Role for Notch4 in Vascular Malformation Pathobiology

Vascular malformations are abnormal vessels that improperly formed during fetal development or shortly after birth. They can include different types of vessels such as arteries, veins, and lymphatics, and are termed arteriovenous, venous, and lymphatic malformations, respectively. Overall, vascular malformations affect 1.2%-1.5% of the general population and range from a prevalence of 0.1% to 1% of births. Vascular malformations can cause complications, some of which can be life-threatening. These can include severe hemorrhaging, infections, clotting abnormalities, deep vein thrombosis, pain, and difficulties with ambulation. Despite these severe complications, the development of U.S. Food and Drug Administration-approved treatment for vascular malformations has been hindered by lack of understanding of all the players that contribute to vascular malformation development.

Notch signaling is an evolutionarily conserved pathway that is known to regulate vascular development. In mice, both loss and gain of Notch signaling the vasculature lead to development of arteriovenous malformations. Numerous therapies that target Notch signaling pathway are in clinical development or trials, and their use for vascular malformations has been proposed. Thus, it is essential to understand the functions of Notch in vascular development and maintenance, and how its misregulation contributes to vascular malformations pathobiology.

We have generated two new mouse models of vascular malformations using a novel Notch4 mutant mouse line (N4Ex1) generated in the Principal Investigator's lab. Mice with only one copy of Notch4 develop many of the hallmarks of the vascular malformation syndrome, Hereditary Hemorrhagic Telangiectasia. This includes telangiectasias in the feet, tongue, and intestines, vascular defects and coagulopathies in the liver, as well as pulmonary arteriovenous shunts. When Notch4 was specifically deleted from the lymphatic vessels, these mice develop lymphatic malformations in the dermis, liver, and lungs. Our studies of human lymphatic malformations identified four candidate mutations in Notch4 predicted to disrupt its normal function. Thus, we hypothesize that Notch4 functions in vascular maturation and maintenance and that its disruption contributes to the development of vascular malformations.

To address this hypothesis, we propose the following aims:

Aim 1: Determine endothelial Notch4 functions in mice.

We will further characterize the vascular malformation phenotypes in N4Ex1 mice with a goal of understanding the roles of Notch4 in vascular maturation and homeostasis.

Aim 2: Characterization of putative lymphatic malformation Notch4 variants.

We will generate endothelial cells that express the Notch4 mutants identified in lymphatic malformations LM patients and determine how the mutations affect the protein and the ability of the cells to develop into arteries, veins, and lymphatics.

Aim 3: Determine the endothelial Notch4 transcriptional profile.

To understand how Notch4 regulates endothelial cells, we will profile the mRNA from blood and lymphatic endothelial cells isolated from N4Ex1and control mice. To complement these studies, endothelial cells with Notch4 knocked down will be stimulated in culture and mRNA sequencing performed. The two datasets will be analyzed together to generate a Notch4-specific endothelial gene expression profile.

The goals of the proposed studies are to understand how Notch4 deficiencies contribute to vascular malformations. Our innovative proposal addresses the PRMRP vascular malformations area of encouragement, as it is a study that will provide the field with new insights into 'the natural history, genetics, and pathogenesis of vascular malformations,' as well as has the potential identify novel drug targets for the treatment of vascular malformations.