Bernardo Tavora

Use of the mouse aortic ring assay to study angiogenesis

Here we provide a protocol for quantitative three-dimensional ex vivo mouse aortic ring angiogenesis assays, in which developing microvessels undergo many key features of angiogenesis over a timescale similar to that observed in vivo. The aortic ring assay allows analysis of cellular proliferation, migration, tube formation, microvessel branching, perivascular recruitment and remodeling—all without the need for cellular dissociation—thus providing a more complete picture of angiogenic processes compared with traditional cell-based assays. Our protocol can be applied to aortic rings from embryonic stage E18 through to adulthood and can incorporate genetic manipulation, treatment with growth factors, drugs or siRNA. This robust assay allows assessment of the salient steps in angiogenesis and quantification of the developing microvessels, and it can be used to identify new modulators of angiogenesis. The assay takes 6–14 d to complete, depending on the age of the mice, treatments applied and whether immunostaining is performed.

Endothelial FAK is required for tumour angiogenesis

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.

αvβ3 Integrin Limits the Contribution of Neuropilin-1 to Vascular Endothelial Growth Factor-induced Angiogenesis

Both vascular endothelial growth factor receptors (VEGFR) and integrins are major regulators of VEGF-induced angiogenesis. Previous work has shown that β3 integrin can regulate negatively VEGFR2 expression. Here we show that β3 integrin can regulate negatively VEGF-mediated angiogenesis by limiting the interaction of the co-receptor NRP1 (neuropilin-1) with VEGFR2. In the presence of αvβ3 integrin, NRP1 contributed minimally to VEGF-induced angiogenic processes in vivo, ex vivo, and in vitro. Conversely, when β3 integrin expression is absent or low or its function is blocked with RGD-mimetic inhibitors, VEGF-mediated responses became NRP1-dependent. Indeed, combined inhibition of β3 integrin and NRP1 decreased VEGF-mediated angiogenic responses further than individual inhibition of these receptors. We also show that αvβ3 integrin can associate with NRP1 in a VEGF-dependent fashion. Our data suggest that β3 integrin may, in part, negatively regulate VEGF signaling by sequestering NRP1 and preventing it from interacting with VEGFR2.

Endothelial-cell FAK targeting sensitizes tumours to DNA-damaging therapy.

Chemoresistance is a serious limitation of cancer treatment. Until recently, almost all the work done to study this limitation has been restricted to tumour cells. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-κB activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically.

Endothelial α3β1-Integrin Represses Pathological Angiogenesis and Sustains Endothelial-VEGF

Integrin alpha3beta1 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of alpha3beta1 can affect angiogenesis either positively or negatively, either a direct in vivo role for alpha3beta1 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of alpha3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where alpha3 is deleted specifically in endothelial cells (ec-alpha3-/-). Here we show that ec-alpha3-/- mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that alpha3beta1 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial alpha3beta1 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.

Genetic ablation of the alpha 6-integrin subunit in Tie1Cre mice enhances tumour angiogenesis†

Laminins are expressed highly in blood vessel basement membranes and have been implicated in angiogenesis. α6β1‐ and α6β4‐integrins are major receptors for laminins in endothelial cells, but the precise role of endothelial α6‐integrin in tumour angiogenesis is not clear. We show that blood vessels in human invasive ductal carcinoma of the breast have decreased expression of the α6‐integrin‐subunit when compared with normal breast tissue. These data suggest that a decrease in α6‐integrin‐subunit expression in endothelial cells is associated with tumour angiogenesis. To test whether the loss of the endothelial α6‐integrin subunit affects tumour growth and angiogenesis, we generated α6fl/fl‐Tie1Cre+ mice and showed that endothelial deletion of α6‐integrin is sufficient to enhance tumour size and tumour angiogenesis in both murine B16F0 melanoma and Lewis cell lung carcinoma. Mechanistically, endothelial α6‐integrin deficiency elevated significantly VEGF‐mediated angiogenesis both in vivo and ex vivo. In particular, α6‐integrin‐deficient endothelial cells displayed increased levels of VEGF‐receptor 2 (VEGFR2) and VEGF‐mediated downstream ERK1/2 activation. By developing the first endothelial‐specific α6‐knockout mice, we show that the expression of the α6‐integrin subunit in endothelial cells acts as a negative regulator of angiogenesis both in vivo and ex vivo. Copyright

FAK-heterozygous mice display enhanced tumour angiogenesis

Genetic ablation of endothelial Focal Adhesion Kinase (FAK) can inhibit pathological angiogenesis, suggesting that loss of endothelial FAK is sufficient to reduce neovascularisation. Here we show that reduced stromal-FAK expression in FAK-heterozygous mice unexpectedly enhances both B16F0 and CMT19T tumour growth and angiogenesis. We further demonstrate that cell proliferation and microvessel sprouting, but not migration, are increased in serum-stimulated FAK-heterozygous endothelial cells. FAK-heterozygous endothelial cells display an imbalance in FAK phosphorylation at pY397 and pY861 without changes in Pyk2 or Erk1/2 activity. By contrast, serum-stimulated phosphorylation of Akt is enhanced in FAK-heterozygous endothelial cells and these cells are more sensitive to Akt inhibition. Additionally, low doses of a pharmacological FAK inhibitor, although too low to affect FAK autophosphorylation in vitro, can enhance angiogenesis ex vivo and tumor growth in vivo. Our results highlight a potential novel role for FAK as a non-linear, dose-dependent regulator of angiogenesis where heterozygous levels of FAK enhance angiogenesis.

Focal Adhesion Kinase (FAK) tyrosine 397E mutation restores the vascular leakage defect in endothelium-specific FAK-kinase dead mice: Endothelial FAKY397E mutation and tumour vascular leakage

Focal adhesion kinase (FAK) inhibitors have been developed as potential anticancer agents and are undergoing clinical trials. In vitro activation of the FAK kinase domain triggers autophosphorylation of Y397, Src activation, and subsequent phosphorylation of other FAK tyrosine residues. However, how FAK Y397 mutations affect FAK kinase‐dead (KD) phenotypes in tumour angiogenesis in vivo is unknown. We developed three Pdgfb‐iCreert‐driven endothelial cell (EC)‐specific, tamoxifen‐inducible homozygous mutant mouse lines: FAK wild‐type (WT), FAK KD, and FAK double mutant (DM), i.e. KD with a putatively phosphomimetic Y397E mutation. These ECCre+;FAKWT/WT, ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre+;FAKWT/WT and ECCre−;FAKWT/WT control mice. In contrast, tumour growth and vessel density were decreased in ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice, as compared with Cre − littermates. Despite no change in the percentage of perfused vessels or pericyte coverage in either genotype, tumour hypoxia was elevated in ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice. Furthermore, although ECCre+;FAKKD/KD mice showed reduced blood vessel leakage, ECCre+;FAKDM/DM and ECCre−;FAKDM/DM mice showed no difference in leakage. Mechanistically, fibronectin‐stimulated Y397 autophosphorylation was reduced in Cre+;FAKKD/KD ECs as compared with Cre+;FAKWT/WT cells, with no change in phosphorylation of the known Src targets FAK‐Y577, FAK‐Y861, FAK‐Y925, paxillin‐Y118, p130Cas‐Y410. Cre+;FAKDM/DM ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE‐cadherin‐pY658 levels in Cre+;FAKKD/KD ECs were rescued in Cre+FAKDM/DM ECs, corresponding with the rescue in vessel leakage in the ECCre+;FAKDM/DM mice. We show that EC‐specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAKDM/DM mice restored the KD‐dependent tumour vascular leakage observed in ECCre+;FAKKD/KD mice in vivo. This study opens new fields in in vivo FAK signalling.

Haematopoietic focal adhesion kinase deficiency alters haematopoietic homeostasis to drive tumour metastasis

Metastasis is the main cause of cancer-related death and thus understanding the molecular and cellular mechanisms underlying this process is critical. Here, our data demonstrate, contrary to established dogma, that loss of haematopoietic-derived focal adhesion kinase (FAK) is sufficient to enhance tumour metastasis. Using both experimental and spontaneous metastasis models, we show that genetic ablation of haematopoietic FAK does not affect primary tumour growth but enhances the incidence of metastasis significantly. At a molecular level, haematopoietic FAK deletion results in an increase in PU-1 levels and decrease in GATA-1 levels causing a shift of hematopoietic homeostasis towards a myeloid commitment. The subsequent increase in circulating granulocyte number, with an increase in serum CXCL12 and granulocyte CXCR4 levels, was required for augmented metastasis in mice lacking haematopoietic FAK. Overall our findings provide a mechanism by which haematopoietic FAK controls cancer metastasis.

Generation of point‐mutant FAK knockin mice

Focal adhesion kinase is a non‐receptor protein tyrosine kinase with signaling functions downstream of integrins and growth factor receptors. In addition to its role in adhesion, migration, and proliferation it also has non‐kinase scaffolding functions in the nucleus. Focal adhesion kinase (FAK) activation involves the following: (1) ligand bound growth factors or clustered integrins activate FAK kinase domain; (2) FAK autophosphorylates tyrosine (Y) 397; (3) Src binds pY397 and phosphorylates FAK at various other sites including Y861; (4) downstream signaling of activated FAK elicits changes in cellular behavior. Although many studies have demonstrated roles for the kinase domain, Y397 and Y861 sites, in vitro much less is known about their functions in vivo. Here, we report the generation of a series of FAK‐mutant knockin mice where mutant FAK, either kinase dead, non‐phosphorylatable mutants Y397F and Y861F, or mutant Y397E—containing a phosphomimetic site that results in a constitutive active Y397, can be expressed in a Cre inducible fashion driven by the ROSA26 promoter. In future studies, intercrossing these mice with FAKflox/flox mice and inducible cre‐expressing mice will enable the in vivo study of mutant FAK function in the absence of endogenous FAK in a spatially and temporally regulated fashion within the whole organism. genesis 52:907–915, 2014.