Limits...
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's localisation in focal adhesions is altered in β3-integrin-heterozygous endothelial cells. (A) β3-WT and β3-HET ECs were subjected to a cell-fractionation experiment following ±VEGF treatment for 10 min. Fractionated samples were then analysed by western blot for the indicated proteins. 1=cytoplasmic extract; 2=membrane extract; 3=soluble nuclear extract; 4=chromatin-bound nuclear extract; 5=cytoskeletal extract. Protein markers for each subcellular compartment were included as controls. Data are representative of two independent experiments. The bar charts represent the relative proportion (%) of VEGFR2 or NRP1 present in each fraction determined by ImageJ™ densitometry. (B) Left and right panels: β3-WT and β3-HET ECs were seeded overnight onto FN-coated glass coverslips and then stimulated with VEGF for 10 min. Cells were fixed and stained with phalloidin for filamentous actin (F-actin; green), and immunostained for neuropilin-1 (NRP1; red). Arrows point to the ends of actin filaments. Middle panel: β3-WT ECs were transfected with a β3-integrin-GFP construct (green) and seeded on FN-coated coverslips. 48 h later, cells were fixed and immunostained for NRP1 (red). Split-channel close-ups are shown to depict β3-integrin/NRP1 colocalisation. Scale bars: 10 μm (middle), or 20 μm. (C) ECs were allowed to adhere to FN-coated dishes for 90 min to establish ‘mature’ integrin-dependent focal adhesions (FAs). FAs were chemically cross-linked to the plates and cells were lysed with RIPA buffer. Non-cross-linked proteins and other cellular components were rinsed away under high-sheer flow. FA-enriched complexes were eluted, subjected to SDS-PAGE and then analysed by label-free, quantitative mass spectometry. The graphs represent log ratio plots of the proteomic data comparing unstimulated β3-WT ECs (y-axis) and β3-HET ECs (x-axis). Proteins above the diagonal line are higher in β3-WT ECs, whereas those below the line are higher in β3-HET ECs (n=3 samples per genotype). NRP1 is present in both adhesomes (red circle). (D) FA-enriched EC samples were processed as in C and then analysed by western blot for the indicated proteins. A total cell lysate (TCL) is shown for comparison. Data are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4582102&req=5

DMM019927F4: NRP1's localisation in focal adhesions is altered in β3-integrin-heterozygous endothelial cells. (A) β3-WT and β3-HET ECs were subjected to a cell-fractionation experiment following ±VEGF treatment for 10 min. Fractionated samples were then analysed by western blot for the indicated proteins. 1=cytoplasmic extract; 2=membrane extract; 3=soluble nuclear extract; 4=chromatin-bound nuclear extract; 5=cytoskeletal extract. Protein markers for each subcellular compartment were included as controls. Data are representative of two independent experiments. The bar charts represent the relative proportion (%) of VEGFR2 or NRP1 present in each fraction determined by ImageJ™ densitometry. (B) Left and right panels: β3-WT and β3-HET ECs were seeded overnight onto FN-coated glass coverslips and then stimulated with VEGF for 10 min. Cells were fixed and stained with phalloidin for filamentous actin (F-actin; green), and immunostained for neuropilin-1 (NRP1; red). Arrows point to the ends of actin filaments. Middle panel: β3-WT ECs were transfected with a β3-integrin-GFP construct (green) and seeded on FN-coated coverslips. 48 h later, cells were fixed and immunostained for NRP1 (red). Split-channel close-ups are shown to depict β3-integrin/NRP1 colocalisation. Scale bars: 10 μm (middle), or 20 μm. (C) ECs were allowed to adhere to FN-coated dishes for 90 min to establish ‘mature’ integrin-dependent focal adhesions (FAs). FAs were chemically cross-linked to the plates and cells were lysed with RIPA buffer. Non-cross-linked proteins and other cellular components were rinsed away under high-sheer flow. FA-enriched complexes were eluted, subjected to SDS-PAGE and then analysed by label-free, quantitative mass spectometry. The graphs represent log ratio plots of the proteomic data comparing unstimulated β3-WT ECs (y-axis) and β3-HET ECs (x-axis). Proteins above the diagonal line are higher in β3-WT ECs, whereas those below the line are higher in β3-HET ECs (n=3 samples per genotype). NRP1 is present in both adhesomes (red circle). (D) FA-enriched EC samples were processed as in C and then analysed by western blot for the indicated proteins. A total cell lysate (TCL) is shown for comparison. Data are representative of three independent experiments.

Mentions: The findings described above suggested that aberrant integrin-directed migration, essential for pathological angiogenesis, correlates with the phenotypic difference in sensitivity to NRP1 perturbations that we observed in β3-HET mice. NRP1 is known to colocalise with a number of FA-associated proteins (Seerapu et al., 2013) and is involved in FA turnover. We therefore took a closer look at the structural and functional characteristics of mature endothelial FAs, focal points of cellular migration. We first examined NRP1's distribution in subcellular compartments after cell fractionation and immunoblotting. Other than the previously noted trend toward overexpression of the protein in β3-HET ECs, we found no obvious differences (Fig. 4A). We next immunolocalised NRP1 in cells plated on FN overnight. Because of the VEGF-dependence of our phenotypic angiogenic responses, we initially concentrated on VEGF-stimulated cells. In β3-WT ECs, NRP1 localised to mature FAs, which were found at the ends of filamentous actin (F-actin) fibres (Fig. 4B, left); as expected from previous studies (Robinson et al., 2009), NRP1 also colocalised with GFP-tagged β3-integrin in cDNA-expression-construct-transfected β3-WT ECs (Fig. 4B, middle). However, in VEGF-stimulated β3-HET ECs, NRP1 no longer localised to these sites (Fig. 4B, right).Fig. 4.


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's localisation in focal adhesions is altered in β3-integrin-heterozygous endothelial cells. (A) β3-WT and β3-HET ECs were subjected to a cell-fractionation experiment following ±VEGF treatment for 10 min. Fractionated samples were then analysed by western blot for the indicated proteins. 1=cytoplasmic extract; 2=membrane extract; 3=soluble nuclear extract; 4=chromatin-bound nuclear extract; 5=cytoskeletal extract. Protein markers for each subcellular compartment were included as controls. Data are representative of two independent experiments. The bar charts represent the relative proportion (%) of VEGFR2 or NRP1 present in each fraction determined by ImageJ™ densitometry. (B) Left and right panels: β3-WT and β3-HET ECs were seeded overnight onto FN-coated glass coverslips and then stimulated with VEGF for 10 min. Cells were fixed and stained with phalloidin for filamentous actin (F-actin; green), and immunostained for neuropilin-1 (NRP1; red). Arrows point to the ends of actin filaments. Middle panel: β3-WT ECs were transfected with a β3-integrin-GFP construct (green) and seeded on FN-coated coverslips. 48 h later, cells were fixed and immunostained for NRP1 (red). Split-channel close-ups are shown to depict β3-integrin/NRP1 colocalisation. Scale bars: 10 μm (middle), or 20 μm. (C) ECs were allowed to adhere to FN-coated dishes for 90 min to establish ‘mature’ integrin-dependent focal adhesions (FAs). FAs were chemically cross-linked to the plates and cells were lysed with RIPA buffer. Non-cross-linked proteins and other cellular components were rinsed away under high-sheer flow. FA-enriched complexes were eluted, subjected to SDS-PAGE and then analysed by label-free, quantitative mass spectometry. The graphs represent log ratio plots of the proteomic data comparing unstimulated β3-WT ECs (y-axis) and β3-HET ECs (x-axis). Proteins above the diagonal line are higher in β3-WT ECs, whereas those below the line are higher in β3-HET ECs (n=3 samples per genotype). NRP1 is present in both adhesomes (red circle). (D) FA-enriched EC samples were processed as in C and then analysed by western blot for the indicated proteins. A total cell lysate (TCL) is shown for comparison. Data are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582102&req=5

DMM019927F4: NRP1's localisation in focal adhesions is altered in β3-integrin-heterozygous endothelial cells. (A) β3-WT and β3-HET ECs were subjected to a cell-fractionation experiment following ±VEGF treatment for 10 min. Fractionated samples were then analysed by western blot for the indicated proteins. 1=cytoplasmic extract; 2=membrane extract; 3=soluble nuclear extract; 4=chromatin-bound nuclear extract; 5=cytoskeletal extract. Protein markers for each subcellular compartment were included as controls. Data are representative of two independent experiments. The bar charts represent the relative proportion (%) of VEGFR2 or NRP1 present in each fraction determined by ImageJ™ densitometry. (B) Left and right panels: β3-WT and β3-HET ECs were seeded overnight onto FN-coated glass coverslips and then stimulated with VEGF for 10 min. Cells were fixed and stained with phalloidin for filamentous actin (F-actin; green), and immunostained for neuropilin-1 (NRP1; red). Arrows point to the ends of actin filaments. Middle panel: β3-WT ECs were transfected with a β3-integrin-GFP construct (green) and seeded on FN-coated coverslips. 48 h later, cells were fixed and immunostained for NRP1 (red). Split-channel close-ups are shown to depict β3-integrin/NRP1 colocalisation. Scale bars: 10 μm (middle), or 20 μm. (C) ECs were allowed to adhere to FN-coated dishes for 90 min to establish ‘mature’ integrin-dependent focal adhesions (FAs). FAs were chemically cross-linked to the plates and cells were lysed with RIPA buffer. Non-cross-linked proteins and other cellular components were rinsed away under high-sheer flow. FA-enriched complexes were eluted, subjected to SDS-PAGE and then analysed by label-free, quantitative mass spectometry. The graphs represent log ratio plots of the proteomic data comparing unstimulated β3-WT ECs (y-axis) and β3-HET ECs (x-axis). Proteins above the diagonal line are higher in β3-WT ECs, whereas those below the line are higher in β3-HET ECs (n=3 samples per genotype). NRP1 is present in both adhesomes (red circle). (D) FA-enriched EC samples were processed as in C and then analysed by western blot for the indicated proteins. A total cell lysate (TCL) is shown for comparison. Data are representative of three independent experiments.
Mentions: The findings described above suggested that aberrant integrin-directed migration, essential for pathological angiogenesis, correlates with the phenotypic difference in sensitivity to NRP1 perturbations that we observed in β3-HET mice. NRP1 is known to colocalise with a number of FA-associated proteins (Seerapu et al., 2013) and is involved in FA turnover. We therefore took a closer look at the structural and functional characteristics of mature endothelial FAs, focal points of cellular migration. We first examined NRP1's distribution in subcellular compartments after cell fractionation and immunoblotting. Other than the previously noted trend toward overexpression of the protein in β3-HET ECs, we found no obvious differences (Fig. 4A). We next immunolocalised NRP1 in cells plated on FN overnight. Because of the VEGF-dependence of our phenotypic angiogenic responses, we initially concentrated on VEGF-stimulated cells. In β3-WT ECs, NRP1 localised to mature FAs, which were found at the ends of filamentous actin (F-actin) fibres (Fig. 4B, left); as expected from previous studies (Robinson et al., 2009), NRP1 also colocalised with GFP-tagged β3-integrin in cDNA-expression-construct-transfected β3-WT ECs (Fig. 4B, middle). However, in VEGF-stimulated β3-HET ECs, NRP1 no longer localised to these sites (Fig. 4B, right).Fig. 4.

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