Candidate's immediate career plans are advancement to Asst Project Scientist with several years'mentored project work to gain skills in transgenic mouse methodology/genetics. Long-term goal: acquire independent RO1 funding and a faculty position with independent research in cardiovascular biology. This proposal aims to identify signaling molecules promoting endothelial barrier integrity. Endothelial cell-cell junction disruption plus contractile stress fiber formation increase vessel permeability. Dysregulation of endothelial barrier function contributes to vessel leak in cardiovascular pathology. Rap1 GTPase stabilizes endothelial junctions. KRIT1 and CCM2 mutations produce "Cerebral Cavernous Malformations" disease characterized by vascular lesions with abnormal endothelium. Preliminary work identified KRIT1 as a Rap1 effectors having beta-integrin binding site, and demonstrated KRIT1 and CCM2 targeting to endothelial junctions that was associated with stabilized cortical actin and decreased permeability. HYPOTHESIS: KRIT1 is a Rap1 effector recruiting ICAP1 modulating beta-integrins and/or CCM2, regulating f-actin mediated barrier function through Rho-GTPases.
SPECIFIC AIMS are to: 1) test whether KRIT1 localization and function are mediated by ICAP1 and CCM2 binding. 2) Evaluate KRIT1 PERM domain interactions with beta-integrins in regulating junctional stability. 3) Examine Rac- and Rho-GTPase interactions with KRIT1 in modulating actin cytoskeleton associated with barrier function. Molecular interactions in cultured cells will be tested with in vitro protein binding assays and co-immunoprecipitations, using siRNA depletion and ectopic expression of mutated binding domain proteins. Permeability will be tested in vivo with pulmonary vascular leak assays using beta-integrin, KRIT1, and CCM2 mutant mice;and in vitro using the animal's cultured endothelial cells in Tran swell filters. Molecular localization will be imaged using confocal immunofluorescence microscopy, plus real-time micro imaging to visualize kinetic localization of labeled ectopic proteins and fluorescent Rho-GTPase biosensors.
This project will increase understanding of vascular permeability in general and CCM disease in particular. It will potentially identify target molecules for pharmacological treatment of pathological leak, and genetic treatment of CCM2 disease.
|Shenkar, Robert; Shi, Changbin; Rebeiz, Tania et al. (2015) Exceptional aggressiveness of cerebral cavernous malformation disease associated with PDCD10 mutations. Genet Med 17:188-96|
|McDonald, David A; Shi, Changbin; Shenkar, Robert et al. (2012) Fasudil decreases lesion burden in a murine model of cerebral cavernous malformation disease. Stroke 43:571-4|
|McDonald, David A; Shenkar, Robert; Shi, Changbin et al. (2011) A novel mouse model of cerebral cavernous malformations based on the two-hit mutation hypothesis recapitulates the human disease. Hum Mol Genet 20:211-22|