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

Simultaneously depleting both β3-integrin and NRP1 blocks growth and angiogenesis in already-established tumours. (A) Primary lung microvascular ECs were isolated from β3-floxed-Pdgfb-iCreERT2-negative and -positive animals. Tamoxifen (OHT) was administered after pure EC populations were achieved. Cells were then plated overnight on FN-coated glass coverslips. Cells were starved for 3 h and then treated ±VEGF for 10 min in serum-free medium. Cells were fixed and stained with phalloidin for F-actin (green), and immunostained for NRP1 (red). White arrows point to the ends of actin filaments. Scale bar: 20 μm. The bar chart shows the percentage of cells within the population showing NRP1 staining at the end of actin filaments (mean+s.e.m.; n≥50 cells per condition). (B) Microvessel sprouting of aortic ring explants of the indicated genotypes. Protein knockout in ECs was induced in culture with 1 μM OHT 4 days after VEGF-induced sprouting had been established. The bar chart shows the total number of microvessel sprouts per aortic ring after an additional 4 days of VEGF-stimulation (mean+s.e.m. from three independent experiments; n≥40 rings per genotype). (C) Tumour growth and angiogenesis were measured in animals of the indicated genotypes. Mice were injected subcutaneously with CMT19T cells and 10 days later OHT was administered. After an additional 10 days (20 days in total) tumours were harvested. Upper panel: the bar chart shows mean tumour volumes measured at days 10 and 20 (+s.e.m. of two or more independent experiments; n≥10 animals per genotype). The western blot to the right shows representative depletion of β3-integrin and NRP1 in tumour endothelial cells (TECs) isolated from Cre-positive animals, compared to Cre-negative littermate controls. Bottom panel: blood-vessel density in 20-day tumours was assessed by counting the total number of endomucin-positive vessels around the periphery (within 150 μm of the edge of the tumour) of midline bisected tumour sections. The bar chart shows mean vessel number per mm2 (+s.e.m.). Representative micrographs of endomucin staining (red) are shown below. Scale bar: 50 μm. Asterisks indicate statistical significance: *P<0.05; ***P<0.001; nsd, not significantly different. Unpaired two-tailed t-test. (D) Schematic representation of the hypothesised participation of NRP1 in focal adhesion (FA) remodelling and migration in β3-WT (left) and β3-suppressed (right) ECs. ITGN, integrin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

DMM019927F6: Simultaneously depleting both β3-integrin and NRP1 blocks growth and angiogenesis in already-established tumours. (A) Primary lung microvascular ECs were isolated from β3-floxed-Pdgfb-iCreERT2-negative and -positive animals. Tamoxifen (OHT) was administered after pure EC populations were achieved. Cells were then plated overnight on FN-coated glass coverslips. Cells were starved for 3 h and then treated ±VEGF for 10 min in serum-free medium. Cells were fixed and stained with phalloidin for F-actin (green), and immunostained for NRP1 (red). White arrows point to the ends of actin filaments. Scale bar: 20 μm. The bar chart shows the percentage of cells within the population showing NRP1 staining at the end of actin filaments (mean+s.e.m.; n≥50 cells per condition). (B) Microvessel sprouting of aortic ring explants of the indicated genotypes. Protein knockout in ECs was induced in culture with 1 μM OHT 4 days after VEGF-induced sprouting had been established. The bar chart shows the total number of microvessel sprouts per aortic ring after an additional 4 days of VEGF-stimulation (mean+s.e.m. from three independent experiments; n≥40 rings per genotype). (C) Tumour growth and angiogenesis were measured in animals of the indicated genotypes. Mice were injected subcutaneously with CMT19T cells and 10 days later OHT was administered. After an additional 10 days (20 days in total) tumours were harvested. Upper panel: the bar chart shows mean tumour volumes measured at days 10 and 20 (+s.e.m. of two or more independent experiments; n≥10 animals per genotype). The western blot to the right shows representative depletion of β3-integrin and NRP1 in tumour endothelial cells (TECs) isolated from Cre-positive animals, compared to Cre-negative littermate controls. Bottom panel: blood-vessel density in 20-day tumours was assessed by counting the total number of endomucin-positive vessels around the periphery (within 150 μm of the edge of the tumour) of midline bisected tumour sections. The bar chart shows mean vessel number per mm2 (+s.e.m.). Representative micrographs of endomucin staining (red) are shown below. Scale bar: 50 μm. Asterisks indicate statistical significance: *P<0.05; ***P<0.001; nsd, not significantly different. Unpaired two-tailed t-test. (D) Schematic representation of the hypothesised participation of NRP1 in focal adhesion (FA) remodelling and migration in β3-WT (left) and β3-suppressed (right) ECs. ITGN, integrin.

Mentions: The identification of the mechanisms underlying increased sensitivity to NRP1 disruption in ECs with reduced β3-integrin expression should enable the rational design of intervention strategies to improve anti-angiogenic outcomes in patients with advanced cancers. To provide evidence in support of this idea, we performed proof-of-concept studies in β3-integrin/NRP1-double-floxed mice crossed to OHT-inducible-Pdgfb-iCreERT2 transgenics. First, though, we confirmed the same mechanistic principle described above in primary lung ECs acutely depleted of β3-integrin (Fig. 6A). We observed NRP1 expression at the ends of F-actin in OHT-treated β3-WT cells with and without VEGF-stimulation, but not in the majority of OHT-treated β3-KO ECs after VEGF treatment. We then initiated VEGF-induced microvessel sprouting in aortic rings isolated from: (1) β3-floxed mice with and without Pdgfb-iCreERT2; (2) NRP1-floxed mice with and without Pdgfb-iCreERT2; or (3) double-floxed mice with and without Pdgfb-iCreERT2. OHT was administered to all rings after 4 days of sprouting, and microvessels were enumerated 4 days later. Only in rings from double-floxed Pdgfb-iCreERT2-positive animals was further sprouting significantly inhibited (Fig. 6B). Finally, we performed intervention CMT19T allograft studies by establishing vascularised tumours in these same animals. After 10 days of growth, all animals were administered OHT and tumours were allowed to grow for another 10 days. In concordance with the intervention aortic ring studies, further tumour growth and angiogenesis were significantly inhibited in double-floxed Pdgfb-iCreERT2-positive animals, but not in any of the other genotypes (Fig. 6C).Fig. 6.


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)

Simultaneously depleting both β3-integrin and NRP1 blocks growth and angiogenesis in already-established tumours. (A) Primary lung microvascular ECs were isolated from β3-floxed-Pdgfb-iCreERT2-negative and -positive animals. Tamoxifen (OHT) was administered after pure EC populations were achieved. Cells were then plated overnight on FN-coated glass coverslips. Cells were starved for 3 h and then treated ±VEGF for 10 min in serum-free medium. Cells were fixed and stained with phalloidin for F-actin (green), and immunostained for NRP1 (red). White arrows point to the ends of actin filaments. Scale bar: 20 μm. The bar chart shows the percentage of cells within the population showing NRP1 staining at the end of actin filaments (mean+s.e.m.; n≥50 cells per condition). (B) Microvessel sprouting of aortic ring explants of the indicated genotypes. Protein knockout in ECs was induced in culture with 1 μM OHT 4 days after VEGF-induced sprouting had been established. The bar chart shows the total number of microvessel sprouts per aortic ring after an additional 4 days of VEGF-stimulation (mean+s.e.m. from three independent experiments; n≥40 rings per genotype). (C) Tumour growth and angiogenesis were measured in animals of the indicated genotypes. Mice were injected subcutaneously with CMT19T cells and 10 days later OHT was administered. After an additional 10 days (20 days in total) tumours were harvested. Upper panel: the bar chart shows mean tumour volumes measured at days 10 and 20 (+s.e.m. of two or more independent experiments; n≥10 animals per genotype). The western blot to the right shows representative depletion of β3-integrin and NRP1 in tumour endothelial cells (TECs) isolated from Cre-positive animals, compared to Cre-negative littermate controls. Bottom panel: blood-vessel density in 20-day tumours was assessed by counting the total number of endomucin-positive vessels around the periphery (within 150 μm of the edge of the tumour) of midline bisected tumour sections. The bar chart shows mean vessel number per mm2 (+s.e.m.). Representative micrographs of endomucin staining (red) are shown below. Scale bar: 50 μm. Asterisks indicate statistical significance: *P<0.05; ***P<0.001; nsd, not significantly different. Unpaired two-tailed t-test. (D) Schematic representation of the hypothesised participation of NRP1 in focal adhesion (FA) remodelling and migration in β3-WT (left) and β3-suppressed (right) ECs. ITGN, integrin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

DMM019927F6: Simultaneously depleting both β3-integrin and NRP1 blocks growth and angiogenesis in already-established tumours. (A) Primary lung microvascular ECs were isolated from β3-floxed-Pdgfb-iCreERT2-negative and -positive animals. Tamoxifen (OHT) was administered after pure EC populations were achieved. Cells were then plated overnight on FN-coated glass coverslips. Cells were starved for 3 h and then treated ±VEGF for 10 min in serum-free medium. Cells were fixed and stained with phalloidin for F-actin (green), and immunostained for NRP1 (red). White arrows point to the ends of actin filaments. Scale bar: 20 μm. The bar chart shows the percentage of cells within the population showing NRP1 staining at the end of actin filaments (mean+s.e.m.; n≥50 cells per condition). (B) Microvessel sprouting of aortic ring explants of the indicated genotypes. Protein knockout in ECs was induced in culture with 1 μM OHT 4 days after VEGF-induced sprouting had been established. The bar chart shows the total number of microvessel sprouts per aortic ring after an additional 4 days of VEGF-stimulation (mean+s.e.m. from three independent experiments; n≥40 rings per genotype). (C) Tumour growth and angiogenesis were measured in animals of the indicated genotypes. Mice were injected subcutaneously with CMT19T cells and 10 days later OHT was administered. After an additional 10 days (20 days in total) tumours were harvested. Upper panel: the bar chart shows mean tumour volumes measured at days 10 and 20 (+s.e.m. of two or more independent experiments; n≥10 animals per genotype). The western blot to the right shows representative depletion of β3-integrin and NRP1 in tumour endothelial cells (TECs) isolated from Cre-positive animals, compared to Cre-negative littermate controls. Bottom panel: blood-vessel density in 20-day tumours was assessed by counting the total number of endomucin-positive vessels around the periphery (within 150 μm of the edge of the tumour) of midline bisected tumour sections. The bar chart shows mean vessel number per mm2 (+s.e.m.). Representative micrographs of endomucin staining (red) are shown below. Scale bar: 50 μm. Asterisks indicate statistical significance: *P<0.05; ***P<0.001; nsd, not significantly different. Unpaired two-tailed t-test. (D) Schematic representation of the hypothesised participation of NRP1 in focal adhesion (FA) remodelling and migration in β3-WT (left) and β3-suppressed (right) ECs. ITGN, integrin.
Mentions: The identification of the mechanisms underlying increased sensitivity to NRP1 disruption in ECs with reduced β3-integrin expression should enable the rational design of intervention strategies to improve anti-angiogenic outcomes in patients with advanced cancers. To provide evidence in support of this idea, we performed proof-of-concept studies in β3-integrin/NRP1-double-floxed mice crossed to OHT-inducible-Pdgfb-iCreERT2 transgenics. First, though, we confirmed the same mechanistic principle described above in primary lung ECs acutely depleted of β3-integrin (Fig. 6A). We observed NRP1 expression at the ends of F-actin in OHT-treated β3-WT cells with and without VEGF-stimulation, but not in the majority of OHT-treated β3-KO ECs after VEGF treatment. We then initiated VEGF-induced microvessel sprouting in aortic rings isolated from: (1) β3-floxed mice with and without Pdgfb-iCreERT2; (2) NRP1-floxed mice with and without Pdgfb-iCreERT2; or (3) double-floxed mice with and without Pdgfb-iCreERT2. OHT was administered to all rings after 4 days of sprouting, and microvessels were enumerated 4 days later. Only in rings from double-floxed Pdgfb-iCreERT2-positive animals was further sprouting significantly inhibited (Fig. 6B). Finally, we performed intervention CMT19T allograft studies by establishing vascularised tumours in these same animals. After 10 days of growth, all animals were administered OHT and tumours were allowed to grow for another 10 days. In concordance with the intervention aortic ring studies, further tumour growth and angiogenesis were significantly inhibited in double-floxed Pdgfb-iCreERT2-positive animals, but not in any of the other genotypes (Fig. 6C).Fig. 6.

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