Tanguy Lechertier

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.

Focal adhesion kinase and tumour angiogenesis

Angiogenesis, the formation of new blood vessels from pre‐existing ones, is essential for tumour development. It is initiated and regulated by growth factors via their surface receptors, which activate several intracellular signalling pathways in endothelial cells. Cell adhesion molecules, such as integrins, also regulate angiogenesis. Despite these facts, inhibitors of endothelial cell growth factor receptors or integrins have not been as effective as initially hoped in the long‐term inhibition of angiogenesis in cancer patients. Signalling downstream of growth factor receptors and integrins converge on the ubiquitously expressed non‐receptor tyrosine kinase focal adhesion kinase (FAK). FAK is involved in endothelial cell proliferation, migration and survival, is up‐regulated in many cancers and has recently been shown to control tumour angiogenesis. Indeed, FAK inhibitors are presently being developed for the treatment of cancer. However, recent studies have indicated the complexities of understanding the precise role for FAK in angiogenesis. Here we have summarized some of the key features of FAK, addressed some of the apparently contradictory roles of this molecule in angiogenesis and provided some perspectives for future studies. Copyright

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.

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.

Gene expression analysis in human breast cancer associated blood vessels.

Angiogenesis is essential for solid tumour growth, whilst the molecular profiles of tumour blood vessels have been reported to be different between cancer types. Although presently available anti-angiogenic strategies are providing some promise for the treatment of some cancers it is perhaps not surprisingly that, none of the anti-angiogenic agents available work on all tumours. Thus, the discovery of novel anti-angiogenic targets, relevant to individual cancer types, is required. Using Affymetrix microarray analysis of laser-captured, CD31-positive blood vessels we have identified 63 genes that are upregulated significantly (5–72 fold) in angiogenic blood vessels associated with human invasive ductal carcinoma (IDC) of the breast as compared with blood vessels in normal human breast. We tested the angiogenic capacity of a subset of these genes. Genes were selected based on either their known cellular functions, their enriched expression in endothelial cells and/or their sensitivity to anti-VEGF treatment; all features implicating their involvement in angiogenesis. For example, RRM2, a ribonucleotide reductase involved in DNA synthesis, was upregulated 32-fold in IDC-associated blood vessels; ATF1, a nuclear activating transcription factor involved in cellular growth and survival was upregulated 23-fold in IDC-associated blood vessels and HEX-B, a hexosaminidase involved in the breakdown of GM2 gangliosides, was upregulated 8-fold in IDC-associated blood vessels. Furthermore, in silico analysis confirmed that AFT1 and HEX-B also were enriched in endothelial cells when compared with non-endothelial cells. None of these genes have been reported previously to be involved in neovascularisation. However, our data establish that siRNA depletion of Rrm2, Atf1 or Hex-B had significant anti-angiogenic effects in VEGF-stimulated ex vivo mouse aortic ring assays. Overall, our results provide proof-of-principle that our approach can identify a cohort of potentially novel anti-angiogenic targets that are likley to be, but not exclusivley, relevant to breast cancer.

Pancreatic stellate cells regulate blood vessel density in the stroma of pancreatic ductal adenocarcinoma

Background/objectives The vascular heterogeneity of pancreatic ductal adenocarcinoma (PDAC) has never been characterised. We analysed the heterogeneous vascular density of human PDAC along with its prognostic correlation. Methods Tissue Microarrays of 87 patients with different pancreatico-biliary pathologies were analysed in an automated manner (Ariol™) after CD31 staining to assess vascular density in juxta-tumoral and panstromal compartments. In vitro and ex vivo assays were carried out to assess the role of PSC. Results PDAC has a distinct vascular density and distribution of vessels compared to cholangiocarcinoma. The PDAC juxta-tumoral stroma was hypovascular and the normal adjacent rim was hypervascular compared to the panstromal compartment. These features adversely affected patient prognosis, suggesting a model for spatio-temporal PDAC evolution. Mice aortic rings and 3D organotypic cultures demonstrated pro- and anti-angiogenic signalling from activated PSC and cancer cells respectively. ATRA-induced quiescence suppressed the pro-angiogenic activity of PSC. Conclusion Human PDAC has variable vascularity at microscopic level suggesting that novel stromal directed therapies would need to be determined by pathological characteristics.

Multiple roles of integrin-α3 at the neuromuscular junction.

ABSTRACT The neuromuscular junction (NMJ) is the synapse between motoneurons and skeletal muscle, and is responsible for eliciting muscle contraction. Neurotransmission at synapses depends on the release of synaptic vesicles at sites called active zones (AZs). Various proteins of the extracellular matrix are crucial for NMJ development; however, little is known about the identity and functions of the receptors that mediate their effects. Using genetically modified mice, we find that integrin-α3 (encoded by Itga3), an adhesion receptor at the presynaptic membrane, is involved in the localisation of AZ components and efficient synaptic vesicle release. Integrin-α3 also regulates integrity of the synapse – mutant NMJs present with progressive structural changes and upregulated autophagy, features commonly observed during ageing and in models of neurodegeneration. Unexpectedly, we find instances of nerve terminal detachment from the muscle fibre; to our knowledge, this is the first report of a receptor that is required for the physical anchorage of pre- and postsynaptic elements at the NMJ. These results demonstrate multiple roles of integrin-α3 at the NMJ, and suggest that alterations in its function could underlie defects that occur in neurodegeneration or ageing. Summary: Adhesion receptor integrin-α3 is involved in presynaptic differentiation, synaptic maintenance and nerve terminal anchorage at the neuromuscular synapse.

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.

203 Hypoxia-inducible Factor-1 Regulates Matrix Metalloproteinase-14 Expression: Underlying Effects of Hypoxia and Statins

Introduction Matrix metalloproteinase-14 (MMP14) plays an important role in atherosclerosis and angiogenesis. There is evidence indicating that hypoxia and inflammatory stimuli up-regulate MMP14 expression whereas statins have an opposite effect. However, the molecular mechanism by which these factors modulate MMP14 expression is unclear. We sought to investigate whether it involves the transcription factor hypoxia-inducible factor-1 (HIF1). Methods and Results A bioinformatics analysis of the MMP14 gene promoter identified two regions matched with the consensus sequence of HIF1-responsive element (HRE). In vitro DNA-protein interaction assay demonstrated the ability of these sequences to complex with HIF1α/HIF1β, and chromatin immunoprecipitation showed HIF1 binding to the MMP14 promoter in vascular endothelial cells under hypoxia. Cell transfection and promoter-reporter assays demonstrated that augmenting HIF1α/HIF1β expression increased MMP14 gene promoter activity and this effect was abolished by mutating the HREs in the MMP14 promoter. MMP14 mRNA and protein assays showed that hypoxia increased MMP14 expression in endothelial cells but this effect diminished in endothelial cells with HIF1α knockdown or knockout. HIF1α knockdown/knockout or MMP14 inhibition by a blocking antibody attenuated hypoxia-induced endothelial cell proliferation/migration and angiogenesis. Simvastatin reduced HIF1α in endothelial cells and mitigated hypoxia- or HIF1-induced MMP14 expression, endothelial cell proliferation/migration, and angiogenesis. Conclusions The results of this study indicate that hypoxia increases MMP14 expression in vascular endothelial cells by inducing HIF1 binding to HREs in the MMP14 gene promoter, and this action is attenuated by simvastatin.

Endogenous ribosomal protein L29 (RPL29): a newly identified regulator of angiogenesis in mice

SUMMARY Cellular ribosomal protein L29 (RPL29) is known to be important in protein synthesis, but its function during angiogenesis has never been described before. We have shown previously that mice lacking β3-integrins support enhanced tumour angiogenesis and, therefore, deletion of endothelial αvβ3 can provide a method for discovery of novel regulators of tumour angiogenesis. Here, we describe significant upregulation of RPL29 in β3-null endothelial cells at both the mRNA and protein level. Ex vivo, we show that VEGF-stimulated microvessel sprouting was reduced significantly in Rpl29-heterozygous and Rpl29-null aortic ring assays compared with wild-type controls. Moreover, we provide in vivo evidence that RPL29 can regulate tumour angiogenesis. Tumour blood vessel density in subcutaneously grown Lewis lung carcinomas was reduced significantly in Rpl29-mutant mice. Additionally, depletion of Rpl29 using RNA interference inhibited VEGF-induced aortic ring sprouting, suggesting that anti-RPL29 strategies might have anti-angiogenic potential. Overall, our results identify that loss or depletion of RPL29 can reduce angiogenesis in vivo and ex vivo.