FLVCR2 Regulation of Intracellular Heme Modulates Vascular Tip/Stalk Specification During Retinal Angiogenesis

Purpose: Fowler syndrome is an embryonic lethal disease with proliferative vasculopathy and hydranencephaly-hydrocephaly of the central nervous system caused by mutations in feline leukemia virus subgroup C receptor-related protein 2 (FLVCR2) characterized by large regions of hypovascularization along with glomeruloid formation of poorly organized vascular endothelial cell masses. The purpose of the current study is to elucidate the mechanisms by which FLVCR2 regulates retinal angiogenesis. Methods: Recent animal models have characterized reduced brain vascularization in gene deletion models of Flvcr2. Here, we use conditional knockout of Flvcr2 in mice and knockdown in primary bovine retinal endothelial cell culture to explore retinal development and associated mechanisms of angiogenic regulation by FLVCR2. Results: Endothelial cell-specific deletion of Flvcr2 or chemical inhibition of heme synthesis during post-natal retinal vascularization led to reduced vascular expansion in both primary (superficial) and secondary (deep) vascular plexuses, and a more severe but partially penetrant phenotype with attenuation of retinal vascular development and formation of abnormal vascular glomeruloids. Mechanistically, Flvcr2 knockdown in retinal endothelial cells led to reduced heme content and promoted NOTCH signaling and hyperproliferation that could be reversed with addition of a heme precursor. Similar effects were observed in cells treated with inhibitors of heme synthesis and reversed with addition of cell-permeable hemin. Furthermore, we found that FLVCR2/heme signaling attenuates NOTCH1 activation and affects downstream tip/stalk cell specification, while also preventing VEGF-induced proliferation. Conclusions: Collectively, these data indicate a newly identified role for FLVCR2 and heme in the regulation of tip/stalk specification in angiogenesis.