Identification of Ubiquitin E3 Ligase c-Cbl as a Novel Angiogenesis-Suppressor Protein
M Mehta, D Husain, E Ahmed, W Pfeifer, N Rahimi. Boston University, Boston, MA; Boston University, Boston
Background: A crucial aspect of many human diseases such as cancer, inflammatory diseases, and age-related macular degeneration is the formation of new blood vessels known as angiogenesis. In angiogenesis, VEGF activation of VEGF receptor-2 (VEGFR-2) leads to tyrosine phosphorylation of Phospholipase C- gamma 1(PLC-g1). Abolishing PLC-g1 binding site on VEGFR-2 has shown to inhibit endothelial cell tube formation, proliferation and angiogenesis. Previous work from our laboratory demonstrates that Casitas B-lineage lymphoma (c-Cbl) promotes ubiquitination of PLCg1 and suppression of its tyrosine phosphorylation (Singh et al., PNAS 2007). This study is designed to evaluate the role of c-Cbl in pathological angiogenesis in null c-Cbl mice.
Design: Endothelial cells were isolated from wild type and c-Cbl null mice and assayed for their ability to proliferate in response to Vascular Endothelial Growth Factor (VEGF). Western blot analysis was used to analyze PLCg1 activation in wild type endothelial cells versus c-Cbl null cells. In vivo angiogenesis assays including laser-induced choroidal neovascularization (CNV) and tumor-induced angiogenesis were performed. CNV was induced by laser photocoagulation (50μ, 0.1 seconds, 200 mw). 14 lesions were induced in the eyes of nude mice and 18 lesions in those of control mice. These lesions were documented at days 7, 10, 14 and 21 with fundus photography and angiography for progression, size and leakage from the CNV. The mice were sacrificed at week 4 and the enucleated eyes were frozen, sectioned and histologically evaluated. Hematoxylin and eosin (H&E) staining and immunoperoxidase staining with CD31 and phospho-PLCg1 antibodies were performed.
Results: Laser-induced choroidal neovascularization (CNV) lesions in the c-Cbl knockout mice were significantly more in number, more confluent and increased in size with time compared to control wild type mice. The enhanced angiogenesis was also observed in VEGF- and tumor-induced angiogenesis in c-Cbl null mice. Immunoperoxidase staining with CD31 (antibodies demonstrating endothelial cells) and phospho-PLCg1 antibodies in the c-Cbl knockout mice confirmed the enhanced activation PLCg1 in endothelial cells.
Conclusions: The data showed that loss of c-Cbl results in increased activation of PLCg1 and enhanced proliferation of endothelial cells in vitro. Altogether, our in vitro and in vivo analysis for the first time identifies c-Cbl as a novel angiogenesis-suppressor protein.
Tuesday, March 23, 2010 2:00 PM
Platform Session: Section H, Tuesday Afternoon