Cerebral Cavernous Malformation (CCM) is the most common inherited vascular malformation of the brain. Molecular genetic studies established linkage of CCM to 3 different loci in the genome (CCM1-3). Mutations in KRIT1, a gene of unknown function, lead to CCM1. KRIT1 was initially cloned through its interaction with Krev1/Rap1A, a small GTPase homologous to Ras. More recent studies demonstrate KRIT1 binding to Integrin Cytoplasmic Domain Associated Protein 1-alpha (ICAP-1) as well as to microtubules, implicating integrin and cytoskeletal interactions with KRIT1. Our preliminary results confirm KRIT1 expression by endothelial cells especially during early angiogenesis and reveal specific KRIT1 expression on the foot processes of astrocytes in the central nervous system (CNS) suggesting that astrocyte-endothelial cell communication might be important in KRIT1 function. The latter finding offers a possible explanation as to why CCM lesions are CNS specific. Based on these results, we hypothesize that KRIT1 is important in early angiogenesis and mediates the communication between the extracellular matrix through beta1 integrin and ICAP-1 to Krev1/Rap1a signaling and thereby influences changes in the cytoskeleton via microtubules. Based on this model, mutations in KRIT1 would cause uncoupling of endothelial cell interactions with the extracellular matrix and other cells such as astrocytes. In this grant application, we propose to activate or inhibit KRIT1 expression with the use of siRNA and various mutational constructs to examine KRIT1 's role in basic endothelial cell functions such as adhesion, proliferation, and apoptosis along with complex functions such as tube formation in 3D cultures. We also propose to use the developing rat retina model to study KRIT1's possible role in endothelial cell-astrocyte interactions. Finally, we propose to use a novel approach of hypothesis-driven genetics research with the use of microarray analysis to identify candidate CCM2 and CCM3 genes by comparing endothelial cell gene expression before and after KRIT1 silencing.
