Heart of Glass (HEG1), a transmembrane receptor, and Rasip1, an endothelial-specific Rap1-binding protein, are both essential for cardiovascular development. Here we performed a proteomic screen for novel HEG1 interactors and report that HEG1 binds directly to Rasip1. Rasip1 localizes to forming endothelial cell (EC) cell-cell junctions and silencing HEG1 prevents this localization. Conversely, mitochondria-targeted HEG1 relocalizes Rasip1 to mitochondria in cells. The Rasip1-binding site in HEG1 contains a 9 residue sequence, deletion of which abrogates HEG1's ability to recruit Rasip1. HEG1 binds to a central region of Rasip1 and deletion of this domain eliminates Rasip1's ability to bind HEG1, to translocate to EC junctions, to inhibit ROCK activity, and to maintain EC junctional integrity. These studies establish that the binding of HEG1 to Rasip1 mediates Rap1-dependent recruitment of Rasip1 to and stabilization of EC cell-cell junctions.

KRIT1 mutations are the most common cause of cerebral cavernous malformation (CCM). Acute Krit1 gene inactivation in mouse brain microvascular endothelial cells (BMECs) changes expression of multiple genes involved in vascular development. These changes include suppression of Thbs1, which encodes thrombospondin1 (TSP1) and has been ascribed to KLF2- and KLF4-mediated repression of Thbs1 In vitro reconstitution of TSP1 with either full-length TSP1 or 3TSR, an anti-angiogenic TSP1 fragment, suppresses heightened vascular endothelial growth factor signaling and preserves BMEC tight junctions. Furthermore, administration of 3TSR prevents the development of lesions in a mouse model of CCM1 (Krit1^ECKO ) as judged by histology and quantitative micro-computed tomography. Conversely, reduced TSP1 expression contributes to the pathogenesis of CCM, because inactivation of one or two copies of Thbs1 exacerbated CCM formation. Thus, loss of Krit1 function disables an angiogenic checkpoint to enable CCM formation. These results suggest that 3TSR, or other angiogenesis inhibitors, can be repurposed for TSP1 replacement therapy for CCMs.