Endothelial Cell Isolation and Quantitative Polymerase Chain Reaction

Cerebral cavernous malformations (CCMs) are common vascular malformations that arise primarily in the central nervous system. CCMs are typically diagnosed in middle age and constitute an important cause of stroke and neurological deficit in younger individuals. One third of CCMs are familiar, and positional cloning studies have identified loss of function mutations in 3 genes, KRIT1, CCM2 and PDCD10, as causal for this disease. KRIT1, CCM2, and PDCD10 encode intracellular adaptor proteins that have been shown to form a single biochemical complex that is bound by the transmembrane receptor Heart of Glass (HEG), but the role of HEG in CCM disease has not been defined. Fish and mouse genetic studies have demonstrated that HEG, KRIT1, CCM2, and PDCD10 function together in endothelial cells during formation of the heart and vasculature. In addition, inducible endothelial deletion of Krit1, Ccm2, or Pdcd10 in neonatal mice results in the formation of retinal and hindbrain CCMs that accurately reproduce the human disease. In the present study, we use genetically modified mice and studies of human patients with familial CCM to test the role of HEG in CCM formation rigorously. Materials and Methods Mice Mutant Heg and Ccm2 mouse alleles and Cre transgenic mice have been described previously. The University of Pennsylvania Institutional Animal Care and Use Committee approved all animal protocols.