Limits...
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.

Show MeSH

Related in: MedlinePlus

Collapsin-1 inhibits the motility of RAEC. RAEC were isolated from rat aortas as described in Materials and Methods and were cultured in DME containing 10% FCS and FGF-2 (1 ng/ml). (A) Northern blot analysis of total RAEC RNA with 32P-labeled rat NRP1 cDNA as a probe. Arrow indicates the position of rat NRP1. (B) The RAEC were seeded in the upper wells of a Boyden chamber and increasing concentrations of collapsin-1 were added to the lower wells. After 4 h, the number of migrated cells per field was counted as in Fig. 2. Each data point represents the mean ± SD of four independent wells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199727&req=5

Figure 6: Collapsin-1 inhibits the motility of RAEC. RAEC were isolated from rat aortas as described in Materials and Methods and were cultured in DME containing 10% FCS and FGF-2 (1 ng/ml). (A) Northern blot analysis of total RAEC RNA with 32P-labeled rat NRP1 cDNA as a probe. Arrow indicates the position of rat NRP1. (B) The RAEC were seeded in the upper wells of a Boyden chamber and increasing concentrations of collapsin-1 were added to the lower wells. After 4 h, the number of migrated cells per field was counted as in Fig. 2. Each data point represents the mean ± SD of four independent wells.

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 inhibits the motility of RAEC. RAEC were isolated from rat aortas as described in Materials and Methods and were cultured in DME containing 10% FCS and FGF-2 (1 ng/ml). (A) Northern blot analysis of total RAEC RNA with 32P-labeled rat NRP1 cDNA as a probe. Arrow indicates the position of rat NRP1. (B) The RAEC were seeded in the upper wells of a Boyden chamber and increasing concentrations of collapsin-1 were added to the lower wells. After 4 h, the number of migrated cells per field was counted as in Fig. 2. Each data point represents the mean ± SD of four independent wells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199727&req=5

Figure 6: Collapsin-1 inhibits the motility of RAEC. RAEC were isolated from rat aortas as described in Materials and Methods and were cultured in DME containing 10% FCS and FGF-2 (1 ng/ml). (A) Northern blot analysis of total RAEC RNA with 32P-labeled rat NRP1 cDNA as a probe. Arrow indicates the position of rat NRP1. (B) The RAEC were seeded in the upper wells of a Boyden chamber and increasing concentrations of collapsin-1 were added to the lower wells. After 4 h, the number of migrated cells per field was counted as in Fig. 2. Each data point represents the mean ± SD of four independent wells.
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
Related in: MedlinePlus