Development of an In Vitro Culture Model of Brain Arteriovenous Malformations (AVMs) to Determine the Role of the Transcription Factor, COUP-TF II, in Human AVM Tissue

Brain arteriovenous malformations (AVMs) are devastating vascular lesions characterized by loss of capillary beds and formation of a nest of abnormal arteries and veins. AVMs, rarely diagnosed early, often present through seizures, migraines or hemorrhages. Our group has human tissue evidence that AVMs express proteins normally present in lymphatic vessels. Specifically, AVMs express high levels of COUP-TFII, a key transcription factor in venous and lymphatic specification during development.  This is a novel finding, as there is normally no lymphatic system within the brain. The expression of lymphatic proteins in the cerebrovasculature may lead to loss of venous and arterial identity, causing some vessels to acquire a lymphatic phenotype.  Research in AVMs is difficult, as there are currently no in vivo or in vitro disease models. My goal is to establish human endothelial cell (EC) tube formation assays as an in vitro model to test the hypothesis that COUP-TFII is a master regulator of this disorder. I can then determine if over-expression of COUP-TFII is necessary and sufficient to initiate the cascade of expression of lymphatic proteins. By over-expressing COUP-TFII in ECs through lentiviral infection, I will determine if downstream lymphatic protein levels are elevated, using qPCR, immunocytochemistry and Western blot. Preliminary work in HEK293 and ECs has validated the over-expression construct by qPCR and Western blot and the subsequent lentiviral over-expression construct has been generated and is currently being tested in both cell lines. If this is confirmed, this may change the traditional view of AVM disease biology and development.