Research Projects
Notch Signaling in Normal and Pathological Angiogenesis
Our overall goal is to define the mechanisms by which Notch signaling controls normal and pathological angiogenesis and identify therapeutic targets to treat human disease.
Notch signaling has essential and diverse functions in angiogenesis. Abnormal Notch signaling is a significant contributor to cardiovascular diseases; including blindness, stroke, ischemia, arteriovenous malformations (AVM) and tumor angiogenesis. Notch acts to regulate expression of downstream targets that carry out Notch functions (Notch effectors). In endothelial cells, the Notch ligand Dll4 signals in normal and pathological angiogenesis to restrict sprout formation and promote perfusion, whereas the Notch ligand Jag1 may have both Notch activating and inhibitory roles.
Our knowledge of Notch-regulated angiogeneic mediators outside of the VEGF family is limited. We use unbiased screening of Dll4- or Jag1-Notch induced endothelial transcription to identify putative direct Notch targets, validate those targets in loss- and gain-of function angiogenic assays, and determine their in vivo relevance.
We have recently identified several novel classes of rapid-response Notch targets and are starting to explore the role(s) of these genes in angiogenesis. Defining such targets is a significant step forward in obtaining a deeper knowledge of the role of Notch in healthy vessels and human disease. Current work in the lab explores the roles of Notch-driven angiogenesis in tumor formation and metastasis (described below), cardiovascular disease, and aging-related cognitive decline. In particular, several projects focus on specific Notch effectors and how they regulate specific aspects of Notch-mediated endothelial phenotypes.
Notch signaling in tumor angiogenesis
Triple-negative breast cancer (TNBC) is diagnosed in approximately 40,000 women each year in the United States. Unlike other forms of breast cancer, TNBC generally requires treatment with chemotherapy, radiation, or surgery. Targeted therapies for TNBC are badly needed to improve the quality of life for patients. TNBC tumors frequently overexpress Notch ligand Jag1, which is thought to promote tumor growth by increasing the tumor’s ability to recruit blood vessels from neighboring tissue so that the tumor receives oxygen and nutrients. Researchers have previously attempted to stop tumor growth by blocking all of Notch signaling, but currently available inhibitors of Notch signaling have failed due to severe gastrointestinal toxicity or vascular malformations.
The lab has previously developed a class of proteins, called Notch decoys which only block specific elements of the Notch signaling pathway and which show little or no toxicity. Jag1-specific Notch decoys reduce tumor growth, tumor blood vessels, and perfusion. Current work in the lab is aimed at elucidating how Jag1 affects the interactions between tumor cells and adjacent blood vessels to control tumor growth and the ability of tumor cells to enter the bloodstream and metastasize.
Most studies on Notch-mediated tumor angiogenesis focus on Notch1 and the ligand Dll4. Current work in the lab is also exploring critical roles of other Notch receptors and ligands in TNBC and other cancer types.
Health disparities in TNBC
Triple-negative breast cancer (TNBC) occurs at disproportionately high rates in Black Americans and contributes to poor clinical outcomes in Black patients with breast cancer. Current work in the lab is exploring selected candidate proteins that may contribute to the differences in incidence and progression of TNBC in different racial and ethnic groups and how these underlying differences may indicate different optimal treatments.
CLIC proteins in vascular function
Blood vessels must maintain a delicate balance between sufficient permeability to pass necessary proteins, nutrients, signaling factors, and cells, while maintaining sufficient barrier function to prevent edema, dysregulated immune response, and thrombosis. Chloride intracellular channels (CLICs) are a poorly understood family of six metamorphic proteins with potential functions either as ion channels or glutathione-S-transferases, however, these activities have not been linked to specific intracellular processes or physiological functions. CLIC1 and CLIC4 are expressed in endothelial cells and are vital for angiogenesis in vitro and in vivo. Recent work in our lab has demonstrated that these proteins play critical roles in endothelial response to S1P, a central signaling factor in the control of endothelial barrier formation and vascular leakiness. Current work in the lab is determining the gene-specific and common functions of CLIC1 and CLIC4 that allow these proteins to link GPCRs, like the S1P receptor and other endothelial receptors, to downstream GTPase and vascular function.
Vascular function of Anthrax Toxin Receptors
The Anthrax Toxin Receptors (ANTXR) AntxR1 and AntxR2 are linked to multiple vascular disease states, including tumor angiogenesis, wound healing, and the human syndromes GAPO and Juvenile Systemic Hyalinosis (JSH), which manifest with damaged vessels. Our lab has established that a key mechanism of AntxR action is to regulate the remodeling and homeostasis of the extracellular matrix and prevent fibrosis. Somatic mouse mutants of AntxR1 and R2 exhibit excessive extracellular matrix, increased fibrosis, and profound vascular defects. Expression studies and in vitro analysis suggest that ANTXRs have functions in both endothelial cell and vascular mural cells. Current work in the lab is determining which cell type(s) require ANTXRs for normal vascular function and the mechanisms of ANTXR regulation of fibrosis and fibrotic disease.
Modeling with RiboTag
Many projects in the lab involve unbiased expression profiling of endothelial cells. Endothelial cells are generally a small component of the overall organ tissue and widely dispersed and isolation of endothelial populations previously required laborious tissue digestion and disaggregation. Unfortunately, tissue digestion has the potential to either artificially disrupt Notch receptor-ligand interactions, which are dependent on cell-cell contacts, or artificially activate Notch signaling, which is strongly activated by trypsin/EDTA.
As a result, our lab has optimized use of the endothelial RiboTag model, which permits cell type-specific RNA isolation without digestion of cell-cell contacts (Sanz et al, PNAS, 2009). RiboTag mice carry a conditional mutation in ribosomal protein L22 (Rpl22) locus which contains a loxP-flanked wildtype exon 4 followed by a modified exon 4 encoding an in- frame fusion of the Rpl22 C-terminus to hemagglutinin (HA) peptide. Cells that express Cre recombinase will express the HA-tagged ribosomal protein, which is incorporated into translating ribosomes and facilitates direct immunoprecipitation (IP) of translated mRNA. Using this technology, our lab has been able to profile high purity endothelial transcriptomes from animals undergoing a variety of treatments.