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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

VEGF-induced migration in β3-integrin-heterozygous endothelial cells is dependent on NRP1. (A) ECs isolated from animals of the four indicated genotypes were seeded on collagen type I (COLI), fibronectin (FN), laminin (LN), vitronectin (VN), or a complex mixture of COLI, FN, VN and gelatin (MIX) for 90 min. Unattached cells were gently washed off, and the remaining cells were fixed and stained. Dye was extracted and measured spectophotometrically. The bar chart shows the percentage of cell adhesion to each component relative to FN (means+s.e.m. from three independent experiments). (B) ECs of the indicated genotypes were plated on increasing concentrations of FN or VN. After 90 min, plates were vigorously washed and remaining cells were fixed and stained. Dye was extracted and measured spectophotometrically. The graph shows the mean (±s.e.m. from ≥two independent experiments) number of cells that remained attached to the plate after the procedure. (C) ECs of the indicated genotypes were measured for their surface expression of endothelial integrin subunits by flow cytometry. Median fluorescence intensity (MFI) was measured after forward versus side scatter data were tightly gated around, and normalised to, an isotype control. The bar chart shows the relative change in MFI of β3-HET compared to β3-WT ECs, or of β3-HET.NRP1Δcyto ECs compared to β3-WT.NRP1Δcyto ECs (means+s.e.m. from three independent experiments). Relative changes were deemed significant with a twofold change. Representative flow-cytometric histogram profiles are shown below for significantly changed integrins. (D) 70×105 cells of the indicated genotypes were plated for 6 h on 10 μg/ml FN in six-well plates. Phase-contrast photographs were taken and cell surface areas were measured using ImageJ™ software. The bar chart represents mean (+s.e.m.) surface area quantified from multiple images (n≥50 cells per genotype). (E) ECs were firmly attached to FN-coated dishes and then imaged live for 15 h in low-serum medium ±VEGF. Individual cells were tracked every 10 min over this period using ImageJ™. The bar chart shows the EC migration speed of each of the indicated genotypes (mean+s.e.m. from three independent experiments; n=50 cells per condition). (F) ECs were plated onto FN-coated dishes overnight. After 3 h of starvation, a scratch wound was created and cells were incubated in low-serum medium ±VEGF for 24 h. The bar chart shows the percentage closure of the scratch ‘wound’ as a result of directed cell migration (means+s.e.m. from three independent experiments; n=27 for each condition). Representative images of scratch-wound closure at 24 h are shown below. Scale bar: 200 μm. Asterisks indicate statistical significance: *P<0.05; **P<0.01; ***P<0.001; nsd, not significantly different. Unpaired two-tailed t-test.
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DMM019927F3: VEGF-induced migration in β3-integrin-heterozygous endothelial cells is dependent on NRP1. (A) ECs isolated from animals of the four indicated genotypes were seeded on collagen type I (COLI), fibronectin (FN), laminin (LN), vitronectin (VN), or a complex mixture of COLI, FN, VN and gelatin (MIX) for 90 min. Unattached cells were gently washed off, and the remaining cells were fixed and stained. Dye was extracted and measured spectophotometrically. The bar chart shows the percentage of cell adhesion to each component relative to FN (means+s.e.m. from three independent experiments). (B) ECs of the indicated genotypes were plated on increasing concentrations of FN or VN. After 90 min, plates were vigorously washed and remaining cells were fixed and stained. Dye was extracted and measured spectophotometrically. The graph shows the mean (±s.e.m. from ≥two independent experiments) number of cells that remained attached to the plate after the procedure. (C) ECs of the indicated genotypes were measured for their surface expression of endothelial integrin subunits by flow cytometry. Median fluorescence intensity (MFI) was measured after forward versus side scatter data were tightly gated around, and normalised to, an isotype control. The bar chart shows the relative change in MFI of β3-HET compared to β3-WT ECs, or of β3-HET.NRP1Δcyto ECs compared to β3-WT.NRP1Δcyto ECs (means+s.e.m. from three independent experiments). Relative changes were deemed significant with a twofold change. Representative flow-cytometric histogram profiles are shown below for significantly changed integrins. (D) 70×105 cells of the indicated genotypes were plated for 6 h on 10 μg/ml FN in six-well plates. Phase-contrast photographs were taken and cell surface areas were measured using ImageJ™ software. The bar chart represents mean (+s.e.m.) surface area quantified from multiple images (n≥50 cells per genotype). (E) ECs were firmly attached to FN-coated dishes and then imaged live for 15 h in low-serum medium ±VEGF. Individual cells were tracked every 10 min over this period using ImageJ™. The bar chart shows the EC migration speed of each of the indicated genotypes (mean+s.e.m. from three independent experiments; n=50 cells per condition). (F) ECs were plated onto FN-coated dishes overnight. After 3 h of starvation, a scratch wound was created and cells were incubated in low-serum medium ±VEGF for 24 h. The bar chart shows the percentage closure of the scratch ‘wound’ as a result of directed cell migration (means+s.e.m. from three independent experiments; n=27 for each condition). Representative images of scratch-wound closure at 24 h are shown below. Scale bar: 200 μm. Asterisks indicate statistical significance: *P<0.05; **P<0.01; ***P<0.001; nsd, not significantly different. Unpaired two-tailed t-test.

Mentions: Prior to its description as a VEGF co-receptor, NRP1 was identified as a surface protein mediating cell adhesion (Takagi et al., 1995). Moreover, evidence has mounted to support a VEGFR2-independent role for NRP1 in regulating EC functions through an FA-dependent mechanism (Fantin et al., 2014; Raimondi et al., 2014; Seerapu et al., 2013). Because NRP1 is known to interact with a number of integrins (Fukasawa et al., 2007; Robinson et al., 2009; Valdembri et al., 2009), we first compared static cell adhesion between β3-WT, β3-HET, β3-WT;NRP1Δcyto and β3-HET;NRP1Δcyto ECs on saturating concentrations of various matrices. The only clear difference noted on saturating matrix concentrations in these assays was an expected reduced adhesion of β3-HET and β3-HET.NRP1Δcyto ECs to matrices containing VN, β3-integrin's canonical ligand (Fig. 3A). A more vigorous examination of adhesions (see Materials and Methods) over a range of matrix concentrations, however, uncovered subtle changes between the genotypes (Fig. 3B). Not surprisingly, compared to β3-WT and β3-WT;NRP1Δcyto ECs, β3-HET and β3-HET;NRP1Δcyto ECs showed reduced adhesion to VN over a range of concentrations tested. β3-WT;NRP1Δcyto EC adhesion to FN was, as expected (Valdembri et al., 2009), somewhat reduced compared to β3-WT ECs. Compared to their WT counterparts, β3-HET and β3-HET;NRP1Δcyto ECs exhibited reduced strength of adhesion to FN over the gradient of concentrations tested.Fig. 3.


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)

VEGF-induced migration in β3-integrin-heterozygous endothelial cells is dependent on NRP1. (A) ECs isolated from animals of the four indicated genotypes were seeded on collagen type I (COLI), fibronectin (FN), laminin (LN), vitronectin (VN), or a complex mixture of COLI, FN, VN and gelatin (MIX) for 90 min. Unattached cells were gently washed off, and the remaining cells were fixed and stained. Dye was extracted and measured spectophotometrically. The bar chart shows the percentage of cell adhesion to each component relative to FN (means+s.e.m. from three independent experiments). (B) ECs of the indicated genotypes were plated on increasing concentrations of FN or VN. After 90 min, plates were vigorously washed and remaining cells were fixed and stained. Dye was extracted and measured spectophotometrically. The graph shows the mean (±s.e.m. from ≥two independent experiments) number of cells that remained attached to the plate after the procedure. (C) ECs of the indicated genotypes were measured for their surface expression of endothelial integrin subunits by flow cytometry. Median fluorescence intensity (MFI) was measured after forward versus side scatter data were tightly gated around, and normalised to, an isotype control. The bar chart shows the relative change in MFI of β3-HET compared to β3-WT ECs, or of β3-HET.NRP1Δcyto ECs compared to β3-WT.NRP1Δcyto ECs (means+s.e.m. from three independent experiments). Relative changes were deemed significant with a twofold change. Representative flow-cytometric histogram profiles are shown below for significantly changed integrins. (D) 70×105 cells of the indicated genotypes were plated for 6 h on 10 μg/ml FN in six-well plates. Phase-contrast photographs were taken and cell surface areas were measured using ImageJ™ software. The bar chart represents mean (+s.e.m.) surface area quantified from multiple images (n≥50 cells per genotype). (E) ECs were firmly attached to FN-coated dishes and then imaged live for 15 h in low-serum medium ±VEGF. Individual cells were tracked every 10 min over this period using ImageJ™. The bar chart shows the EC migration speed of each of the indicated genotypes (mean+s.e.m. from three independent experiments; n=50 cells per condition). (F) ECs were plated onto FN-coated dishes overnight. After 3 h of starvation, a scratch wound was created and cells were incubated in low-serum medium ±VEGF for 24 h. The bar chart shows the percentage closure of the scratch ‘wound’ as a result of directed cell migration (means+s.e.m. from three independent experiments; n=27 for each condition). Representative images of scratch-wound closure at 24 h are shown below. Scale bar: 200 μm. Asterisks indicate statistical significance: *P<0.05; **P<0.01; ***P<0.001; nsd, not significantly different. Unpaired two-tailed t-test.
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DMM019927F3: VEGF-induced migration in β3-integrin-heterozygous endothelial cells is dependent on NRP1. (A) ECs isolated from animals of the four indicated genotypes were seeded on collagen type I (COLI), fibronectin (FN), laminin (LN), vitronectin (VN), or a complex mixture of COLI, FN, VN and gelatin (MIX) for 90 min. Unattached cells were gently washed off, and the remaining cells were fixed and stained. Dye was extracted and measured spectophotometrically. The bar chart shows the percentage of cell adhesion to each component relative to FN (means+s.e.m. from three independent experiments). (B) ECs of the indicated genotypes were plated on increasing concentrations of FN or VN. After 90 min, plates were vigorously washed and remaining cells were fixed and stained. Dye was extracted and measured spectophotometrically. The graph shows the mean (±s.e.m. from ≥two independent experiments) number of cells that remained attached to the plate after the procedure. (C) ECs of the indicated genotypes were measured for their surface expression of endothelial integrin subunits by flow cytometry. Median fluorescence intensity (MFI) was measured after forward versus side scatter data were tightly gated around, and normalised to, an isotype control. The bar chart shows the relative change in MFI of β3-HET compared to β3-WT ECs, or of β3-HET.NRP1Δcyto ECs compared to β3-WT.NRP1Δcyto ECs (means+s.e.m. from three independent experiments). Relative changes were deemed significant with a twofold change. Representative flow-cytometric histogram profiles are shown below for significantly changed integrins. (D) 70×105 cells of the indicated genotypes were plated for 6 h on 10 μg/ml FN in six-well plates. Phase-contrast photographs were taken and cell surface areas were measured using ImageJ™ software. The bar chart represents mean (+s.e.m.) surface area quantified from multiple images (n≥50 cells per genotype). (E) ECs were firmly attached to FN-coated dishes and then imaged live for 15 h in low-serum medium ±VEGF. Individual cells were tracked every 10 min over this period using ImageJ™. The bar chart shows the EC migration speed of each of the indicated genotypes (mean+s.e.m. from three independent experiments; n=50 cells per condition). (F) ECs were plated onto FN-coated dishes overnight. After 3 h of starvation, a scratch wound was created and cells were incubated in low-serum medium ±VEGF for 24 h. The bar chart shows the percentage closure of the scratch ‘wound’ as a result of directed cell migration (means+s.e.m. from three independent experiments; n=27 for each condition). Representative images of scratch-wound closure at 24 h are shown below. Scale bar: 200 μm. Asterisks indicate statistical significance: *P<0.05; **P<0.01; ***P<0.001; nsd, not significantly different. Unpaired two-tailed t-test.
Mentions: Prior to its description as a VEGF co-receptor, NRP1 was identified as a surface protein mediating cell adhesion (Takagi et al., 1995). Moreover, evidence has mounted to support a VEGFR2-independent role for NRP1 in regulating EC functions through an FA-dependent mechanism (Fantin et al., 2014; Raimondi et al., 2014; Seerapu et al., 2013). Because NRP1 is known to interact with a number of integrins (Fukasawa et al., 2007; Robinson et al., 2009; Valdembri et al., 2009), we first compared static cell adhesion between β3-WT, β3-HET, β3-WT;NRP1Δcyto and β3-HET;NRP1Δcyto ECs on saturating concentrations of various matrices. The only clear difference noted on saturating matrix concentrations in these assays was an expected reduced adhesion of β3-HET and β3-HET.NRP1Δcyto ECs to matrices containing VN, β3-integrin's canonical ligand (Fig. 3A). A more vigorous examination of adhesions (see Materials and Methods) over a range of matrix concentrations, however, uncovered subtle changes between the genotypes (Fig. 3B). Not surprisingly, compared to β3-WT and β3-WT;NRP1Δcyto ECs, β3-HET and β3-HET;NRP1Δcyto ECs showed reduced adhesion to VN over a range of concentrations tested. β3-WT;NRP1Δcyto EC adhesion to FN was, as expected (Valdembri et al., 2009), somewhat reduced compared to β3-WT ECs. Compared to their WT counterparts, β3-HET and β3-HET;NRP1Δcyto ECs exhibited reduced strength of adhesion to FN over the gradient of concentrations tested.Fig. 3.

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