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Suppression of β3-integrin in mice triggers a neuropilin-1-dependent change in focal adhesion remodelling that can be targeted to block pathological angiogenesis.

Ellison TS, Atkinson SJ, Steri V, Kirkup BM, Preedy ME, Johnson RT, Ruhrberg C, Edwards DR, Schneider JG, Weilbaecher K, Robinson SD - Dis Model Mech (2015)

Bottom Line: Anti-angiogenic treatments against αvβ3-integrin fail to block tumour growth in the long term, which suggests that the tumour vasculature escapes from angiogenesis inhibition through αvβ3-integrin-independent mechanisms.The simultaneous genetic targeting of both molecules significantly impairs paxillin-1 activation and focal adhesion remodelling in endothelial cells, and therefore inhibits tumour angiogenesis and the growth of already established tumours.These findings provide a firm foundation for testing drugs against these molecules in combination to treat patients with advanced cancers.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

No MeSH data available.


Related in: MedlinePlus

NRP1-dependent functions of VEGFR-2 are normal in β3-integrin-heterozygous endothelial cells. (A) Lung microvascular endothelial cells (ECs) were isolated and immortalised (polyoma-middle-T-antigen) from β3-WT and β3-HET mice that were expressing either normal (WT NRP1) or cytoplasmic-tail-deleted (NRP1 Δcyto) NRP1. Multiple EC lysates of each genotype were western blotted (WB) to examine total cellular levels of VEGFR2 and β3-integrin. Bar charts of densitometric analysis of mean (+s.e.m.) changes between the four genotypes are shown to the right. Asterisks indicate statistical significance: *P<0.05. (B-E) Representative of ≥three independent experiments per blotted protein. ECs were seeded overnight on a complex matrix containing gelatin, collagen, fibronectin and vitronectin, and were then stimulated with VEGF over the indicated time courses. (B) ECs were lysed and blotted for levels of phosphorylated (phospho) and total VEGFR2, NRP1, and phospho (pERK) and total (tERK) ERK1/2. (C) To examine protein degradation, the VEGF time course was extended and EC lysates were blotted for levels of total VEGFR2, NRP1 and β3-integrin. (D) Following a VEGF time course, ECs were trypsinised and analysed by flow cytometry for surface levels of VEGFR2. Median fluorescence intensity was measured after forward versus side scatter data were tightly gated around, and normalised to, an isotype control. The graph shows the relative surface level of VEGFR2 (means±s.e.m.) relative to the 0 (non-stimulated) time point. (E) ECs were stimulated with VEGF for the indicated amounts of time and then lysed and immunoprecipitated for VEGFR2 (VEGFR2 IP), before being blotted for NRP1 association. A total cell lysate (TCL) is shown for comparison. (A-E) HSC-70 served as a loading control. Data are representative of three independent experiments.
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DMM019927F2: NRP1-dependent functions of VEGFR-2 are normal in β3-integrin-heterozygous endothelial cells. (A) Lung microvascular endothelial cells (ECs) were isolated and immortalised (polyoma-middle-T-antigen) from β3-WT and β3-HET mice that were expressing either normal (WT NRP1) or cytoplasmic-tail-deleted (NRP1 Δcyto) NRP1. Multiple EC lysates of each genotype were western blotted (WB) to examine total cellular levels of VEGFR2 and β3-integrin. Bar charts of densitometric analysis of mean (+s.e.m.) changes between the four genotypes are shown to the right. Asterisks indicate statistical significance: *P<0.05. (B-E) Representative of ≥three independent experiments per blotted protein. ECs were seeded overnight on a complex matrix containing gelatin, collagen, fibronectin and vitronectin, and were then stimulated with VEGF over the indicated time courses. (B) ECs were lysed and blotted for levels of phosphorylated (phospho) and total VEGFR2, NRP1, and phospho (pERK) and total (tERK) ERK1/2. (C) To examine protein degradation, the VEGF time course was extended and EC lysates were blotted for levels of total VEGFR2, NRP1 and β3-integrin. (D) Following a VEGF time course, ECs were trypsinised and analysed by flow cytometry for surface levels of VEGFR2. Median fluorescence intensity was measured after forward versus side scatter data were tightly gated around, and normalised to, an isotype control. The graph shows the relative surface level of VEGFR2 (means±s.e.m.) relative to the 0 (non-stimulated) time point. (E) ECs were stimulated with VEGF for the indicated amounts of time and then lysed and immunoprecipitated for VEGFR2 (VEGFR2 IP), before being blotted for NRP1 association. A total cell lysate (TCL) is shown for comparison. (A-E) HSC-70 served as a loading control. Data are representative of three independent experiments.

Mentions: Given that NRP1's cytoplasmic tail is important for the regulation of VEGFR2 signalling and trafficking (Ballmer-Hofer et al., 2011; Herzog et al., 2011), we wanted to investigate whether the NRP1Δcyto mutation differentially affects VEGFR2 function in β3-WT versus β3-HET lung microvascular ECs. We employed polyoma-middle-T-antigen immortalised ECs, isolated from the mutant mice described above, because we and others have shown that they present good models to study angiogenesis (Ni et al., 2014; Robinson et al., 2009; Steri et al., 2014; Tavora et al., 2014). We first compared total VEGFR2 levels in the four genotypes (β3-WT, β3-HET, β3-WT;NRP1Δcyto and β3-HET;NRP1Δcyto). Unlike β3-KO ECs, we noted only a small trend of increased VEGFR2 levels in β3-HET and β3-HET;NRP1Δcyto ECs (Fig. 2A).Fig. 2.


Suppression of β3-integrin in mice triggers a neuropilin-1-dependent change in focal adhesion remodelling that can be targeted to block pathological angiogenesis.

Ellison TS, Atkinson SJ, Steri V, Kirkup BM, Preedy ME, Johnson RT, Ruhrberg C, Edwards DR, Schneider JG, Weilbaecher K, Robinson SD - Dis Model Mech (2015)

NRP1-dependent functions of VEGFR-2 are normal in β3-integrin-heterozygous endothelial cells. (A) Lung microvascular endothelial cells (ECs) were isolated and immortalised (polyoma-middle-T-antigen) from β3-WT and β3-HET mice that were expressing either normal (WT NRP1) or cytoplasmic-tail-deleted (NRP1 Δcyto) NRP1. Multiple EC lysates of each genotype were western blotted (WB) to examine total cellular levels of VEGFR2 and β3-integrin. Bar charts of densitometric analysis of mean (+s.e.m.) changes between the four genotypes are shown to the right. Asterisks indicate statistical significance: *P<0.05. (B-E) Representative of ≥three independent experiments per blotted protein. ECs were seeded overnight on a complex matrix containing gelatin, collagen, fibronectin and vitronectin, and were then stimulated with VEGF over the indicated time courses. (B) ECs were lysed and blotted for levels of phosphorylated (phospho) and total VEGFR2, NRP1, and phospho (pERK) and total (tERK) ERK1/2. (C) To examine protein degradation, the VEGF time course was extended and EC lysates were blotted for levels of total VEGFR2, NRP1 and β3-integrin. (D) Following a VEGF time course, ECs were trypsinised and analysed by flow cytometry for surface levels of VEGFR2. Median fluorescence intensity was measured after forward versus side scatter data were tightly gated around, and normalised to, an isotype control. The graph shows the relative surface level of VEGFR2 (means±s.e.m.) relative to the 0 (non-stimulated) time point. (E) ECs were stimulated with VEGF for the indicated amounts of time and then lysed and immunoprecipitated for VEGFR2 (VEGFR2 IP), before being blotted for NRP1 association. A total cell lysate (TCL) is shown for comparison. (A-E) HSC-70 served as a loading control. Data are representative of three independent experiments.
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Related In: Results  -  Collection

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DMM019927F2: NRP1-dependent functions of VEGFR-2 are normal in β3-integrin-heterozygous endothelial cells. (A) Lung microvascular endothelial cells (ECs) were isolated and immortalised (polyoma-middle-T-antigen) from β3-WT and β3-HET mice that were expressing either normal (WT NRP1) or cytoplasmic-tail-deleted (NRP1 Δcyto) NRP1. Multiple EC lysates of each genotype were western blotted (WB) to examine total cellular levels of VEGFR2 and β3-integrin. Bar charts of densitometric analysis of mean (+s.e.m.) changes between the four genotypes are shown to the right. Asterisks indicate statistical significance: *P<0.05. (B-E) Representative of ≥three independent experiments per blotted protein. ECs were seeded overnight on a complex matrix containing gelatin, collagen, fibronectin and vitronectin, and were then stimulated with VEGF over the indicated time courses. (B) ECs were lysed and blotted for levels of phosphorylated (phospho) and total VEGFR2, NRP1, and phospho (pERK) and total (tERK) ERK1/2. (C) To examine protein degradation, the VEGF time course was extended and EC lysates were blotted for levels of total VEGFR2, NRP1 and β3-integrin. (D) Following a VEGF time course, ECs were trypsinised and analysed by flow cytometry for surface levels of VEGFR2. Median fluorescence intensity was measured after forward versus side scatter data were tightly gated around, and normalised to, an isotype control. The graph shows the relative surface level of VEGFR2 (means±s.e.m.) relative to the 0 (non-stimulated) time point. (E) ECs were stimulated with VEGF for the indicated amounts of time and then lysed and immunoprecipitated for VEGFR2 (VEGFR2 IP), before being blotted for NRP1 association. A total cell lysate (TCL) is shown for comparison. (A-E) HSC-70 served as a loading control. Data are representative of three independent experiments.
Mentions: Given that NRP1's cytoplasmic tail is important for the regulation of VEGFR2 signalling and trafficking (Ballmer-Hofer et al., 2011; Herzog et al., 2011), we wanted to investigate whether the NRP1Δcyto mutation differentially affects VEGFR2 function in β3-WT versus β3-HET lung microvascular ECs. We employed polyoma-middle-T-antigen immortalised ECs, isolated from the mutant mice described above, because we and others have shown that they present good models to study angiogenesis (Ni et al., 2014; Robinson et al., 2009; Steri et al., 2014; Tavora et al., 2014). We first compared total VEGFR2 levels in the four genotypes (β3-WT, β3-HET, β3-WT;NRP1Δcyto and β3-HET;NRP1Δcyto). Unlike β3-KO ECs, we noted only a small trend of increased VEGFR2 levels in β3-HET and β3-HET;NRP1Δcyto ECs (Fig. 2A).Fig. 2.

Bottom Line: Anti-angiogenic treatments against αvβ3-integrin fail to block tumour growth in the long term, which suggests that the tumour vasculature escapes from angiogenesis inhibition through αvβ3-integrin-independent mechanisms.The simultaneous genetic targeting of both molecules significantly impairs paxillin-1 activation and focal adhesion remodelling in endothelial cells, and therefore inhibits tumour angiogenesis and the growth of already established tumours.These findings provide a firm foundation for testing drugs against these molecules in combination to treat patients with advanced cancers.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

No MeSH data available.


Related in: MedlinePlus