Angiogenesis is required for the growth and metastasis of cancers. One of the critical events that occurs during tumor formation, is the remodeling of the angiostatic vascular microenvironment to a pro-angiogenic microenvironment. This remodeling is essential to provide the proper communications between vascular endothelial cells (ECs) and the vascular microenvironment that is critical for proper angiogenesis. Therefore, therapeutic interventions that disrupt communications between ECs and their microenvironment represents a potential approach for inhibiting angiogenesis and cancer. Unfortunately, there remains a great deal to learn about the microenvironment molecules that regulate angiogenesis and the molecular mechanisms by which these molecules functions. To address this problem, we recently conducted a microarray based transcriptome analysis of ECs undergoing angiogenesis in vitro. We identified 39 secreted proteins that were not previously associated with angiogenesis and characterized the angiogenic activity of several of these proteins. In particular, MAGP-2 expression was increased in activated ECs and was found to promote angiogenic activities in vitro and angiogenesis in vivo. We concluded that activated ECs secrete MAGP-2 into the vascular microenvironment to promote angiogenesis. These investigations however did not address the molecular mechanism(s) by which MAGP-2 promotes angiogenesis. Our preliminary results now demonstrate that MAGP-2 promotes angiogenesis by blocking Notch signaling in endothelial cells. Paradoxically, we also find that MAGP-2 promotes Notch signaling in non-endothelial cell types. Simultaneously, we have also found that MAGP-2 is specifically localized to endothelial cytoplasmic granules. Based on these key observations, we hypothesize that MAGP-2 promotes angiogenesis by interfering with EC specific angiostatic Notch receptor/ligand interactions, and that MAGP-2 is secreted from EC granules in response to specific pro-angiogenic chemical stimuli. We will address this hypothesis with three specific aims.
Specific aim 1 will take advantage of our observations that MAGP-2 inhibits Notch signaling in ECs but promotes Notch signaling in other cell types. Using this system, we will identify which Notch receptors and ligands are expressed by these cell types, characterize how MAGP-2 interacts with these receptors, ligands, and receptor/ligand pairs, and finally examine the angiogenic consequences of MAGP-2 interaction with various Notch receptor/ligand pairs.
In specific aim 2, we will first determine if MAGP-2 is stored in the common EC specific storage granules (Weibel-Palade bodies) or alternative storage granules. We will then proceed to identify MAGP-2 sequences that are necessary to direct granule storage, and the chemical signals that stimulate MAGP-2 exocytosis from ECs. By investigating these molecular mechanisms by which MAGP-2 may promote angiogenesis, we will learn valuable information about how remodeling of vascular microenvironments contributes to angiogenesis and tumorigenesis. Since the development of Notch and integrin inhibitors are currently being developed as anti-angiogenesis therapeutics, and regulated EC exocytosis is hypothesized to play an important role during tumorigenesis, these investigations will make direct contributions to understanding human health and how we treat human disease.
Cancer continues to be a leading cause of death and suffering in the United States. Since all solid tumors depend on angiogenesis (i.e., infiltration of new blood vessels) strategies to block it may provide therapeutic opportunities to treat cancer. To address this medical need, this proposal seeks to characterize new molecules and mechanisms by which angiogenesis is controlled so that we can one day use this information to block angiogenesis and combat cancer.
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