Vascular anomalies include a range of mild to severe defects that can be life-threatening, debilitating, or disfiguring. Mutation in the RASA1 gene was found to underlie capillary malformation-arteriovenous malformation; however, there has been no mechanistic understanding of how the loss of function of RASA1 contributes to this disease. In human endothelial cells, presence of ephrin-B2 dominated over serum stimulation to reduce MAPK and PI3K-mTORC1 pathway activation, only when EphB4 and RASA1 were both present. Indeed, zebrafish model showed that vascular defects in EphB4 knockdown embryos were prevented by EphB4 mRNA only when RASA1 binding sites were present. Moreover, reduced function of either EphB4 or RASA1 gene product can generate comparable defects in blood vessel formation and function. Increased TORC1 activity was observed in the affected caudal tail region, which was rescued by treatment with the small molecule inhibitors of PI3K and/or mTORC1 kinases. To further examine whether endothelial specific activation of TORC1 was responsible for these vascular defects, a transgenic zebrafish line, expressing a gain-of-function mutant RhebS16H under endothelial specific promoter, was generated. Transgene-positive embryos augmented the vascular phenotypes with the low doses of RASA1 or EphB4 morpholino, which showed no phenotypes in their wild-type siblings. These data demonstrate a distinct angiogenic role for EphB4 and RASA1 as vascular suppressors of TORC1 activity. To investigate the translational value of this finding, mouse retinal angiogenesis assay was performed by intravitereally injecting RASA1 siRNA. This resulted in a significant increase in vascular density in the retina. Moreover, immunohistochemistry was performed on resected tissues from patients with RASA1 mutations and RASA1-related vascular malformations. Remarkably, consistent and robust phospho-S6 staining was restricted to endothelial cells in these patient blood vessels. This study provides a molecular rationale for the use of FDA-approved mTORC1 inhibitory drugs in the treatment of RASA1-deficient vascular pathologies. A new therapeutic use of an approved drug reduces the time and cost required for drug development, while providing patients with a targeted treatment. Furthermore, we demonstrate the translational value of the zebrafish model as an alternative vertebrate system for the investigation of endogenous endothelial functions of mTORC1 and its cardiovascular consequences.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/10304427 |
| Date | January 2012 |
| Creators | Kawasaki, Jun |
| Contributors | Chan, Joanne |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
Page generated in 0.0077 seconds