Inactivation of HIF-prolyl 4-hydroxylases 1, 2 and 3 in NG2-expressing cells induces HIF2-mediated neurovascular expansion independent of erythropoietin

Aim NG2 cells in the brain are comprised of pericytes and NG2 glia and play an important role in the execution of cerebral hypoxia responses, including the induction of erythropoietin (EPO) in pericytes. Oxygen-dependent angiogenic responses are regulated by hypoxia-inducible factor (HIF), the activity of which is controlled by prolyl 4-hydroxylase domain (PHD) dioxygenases and the von Hippel-Lindau (VHL) tumour suppressor. However, the role of NG2 cells in HIF-regulated cerebral vascular homeostasis is incompletely understood. Methods To examine the HIF/PHD/VHL axis in neurovascular homeostasis, we used a Cre-loxP-based genetic approach in mice and targetedVhl,Epo,Phd1,Phd2,Phd3andHif2ain NG2 cells. Cerebral vasculature was assessed by immunofluorescence, RNA in situ hybridization, gene and protein expression analysis, gel zymography and in situ zymography. Results Vhlinactivation led to a significant increase in angiogenic gene andEpoexpression. This was associated with EPO-independent expansion of capillary networks in cortex, striatum and hypothalamus, as well as pericyte proliferation. A comparable phenotype resulted from the combined inactivation ofPhd2andPhd3, but not fromPhd2inactivation alone. Concomitant PHD1 function loss led to further expansion of the neurovasculature. Genetic inactivation ofHif2ainPhd1/Phd2/Phd3triple mutant mice resulted in normal cerebral vasculature. Conclusion Our studies establish (a) that HIF2 activation in NG2 cells promotes neurovascular expansion and remodelling independently of EPO, (b) that HIF2 activity in NG2 cells is co-controlled by PHD2 and PHD3 and (c) that PHD1 modulates HIF2 transcriptional responses when PHD2 and PHD3 are inactive.

Automated summary

The study investigates the role of NG2 cells in HIF-regulated cerebral vascular homeostasis and finds that HIF2 activation in NG2 cells promotes neurovascular expansion independently of EPO. The activity of HIF2 in NG2 cells is controlled by both PHD2 and PHD3, and PHD1 modulates HIF2 transcriptional responses when PHD2 and PHD3 are inactive.