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Alk1 and Alk5 inhibition by Nrp1 controls vascular sprouting downstream of Notch.

Aspalter IM, Gordon E, Dubrac A, Ragab A, Narloch J, Vizán P, Geudens I, Collins RT, Franco CA, Abrahams CL, Thurston G, Fruttiger M, Rosewell I, Eichmann A, Gerhardt H - Nat Commun (2015)

Bottom Line: We show this is a key endothelial function of neuropilin-1 (Nrp1), which suppresses the stalk-cell phenotype by limiting Smad2/3 activation through Alk1 and Alk5.Notch downregulates Nrp1, thus relieving the inhibition of Alk1 and Alk5, thereby driving stalk-cell behaviour.Conceptually, our work shows that the heterogeneity between neighbouring endothelial cells established by the lateral feedback loop of Dll4/Notch utilizes Nrp1 levels as the pivot, which in turn establishes differential responsiveness to TGF-β/BMP signalling.

View Article: PubMed Central - PubMed

Affiliation: Vascular Biology Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.

ABSTRACT
Sprouting angiogenesis drives blood vessel growth in healthy and diseased tissues. Vegf and Dll4/Notch signalling cooperate in a negative feedback loop that specifies endothelial tip and stalk cells to ensure adequate vessel branching and function. Current concepts posit that endothelial cells default to the tip-cell phenotype when Notch is inactive. Here we identify instead that the stalk-cell phenotype needs to be actively repressed to allow tip-cell formation. We show this is a key endothelial function of neuropilin-1 (Nrp1), which suppresses the stalk-cell phenotype by limiting Smad2/3 activation through Alk1 and Alk5. Notch downregulates Nrp1, thus relieving the inhibition of Alk1 and Alk5, thereby driving stalk-cell behaviour. Conceptually, our work shows that the heterogeneity between neighbouring endothelial cells established by the lateral feedback loop of Dll4/Notch utilizes Nrp1 levels as the pivot, which in turn establishes differential responsiveness to TGF-β/BMP signalling.

No MeSH data available.


Nrp1 deficiency overrules Vegfr2 deficiency during tip cell competition.(a,b,d,e) Representative confocal images of the sprouting vasculature of a chimeric EB composed of Vegfr2GFP/+ cells and Nrp1Lacz/+ cells. Untreated (a,b) or treated with 5 μM DAPT for 5 days until harvesting at day 10 (d,e). (a,d) Tip cells derived from Vegfr2GFP/+ cells are indicated by green arrowheads and Nrp1Lacz/+ tip cells by blue arrowheads; scale bar, 130 μm. Magnifications of individual sprouts; scale bar, 36 μm (b,e). (c,f) Quantification of tip cells from Vegfr2GFP/+:Nrp1LacZ/+ chimeric EBs with equal input levels, either untreated (c) or treated with 5 μM DAPT (f). n=number of EBs analysed; number of counted tips: 458; n=7 (c) and 940; n=5 (f). P values were calculated using a Student's unpaired t-test by comparing quantified contribution with initial percentage of input levels; P<0.0001 (c,f). Values represent mean±s.e.m.
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f4: Nrp1 deficiency overrules Vegfr2 deficiency during tip cell competition.(a,b,d,e) Representative confocal images of the sprouting vasculature of a chimeric EB composed of Vegfr2GFP/+ cells and Nrp1Lacz/+ cells. Untreated (a,b) or treated with 5 μM DAPT for 5 days until harvesting at day 10 (d,e). (a,d) Tip cells derived from Vegfr2GFP/+ cells are indicated by green arrowheads and Nrp1Lacz/+ tip cells by blue arrowheads; scale bar, 130 μm. Magnifications of individual sprouts; scale bar, 36 μm (b,e). (c,f) Quantification of tip cells from Vegfr2GFP/+:Nrp1LacZ/+ chimeric EBs with equal input levels, either untreated (c) or treated with 5 μM DAPT (f). n=number of EBs analysed; number of counted tips: 458; n=7 (c) and 940; n=5 (f). P values were calculated using a Student's unpaired t-test by comparing quantified contribution with initial percentage of input levels; P<0.0001 (c,f). Values represent mean±s.e.m.

Mentions: To directly determine whether Nrp1/Vegfr2 co-receptor activity is functionally involved in tip cell competition, we generated chimeric EBs mixing Vegfr2 (ref. 7) and Nrp1 heterozygous cells. If Vegfr2 co-receptor function accounted for tip cell formation, we expected equal handicap of Vegfr2+/− and Nrp1+/− cells to reach the tip. Surprisingly, we found that Nrp1 heterozygous cells are not able to acquire the tip position even when competing against Vegfr2 heterozygous cells (Fig. 4a–c). Again Notch inhibition was ineffective in restoring the balance at the tip (Fig. 4d–f). Thus a 50% gene dose reduction in Nrp1 impedes tip cell formation even when cells have twice the Vegfr2 level than their competing neighbours and when Notch is inactive. We conclude that unlike Vegfr2, Nrp1 does not operate upstream of Dll4/Notch and that Vegfr2 co-receptor function is unlikely to contribute to Nrp1 signalling events that regulate tip cell competition.


Alk1 and Alk5 inhibition by Nrp1 controls vascular sprouting downstream of Notch.

Aspalter IM, Gordon E, Dubrac A, Ragab A, Narloch J, Vizán P, Geudens I, Collins RT, Franco CA, Abrahams CL, Thurston G, Fruttiger M, Rosewell I, Eichmann A, Gerhardt H - Nat Commun (2015)

Nrp1 deficiency overrules Vegfr2 deficiency during tip cell competition.(a,b,d,e) Representative confocal images of the sprouting vasculature of a chimeric EB composed of Vegfr2GFP/+ cells and Nrp1Lacz/+ cells. Untreated (a,b) or treated with 5 μM DAPT for 5 days until harvesting at day 10 (d,e). (a,d) Tip cells derived from Vegfr2GFP/+ cells are indicated by green arrowheads and Nrp1Lacz/+ tip cells by blue arrowheads; scale bar, 130 μm. Magnifications of individual sprouts; scale bar, 36 μm (b,e). (c,f) Quantification of tip cells from Vegfr2GFP/+:Nrp1LacZ/+ chimeric EBs with equal input levels, either untreated (c) or treated with 5 μM DAPT (f). n=number of EBs analysed; number of counted tips: 458; n=7 (c) and 940; n=5 (f). P values were calculated using a Student's unpaired t-test by comparing quantified contribution with initial percentage of input levels; P<0.0001 (c,f). Values represent mean±s.e.m.
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Related In: Results  -  Collection

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f4: Nrp1 deficiency overrules Vegfr2 deficiency during tip cell competition.(a,b,d,e) Representative confocal images of the sprouting vasculature of a chimeric EB composed of Vegfr2GFP/+ cells and Nrp1Lacz/+ cells. Untreated (a,b) or treated with 5 μM DAPT for 5 days until harvesting at day 10 (d,e). (a,d) Tip cells derived from Vegfr2GFP/+ cells are indicated by green arrowheads and Nrp1Lacz/+ tip cells by blue arrowheads; scale bar, 130 μm. Magnifications of individual sprouts; scale bar, 36 μm (b,e). (c,f) Quantification of tip cells from Vegfr2GFP/+:Nrp1LacZ/+ chimeric EBs with equal input levels, either untreated (c) or treated with 5 μM DAPT (f). n=number of EBs analysed; number of counted tips: 458; n=7 (c) and 940; n=5 (f). P values were calculated using a Student's unpaired t-test by comparing quantified contribution with initial percentage of input levels; P<0.0001 (c,f). Values represent mean±s.e.m.
Mentions: To directly determine whether Nrp1/Vegfr2 co-receptor activity is functionally involved in tip cell competition, we generated chimeric EBs mixing Vegfr2 (ref. 7) and Nrp1 heterozygous cells. If Vegfr2 co-receptor function accounted for tip cell formation, we expected equal handicap of Vegfr2+/− and Nrp1+/− cells to reach the tip. Surprisingly, we found that Nrp1 heterozygous cells are not able to acquire the tip position even when competing against Vegfr2 heterozygous cells (Fig. 4a–c). Again Notch inhibition was ineffective in restoring the balance at the tip (Fig. 4d–f). Thus a 50% gene dose reduction in Nrp1 impedes tip cell formation even when cells have twice the Vegfr2 level than their competing neighbours and when Notch is inactive. We conclude that unlike Vegfr2, Nrp1 does not operate upstream of Dll4/Notch and that Vegfr2 co-receptor function is unlikely to contribute to Nrp1 signalling events that regulate tip cell competition.

Bottom Line: We show this is a key endothelial function of neuropilin-1 (Nrp1), which suppresses the stalk-cell phenotype by limiting Smad2/3 activation through Alk1 and Alk5.Notch downregulates Nrp1, thus relieving the inhibition of Alk1 and Alk5, thereby driving stalk-cell behaviour.Conceptually, our work shows that the heterogeneity between neighbouring endothelial cells established by the lateral feedback loop of Dll4/Notch utilizes Nrp1 levels as the pivot, which in turn establishes differential responsiveness to TGF-β/BMP signalling.

View Article: PubMed Central - PubMed

Affiliation: Vascular Biology Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.

ABSTRACT
Sprouting angiogenesis drives blood vessel growth in healthy and diseased tissues. Vegf and Dll4/Notch signalling cooperate in a negative feedback loop that specifies endothelial tip and stalk cells to ensure adequate vessel branching and function. Current concepts posit that endothelial cells default to the tip-cell phenotype when Notch is inactive. Here we identify instead that the stalk-cell phenotype needs to be actively repressed to allow tip-cell formation. We show this is a key endothelial function of neuropilin-1 (Nrp1), which suppresses the stalk-cell phenotype by limiting Smad2/3 activation through Alk1 and Alk5. Notch downregulates Nrp1, thus relieving the inhibition of Alk1 and Alk5, thereby driving stalk-cell behaviour. Conceptually, our work shows that the heterogeneity between neighbouring endothelial cells established by the lateral feedback loop of Dll4/Notch utilizes Nrp1 levels as the pivot, which in turn establishes differential responsiveness to TGF-β/BMP signalling.

No MeSH data available.