Direct Supervisor: Bart Vanhaesebroeck, PhD
Location: Ludwig Institute for Cancer Research - London
Descriptive title of research activity: Roles of PI 3-kinase isoforms in angiogenesis
Overall goals: This proposal focuses on the role of PI3Ks in angiogenesis (the growth of new blood vessels), a process critical for normal development and physiology but also in several pathological processes. We seek to uncover the roles of a subset of PI3K isoforms in angiogenesis and in signalling by vascular endothelial growth factors (VEGFs), using a combination of pharmacological and genetic approaches, including gene-targeted mice with isoform-selective inactivation of PI3Ks.
Rationale and significance: This project focuses on p110 and p110, catalytic isoforms of PI3K that signal downstream of tyrosine kinases and Ras. In order to uncover the physiological role of these PI3Ks in the organism, we are in the process of generating various mutant mice in which these PI3Ks are inactivated by gene targeting, in a constitutive or conditional manner (our unpublished results). Using some of these mouse lines, we have obtained good preliminary evidence that p110 and p110 are involved in angiogenesis, and this proposal aims at a detailed molecular characterization of the function and signalling of these PI3K isoforms in endothelial cells. This work will uncover physiological roles of enzymes that have remained elusive for over a decade.
This will not only increase the fundamental understanding of PI3K signalling, vascular biology and angiogenesis but will also open new avenues for therapeutic interference with the PI3K pathway in cancer and other diseases characterised by excessive angiogenesis such as rheumatoid arthritis, atherosclerosis, diabetic retinopathy, endometriosis, psoriasis and obesity.
Description of work and methodology: We have used a gene targeting strategy that does not lead to alterations in the expression of the remaining PI3K subunits. Our gene inactivation strategy is to replace the wild-type p110 gene by a knock-in (KI) allele that encodes a kinase-dead p110 mutated in the ATP binding site. This approach more faithfully mimics the action of small molecule chemical inhibitors than classical gene KO or transgenic approaches and assesses the kinase-dependent activities of p110 isoforms. Our p110 KI mice have been published (Science. 2002:297:1031 and Nature 2004:431:1007). For this proposal, we will use mice in which we have inactivated p110 or p110 in a constitutive or conditional manner (our unpublished results).
a. studies in p110 and p110 KI mice – this part focuses on mice in which p110 and p110 have been inactivated in a constitutive manner in all tissues. Given that mice homozygous for these mutated PI3K alleles are embryonic lethal, heterozygous mice will be used for these studies.
b. endothelium-selective inactivation of p110 and p110 in adult mice. In order to investigate whether neo-angiogenesis defects are intrinsic to the endothelial cells, or related to alterations in the vessel environment, we will inactivate p110 and p110 in the adult vascular system by breeding mice that carry conditional p110 or p110 alleles with transgenic mice expressing Cre recombinase under the control of endothelial-specific promoters. For these studies, we will make use of models in which vascular-specific tamoxifen-inducible Cre-ER(T) recombinase can be induced in a temporal manner.
c. roles of p110 and p110 in vascular endothelial growth factor (VEGF) signalling in cell-based models. Endothelial cell lines have been established from gene-targeted mice, allowing detailed signalling and cell biological investigations in order to gain mechanistic insights into the defects observed in the intact animal.
Methodology: Our proposal makes use of a unique, unpublished panel of gene-targeted mice, which are the results of several years of efforts, amongst other funded by MAIN.
a. neo-angiogenesis in implanted matrigel matrix: in these assays angiogenesis is assessed by recruitment of blood vessel networks into matrigel plugs (supplemented with growth factors or vehicle) implanted into mice, followed by vessel counts and detailed morphological analysis.
b. growth of implanted tumour cells: B16 melanoma and 3 LL (Lewis Lung) carcinoma cells will be implanted subcutaneously or intradermally into p110 mutant mice. Assessment will include tumor progression analysis (growth rate, tumor volume and mass, as well as vessel density determined using H&E-stained paraffin sections) and measurement of apoptosis and proliferation of the host endothelium.
c. aortic ring assay: the aortic ring model, in which thoracic aortic rings are placed between two layers of matrigel overlaid with medium with or without VEGF, will be performed, with assessment and quantification of microvessel outgrowth by phase contrast microscopy.