Fig. F). DAPI staining of nuclei showed no DNA breakage, indicating that collapsin-1 did not induce apoptosis in the RAEC (Fig. 7, G–I).

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