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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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Effect of collapsin-1 on microvessel outgrowth in vitro. Rat aortic rings were embedded in type I collagen gels. Serum-free endothelial growth medium was added and replaced every other day with fresh medium. Microvessel structure was observed by phase microscopy on day 14 at low (A, C, and E) or high (B, D, and F) magnification. A and B, no addition; C and D, collapsin-1 (300 ng/ml) was added on day two and every second day thereafter; E and F, collapsin-1 was added on day two and the medium was replaced on day four without further addition of collapsin-1.
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Figure 4: Effect of collapsin-1 on microvessel outgrowth in vitro. Rat aortic rings were embedded in type I collagen gels. Serum-free endothelial growth medium was added and replaced every other day with fresh medium. Microvessel structure was observed by phase microscopy on day 14 at low (A, C, and E) or high (B, D, and F) magnification. A and B, no addition; C and D, collapsin-1 (300 ng/ml) was added on day two and every second day thereafter; E and F, collapsin-1 was added on day two and the medium was replaced on day four without further addition of collapsin-1.

Mentions: The effects of collapsin-1 were tested on EC sprouting and microvessel formation in rat aortic ring segments that were embedded in collagen gels and maintained in a serum-free medium that favored microvessel outgrowth (Fig. 4). As observed by phase-contrast microscopy, branching microvessels formed a capillary network of tubes and loops with lumen-like structures at the periphery of the aortic rings, starting on day four and reaching a maximal degree of sprouting on days 12–15 (Fig. 4A and Fig. B). The identity of the EC in the sprouts was confirmed by immunohistochemical detection of von Willebrand factor and by morphological analysis using EM (not shown). When collapsin-1 (300 ng/ml) was added on day two after embedding of the aortic rings and thereafter every second day, the outgrowth of sprouts was strongly inhibited (Fig. 4C and Fig. D). A quantitative analysis demonstrated that collapsin-1 inhibited microvessel EC outgrowth by 80–90% (Fig. 5). When collapsin-1 was added into the culture medium on day two and washed away on day four, normal outgrowth of microvessels was observed on day 12 (Fig. 4E and Fig. F). The reversibility of the microvessel outgrowth demonstrated that collapsin-1 inhibition of sprouting was not due to cell toxicity.


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)

Effect of collapsin-1 on microvessel outgrowth in vitro. Rat aortic rings were embedded in type I collagen gels. Serum-free endothelial growth medium was added and replaced every other day with fresh medium. Microvessel structure was observed by phase microscopy on day 14 at low (A, C, and E) or high (B, D, and F) magnification. A and B, no addition; C and D, collapsin-1 (300 ng/ml) was added on day two and every second day thereafter; E and F, collapsin-1 was added on day two and the medium was replaced on day four without further addition of collapsin-1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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Figure 4: Effect of collapsin-1 on microvessel outgrowth in vitro. Rat aortic rings were embedded in type I collagen gels. Serum-free endothelial growth medium was added and replaced every other day with fresh medium. Microvessel structure was observed by phase microscopy on day 14 at low (A, C, and E) or high (B, D, and F) magnification. A and B, no addition; C and D, collapsin-1 (300 ng/ml) was added on day two and every second day thereafter; E and F, collapsin-1 was added on day two and the medium was replaced on day four without further addition of collapsin-1.
Mentions: The effects of collapsin-1 were tested on EC sprouting and microvessel formation in rat aortic ring segments that were embedded in collagen gels and maintained in a serum-free medium that favored microvessel outgrowth (Fig. 4). As observed by phase-contrast microscopy, branching microvessels formed a capillary network of tubes and loops with lumen-like structures at the periphery of the aortic rings, starting on day four and reaching a maximal degree of sprouting on days 12–15 (Fig. 4A and Fig. B). The identity of the EC in the sprouts was confirmed by immunohistochemical detection of von Willebrand factor and by morphological analysis using EM (not shown). When collapsin-1 (300 ng/ml) was added on day two after embedding of the aortic rings and thereafter every second day, the outgrowth of sprouts was strongly inhibited (Fig. 4C and Fig. D). A quantitative analysis demonstrated that collapsin-1 inhibited microvessel EC outgrowth by 80–90% (Fig. 5). When collapsin-1 was added into the culture medium on day two and washed away on day four, normal outgrowth of microvessels was observed on day 12 (Fig. 4E and Fig. F). The reversibility of the microvessel outgrowth demonstrated that collapsin-1 inhibition of sprouting was not due to cell toxicity.

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