Notch Peptibodies are Anti-Angiogenic and Signaling via Notch Proteins Regulate CC-Chemokine Production

Angiogenesis is a tightly regulated multi-step process defined as the development of new blood vessels emanating from existing vessels. Under physiological conditions, this activity is confined to embryonic and post-natal development as well as tissue growth and wound healing during the adult life. Angiogenic aberrations have been implicated in several pathologies such as in cancer, vascular malformations, and retinopathy. Understanding the role of Notch in pathological angiogenesis has long been a key area of interest for therapeutic intervention. Notch signaling functions in angiogenesis through the regulation of endothelial cell-fate decisions as well as crosstalk with other angiogenic pathways. Mammals express four homologous notch receptors (Notch 1-4) and five ligands (Jagged (JAG) 1, 2 and delta-like ligand (DLL) 1,3, 4). The critical role of Notch1 and its interacting partners has been well described in the vasculature and angiogenesis. In contrast, Notch4, which is exclusively expressed in the vasculature, remains an enigma as little is known about which, if any, Notch ligands bind to Notch4. Further, the role of Notch4 signaling in angiogenesis is not fully understood. It has been reported that in developmental angiogenesis, Notch4 has an overlapping role with Notch1, but more c has recently emerged demonstrating a unique role for endothelial Notch4 as pro-angiogenic in pathological settings. Thus, uncovering the role of Notch4 signaling, and its binding partners, is of critical importance in order to paint a complete picture of Notch signaling in angiogenesis and endothelial function. To investigate the distinct or overlapping functions of the extracellular domains, I compared core binding domains of the Notch1 and Notch4 receptor and identified potential binding partners (Chapter 3). To achieve this, I investigated a novel alternative means to targeting the Notch signaling pathway using a peptibody-based approach by combining the Notch core binding domain with the Fc domain of human IgG. Using these constructs, I report here for the first time a conserved binding domain within Notch4 that promotes interaction with DLL4 and JAG1. Further, I demonstrated that Notch peptibodies act as decoys by binding to Notch ligands, downregulating Notch signaling, and decreasing sprout formation, demonstrating the anti-angiogenic effects of these agents. To investigate the function of the intracellular domains (ICDs), I studied whether distinct Notch intracellular domains regulate different sets of endothelial transcriptional targets (Chapter 4). Variants of human intracellular Notch1 and intracellular Notch4 proteins were overexpressed in HUVECs to activate Notch signaling and transcriptional profiling was performed. This experimental approach revealed upregulation of CC-chemokines, suggesting a direct role of Notch in inflammatory regulation. This regulation was further confirmed by ELISA of secreted proteins found in conditioned media as well as in in vivo mouse models. Taken together, these findings shed light on Notch4 binding partners and signaling capacity and identified Notch signaling as a novel regulator of inflammatory chemokines. Further, this body of work demonstrates that Notch peptibodies may provide new therapeutic opportunities based upon their unique Notch inhibitory profile and anti-angiogenic properties.