Lamis Hammoud

Identifying and characterizing novel modulators of blood vessel formation using embryonic stem cells

Mammalian vascular development begins shortly after the initiation of gastrulation. Blood vessels develop by two processes: vasculogenesis and angiogenesis. Vasculogenesis occurs in both the embryo and yolk sac by aggregation of de-novo-forming angioblasts (endothelial precursors) into a primitive network of endothelial tubes known as the primitive vascular plexus. This plexus then undergoes angiogenesis: remodeling involving growth, proliferation, regression, migration and sprouting of new vessels from existing ones, resulting in the formation of the mature circulatory network.

In recent years characterization of vascular mutant phenotypes in mice has improved our understanding of vascular development. Embryonic lethal phenotypes associated with failure to develop different phases of the vasculature have been reported for mutations in VEGF and its receptor VEGFR2 (Flk1), Angiopoietin/Tie, PDGF, TGFB, Ephrin, Notch, Hedgehog, and plexin/semaphorins signaling pathways. However the exact hierarchy of action of these pathways remains to be elucidated and downstream effectors remain to be identified.

The goal of my research is to gain a better understanding of the downstream effectors of the VEGF/Flk1 pathway using mouse embryonic stem (ES) cells. ES cells have the ability to differentiate into many tissues, such as blood vessels, in a manner that resembles embryonic development. To that end I have recently optimized an in vitro mouse ES cell-based differentiation assay using Flk-eGFP ES cells. This assay has been validated using a variety of vascular modulators (see images below). I will next be introducing a Notch-responsive reporter linked to red fluorescent protein into these cells. The development of these cell lines will enable me to do high-throughput screens using RNAi and/or small molecule libraries to further dissect these pathways and possibly identify novel candidates involved in modulating blood vessel formation.

Mouse ES cells containing a Flk1-eGFP reporter were aggregated in suspension for 4 days using the hanging drop method, and then embedded in a collagen type I matrix. EBs were grown in control media (A), or in the presence of VEGF (B), L685 (C), SU5416 (D), VEGF+L685 (E), VEGF+SU5416 (F). The magnification was 5x. Top panel consists of phase-contrast images, while bottom panel consists of the corresponding fluorescence images.

Many of the events that occur during embryonic vascular development are recapitulated in the adult during neoangiogenesis, a central component of tumour growth and metastasis. While the latest generation of antiangiogenic drugs developed for cancer therapy (e.g. avastin, an antibody that inhibits VEGF-A), have been shown to prolong life expectancy, they have serious side effects and relapse associated with more aggressive tumors often occurs due to activation of other VEGF-responsive pathways. The identification of novel angiogenic /antiangiogenic pathways through the kind of study I am undertaking may provide novel candidates for modulating blood vessel formation and thus better therapeutic approaches for treating these cancers.