Over the past four decades, endothelial cells lining the vessel wall have become an intense subject of clinical interest since ease of accessibility allows for effective drug targeting. Dysregulation of vessel growth is associated with wide range of disease states such as diabetic retinopathy, wet form of age related macular degeneration, and tumor growth. Tumor growth is angiogenesis dependent. The first step in tumor angiogenesis is endothelial cell activation and directional migration of endothelial cells towards the tumor. Therefore, understanding the mechanisms of directional migration is critical for development of therapeutics that can selectively target tumor endothelial cells from taking this first step. The goal of this laboratory is to identify and study candidate genes that are selectively upregulated in tumor vasculature. Robo4 is an excellent target since it is expressed selectively in tumor vessels and is critical for directional migration of endothelial cells. Further, the cell surface expression of Robo4 allows easy access to potential drugs. Importantly, specific signaling complex assembly on Robo4, if identified to be responsible for directional migration can also be targeted. Previously, we have shown that robo4 is essential for angiogenesis in vivo in zebrafish and activates Rho GTPases in endothelial cells in vitro. Preliminary results suggest that: (A) Robo1 and Robo4 interact and share common signaling molecules at both the cell surface and intracellular level;(B) A complex of proteins including IRSp53, Mena, Cdc42-GTP, and Vilse together assemble in endothelial cells in response to Robo4; (C) Members of the Sry-related high-mobility-group (Sox) family interact with Robo4's cytoplasmic tail. We hypothesize that: """"""""Endothelial cell surface receptors Robo1 and Robo4 co-operatively interact with Slit2 to direct cell migration via a specific complex assembly of intracellular signaling proteins (Cdc42-GTP, Vilse, IRSp53, Mena, Sox)."""""""" To test this hypothesis in aim 1, we will investigate whether Robo1 and Robo4 co- operatively interact with Slit2 and direct signaling complex assembly of Vilse and Mena in endothelial cells using a combination of molecular, biochemical and imaging methods.
In aim 2, we will identify the mechanism of assembly of IRSp53, Mena, Cdc42-GTP, Vilse and Sox with Robo1 and Robo4's cytoplasmic tail in a signaling complex responsible for directing endothelial cell migration using a combination of in vitro biochemical and in vivo functional assays in zebrafish. We anticipate that by accomplishing aims 1 and 2, we will understand how Robo1 and Robo4 dictate the endothelial tip cell to navigate through complex milieu by unraveling the similarities between the two molecules in triggering signaling in endothelial cells, the order in which the different signaling molecules assemble on Robo1 and Robo4's cytoplasmic tail, the cues they respond to, and the integration of different signaling components to form filopodia. Each mechanistic step is a putative target and drugs targeting these steps will benefit diseases characterized by excessive angiogenesis and vascular leakage.

Public Health Relevance

Tumor growth is angiogenesis dependant. We are interested in identifying vascular targets preferably cell surface molecules that are differentially expressed in tumor versus normal endothelial cells. This proposal studies one such target namely Robo4. Robo4 is highly expressed in tumor vessels. Tumor vessels are leaky, and the vascular bed often has a chaotic appearance. The first step in tumor angiogenesis is endothelial cell activation and directional migration of endothelial cells towards the tumor. Therefore, understanding the mechanisms of directional migration is critical for development of therapeutics that can selectively target tumor endothelial cells from taking this first step. This proposal will identify the mechanisms utilized by Robos in mediating directional migration in endothelial cells. We propose two aims to investigate mechanisms used by Robos to mediate directional migration. Accomplishing both aims will determine how Robo1 and Robo4 mechanistically assemble a signaling complex inside endothelial cells in response to Slit2 ligand, thereby co-ordinating directional migration of endothelial cells. This study will identify intracellular molecules common to the Robo1 and Robo4 signaling apparatus that serve as targets for drug design benefiting conditions associated with deregulated endothelial cell migration such as those associated with tumor growth.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
Project #
Application #
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Gao, Yunling
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Medical College of Wisconsin
Schools of Medicine
United States
Zip Code
Neumann, Terrence S; Span, Elise A; Kalous, Kelsey S et al. (2015) Identification of inhibitors that target dual-specificity phosphatase 5 provide new insights into the binding requirements for the two phosphate pockets. BMC Biochem 16:19
Schupp, Marcus-Oliver; Waas, Matthew; Chun, Chang-Zoon et al. (2014) Transcriptional inhibition of etv2 expression is essential for embryonic cardiac development. Dev Biol 393:71-83
Nayak, Jaladhi; Gastonguay, Adam J; Talipov, Marat R et al. (2014) Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins. BMC Biochem 15:27
Zhou, Xia; Fan, Lucy X; Li, Keguo et al. (2014) SIRT2 regulates ciliogenesis and contributes to abnormal centrosome amplification caused by loss of polycystin-1. Hum Mol Genet 23:1644-55
Wang, Ling; Cossette, Stephanie M; Rarick, Kevin R et al. (2013) Astrocytes directly influence tumor cell invasion and metastasis in vivo. PLoS One 8:e80933
Leigh, Noah R; Schupp, Marcus-Oliver; Li, Keguo et al. (2013) Mmp17b is essential for proper neural crest cell migration in vivo. PLoS One 8:e76484
Francois, Mathias; Ramchandran, Ramani (2012) Studies on Axenfeld-Rieger syndrome patients and mice reveal Foxc1's role in corneal neovascularization. Proc Natl Acad Sci U S A 109:1818-9
Lakshmikanthan, Sribalaji; Sobczak, Magdalena; Chun, Changzoon et al. (2011) Rap1 promotes VEGFR2 activation and angiogenesis by a mechanism involving integrin ýývýýýýý. Blood 118:2015-26
Samant, Ganesh V; Schupp, Marcus O; Francois, Mathias et al. (2011) Sox factors transcriptionally regulate ROBO4 gene expression in developing vasculature in zebrafish. J Biol Chem 286:30740-7
Chun, Chang Zoon; Remadevi, Indu; Schupp, Marcus-Oliver et al. (2011) Fli+ etsrp+ hemato-vascular progenitor cells proliferate at the lateral plate mesoderm during vasculogenesis in zebrafish. PLoS One 6:e14732

Showing the most recent 10 out of 19 publications