Endothelial FAK is required for tumour angiogenesis

Abstract

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a fundamental role in integrin and growth factor mediated signalling and is an important player in cell migration and proliferation, processes vital for angiogenesis. However, the role of FAK in adult pathological angiogenesis is unknown. We have generated endothelial-specific tamoxifen-inducible FAK knockout mice by crossing FAK-floxed (FAKfl/fl) mice with the platelet derived growth factor b (Pdgfb)-iCreER mice. Tamoxifen-treatment of Pdgfb-iCreER;FAKfl/fl mice results in FAK deletion in adult endothelial cells (ECs) without any adverse effects. Importantly however, endothelial FAK-deletion in adult mice inhibited tumour growth and reduced tumour angiogenesis. Furthermore, in in vivo angiogenic assays FAK deletion impairs vascular endothelial growth factor (VEGF)-induced neovascularization. In addition, in vitro deletion of FAK in ECs resulted in reduced VEGF-stimulated Akt phosphorylation and correlating reduced cellular proliferation as well as increased cell death. Our data suggest that FAK is required for adult pathological angiogenesis and validates FAK as a possible target for anti-angiogenic therapies.

Figure 1. Endothelial-specific deletion of FAK in cells isolated from adult mice

1. Immunofluorescence analysis of endothelial cells isolated from Pdgfb-iCreER;FAKfl/fl mice shows loss of FAK expression in OHT treated cells (+OHT) but not untreated (Control) cells. FAK (green), DAPI (blue). Scale bar = 50 µm.

2. Flow cytometric analysis for the endothelial markers intercellular adhesion molecule-2 (ICAM-2) and VE-cadherin (VECAD) was performed to confirm the presence of endothelial enriched populations in cell cultures isolated from the lungs of Pdgfb-iCreER;FAKfl/fl mice. Western blot analysis of endothelial cells (EC) isolated from the lungs of Pdgfb-iCreER;FAKfl/fl mice shows that FAK is deleted in ECs upon 4-hydroxytamoxifen treatment (OHT). HSC70 acts as loading control. Bar chart represents mean densitometric values +s.e.m. **p < 0.01. n = 3 independent experiments.

3. Flow cytometric analysis for the endothelial markers ICAM-2 and VECAD was performed to confirm the presence of a non-endothelial cell populations in cell cultures isolated from lungs of adult Pdgfb-iCreER;FAKfl/fl mice. Western blot analysis confirms that FAK levels do not change in non-endothelial (non-EC) cells isolated from the lungs of Pdgfb-iCreER;FAKfl/fl mice. Bar chart represents mean densitometric values +s.e.m. n.s. = not statistically significant. n = 3 independent experiments.

Figure 2. Endothelial-specific deletion of FAK in adult mice inhibits tumour growth and angiogenesis

1. ECFAK^WT and ECFAK^KO mice were given subcutaneous injections of syngeneic tumour cell lines, B16F0 melanoma and CMT19T carcinoma. Twelve-day-old tumour size was decreased significantly in ECFAK^KO mice when compared with ECFAK^WT controls. Pictures of representative tumours are shown. Bar charts show mean tumour volume +s.e.m. Scale bar = 1 cm; n = 11–22 mice per genotype. **p < 0.01.

2. Tumour blood vessel density is reduced in ECFAK^KO mice. Representative immunofluorescence micrographs identifying blood vessels (PECAM) in midline sections of tumours. Blood vessel density was assessed by counting the total number of blood vessels per mm² across entire midline sections of size-matched tumours. DAPI (blue) was used as a nuclear counterstain. Bar charts represent mean tumour blood vessel density +s.e.m. for both tumour types. Scale bar = 200 µm; *p < 0.05; **p < 0.01.

3. Double immunofluorescence staining for endomucin (red) and FAK (green) of tumour sections from ECFAK^WT and ECFAK^KO mice shows loss of FAK in vivo in ECFAK^KO mice but not ECFAK^WT controls. DAPI (blue) was used as a nuclear counterstain. Bar chart represents the percentage of FAK-positive vessels. Scale bar = 100µm; **p < 0.01.

Figure 3. Endothelial-specific deletion of FAK in adult mice reduces VEGF-induced angiogenic responses and migration in vivo

1. In in vivo growth factor-induced angiogenic assays, ECFAK^WT and ECFAK^KO mice were given subcutaneous sponge implants impregnated with either PBS or VEGF. Blood vessel infiltration was examined 14 days postimplantation by immunohistochemical analysis of endomucin-positive blood vessels. Although ECFAK^WT and ECFAK^KO showed a similar lack of response to PBS treatment, VEGF treatment induced an angiogenic response in ECFAK^WT mice but not in ECFAK^KO mice. Bar chart represents mean number of infiltrated blood vessels/20× field of view of sponge section + s.e.m. n = 7–14 mice per genotype. *p < 0.05, n.s. = not statistically significant. Black arrows, endomucin-stained blood vessels; red*, fragments of synthetic sponge.

2. Retinal vasculature at P5 was observed by staining with fluorescein-isolectin B4 (isolectin) and Cy3-coupled antibody against α-SMA. Retinal vasculature outgrowth (double headed arrow) was reduced in the ECFAK^KO retinas when compared with ECFAK^WT controls. The fine filopodial extensions (white arrow heads) observed in ECFAK^WT tip cells were not present in ECFAK^KO retinae where the ‘tip-cells’ appeared blunt ended and thick (white arrows). Top bar chart represents the mean vascular network spread *p < 0.05; n.s. = not statistically significant; n = 8 retinae/genotype. Bottom bar chart represents the mean thickness of endothelial tip sprouts. **p < 0.01. n = 12 ECFAK^KO; n = 11 ECFAK^WT retinae/genotype; Scale bars = 50 µm.

Figure 4. Endothelial FAK deficiency reduces VEGF-induced migration and proliferation but enhances apoptosis in vitro

1. In Dunn chamber migration assays, directional migration and single cell speed of migration were quantified. VEGF-induced directional migration is reduced in FAK−/− endothelial cells. Green area denotes statistically significant directional movement according to Rayleigh testing for clustering in a unidirectional manner. Gradient of the red arrow denotes the trend in direction for movement. Lower track plots represent examples of individual cell migration paths. Bar chart represents the quantification of mean cell migration speed in µm/min +s.e.m. FAK+/+ (n = 74); FAK−/− (n = 67); **p < 0.01.

2. Graph represents the mean % of FAK+/+ and FAK−/− endothelial cells that are BrdU-positive after VEGF stimulation + s.e.m. *p < 0.05, n = 1001 FAK+/+ cells and n = 785 FAK−/− cells.

3. Graph represents the mean % of FAK+/+ and FAK−/− endothelial cells that are TUNEL-positive after VEGF stimulation + s.e.m. **p < 0.01, n = 639 FAK+/+ cells and n = 596 FAK−/− cells.

Figure 5. Endothelial FAK deletion impairs Akt phosphorylation but not ERK1/2 phosphorylation

1. Phosphorylation of Akt (Ser 437) upon VEGF-stimulation was reduced in FAK−/− endothelial cells when compared with FAK+/+ cells. Bar chart represents the mean densitometric quantification of phosphorylated Akt divided by total Akt, normalized to non-VEGF treated samples +s.e.m. n = 3 independent experiments.

2. FAK+/+ cells were either treated or not with OHT to test whether OHT treatment alone could affect Akt phosphorylation. Phosphorylation of Akt (Ser 437) upon VEGF-stimulation was not affected by OHT treatment. Bar chart represents the mean densitometric quantification of phosphorylated Akt divided by total Akt and normalized to non-VEGF treated samples +s.e.m. n = 3 independent experiments.

3. Phosphorylation of ERK1/2 upon VEGF-stimulation was similar for FAK+/+ and FAK−/− cells. Bar chart represents the mean densitometric quantification of phosphorylated ERK1/2 divided by total ERK1/2 and normalized to non-VEGF treated samples +s.e.m. n = 3 independent experiments.

4. Western blot analysis for Pyk2 levels in FAK+/+ and FAK−/− endothelial cells reveal no difference in the amount of FAK protein levels. Bar chart represents the mean densitometric reading divided by HSC70 +s.e.m. n = 3 independent experiments.