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LYMPHANGIOGENOMICS: Genome-Wide Discovery and Functional Analysis of Novel Genes in Lymphangiogenesis

Final Report Summary - LYMPHANGIOGENOMICS (Genome-Wide Discovery and Functional Analysis of Novel Genes in Lymphangiogenesis)

The aim of this project was to discover novel genes important for lymphatic vascular versus blood vascular development and function and to study the functional role and therapeutic potential of their gene products in lymphangiogenesis using state-of-the-art technologies. The lymphatic vasculature is essential for the maintenance of fluid balance in the body, for immune defence, and for the uptake of dietary fat. Absent or damaged lymphatic vessels may lead to lymphoedema, a chronic and disfiguring swelling of the extremities, sometimes necessitating the amputation of the affected limb.

In addition, lymphatic vessels promote metastatic spread of cancer cells to distant organs - a leading cause of death in patients with cancer, and a major obstacle in the design of effective therapies. Lymphatic vessels were identified hundreds of years ago, yet very limited understanding exists of their development, function, and the molecular mechanisms underlying their disease process.

The methods used included large-scale knockout and knock-down of the mouse genome, the embryonic stem (ES) cell technology, knock-down of zebrafish genes by morpholino-antisense and positional cloning of disease susceptibility genes involved in lymphangiogenesis.

These studies provided a new and fundamental understanding of the molecular and cellular basis of lymphangiogenesis and therefore enabled scientists to develop therapies to suppress the growth of lymphatic vessels (e.g. for cancer, inflammatory diseases) or to stimulate their growth (e.g. for tissue ischemia, lymphoedema).

The project achieved many results, one of which was in sprouting angiogenesis - specialised endothelial tip cells lead the outgrowth of blood-vessel sprouts towards gradients of vascular endothelial growth factor (VEGF)-A. VEGF-A is also essential for the induction of endothelial tip cells, but it is not known how single tip cells are selected to lead each vessel sprout, and how tip-cell numbers are determined.

The team presented evidence that delta-like 4 (Dll4)-Notch1 signalling regulates the formation of appropriate numbers of tip cells to control vessel sprouting and branching in the mouse retina. They showed that inhibition of Notch signalling using gamma-secretase inhibitors, genetic inactivation of one allele of the endothelial Notch ligand Dll4, or endothelial-specific genetic deletion of Notch1, all promote increased numbers of tip cells. Conversely, activation of Notch by a soluble jagged1 peptide leads to fewer tip cells and vessel branches.

Dll4 and reporters of Notch signalling are distributed in a mosaic pattern among endothelial cells of actively sprouting retinal vessels. At this location, Notch1-deleted endothelial cells preferentially assume tip-cell characteristics.

Together, results suggest that Dll4-Notch1 signalling between the endothelial cells within the angiogenic sprout serves to restrict tip-cell formation in response to VEGF, thereby establishing the adequate ratio between tip and stalk cells required for correct sprouting and branching patterns. This model offers an explanation for the dose-dependency and haploinsufficiency of the Dll4 gene, and indicates that modulators of Dll4 or Notch signalling, such as gamma-secretase inhibitors developed for Alzheimer's disease, might find usage as pharmacological regulators of angiogenesis.

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