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

Abstract

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 targeted Vhl, Epo, Phd1, Phd2, Phd3 and Hif2a in NG2 cells. Cerebral vasculature was assessed by immunofluorescence, RNA in situ hybridization, gene and protein expression analysis, gel zymography and in situ zymography. Results Vhl inactivation led to a significant increase in angiogenic gene and Epo expression. 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 of Phd2 and Phd3, but not from Phd2 inactivation alone. Concomitant PHD1 function loss led to further expansion of the neurovasculature. Genetic inactivation of Hif2a in Phd1/Phd2/Phd3 triple 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.

Hypoxia-Mediated Complement 1q Binding Protein Regulates Metastasis and Chemoresistance in Triple-Negative Breast Cancer and Modulates the PKC-NF-κB-VCAM-1 Signaling Pathway

“…Both the mRNA and protein levels of C1QBP increased under hypoxic conditions and decreased upon treatment with the HIF-1α inhibitor as expected ( Figure 1E ). Accordingly, we further detected the protein level of vascular endothelial growth factor (VEGF), von Hippel-Lindau tumor suppressor (VHL), E-cadherin 1 (E-cad), and β-tubulin, that are reported to be regulated by hypoxia and/or HIF-1α ( Bordji et al, 2014 ; Ning et al, 2018 ; Ibuki et al, 2020 ; Urrutia et al, 2020 ; Yang et al, 2020 ) ( Supplementary Figure 2B ). Based on the data from the JASPAR database, we generated several primers for HIF-1α containing multiple binding sites on the C1QBP promoter ( Figure 1F ).…”

A triple sense of oxygen promotes neurovascular angiogenesis in NG2‐derived cells

“…17 Hypoxia response, thus, varies between underlying conditions, tissues and even individual cells. Altogether, interventions into the HIF-PHD system, whether pharmacological or genetical, are nearly unpredictable, which brings us back to the in vivo studies of Urrutia et al 1 EPO. In the past, the latter has been attributed angiogenic effects in the brain, but a plethora of clinical trials (review in Ref.…”

“…18) proved that exogenous EPO was unable to ameliorate brain damage. By double knockout (VHL/EPO) Urrutia et al 1 show that the angiogenic phenotype is independent of EPO. Interestingly, knockout of PHD-2, which carries the workload of HIF degradation in normoxia, did not lead to angiogenesis while double knockout (PHD-2/PHD-3) did.…”

“…Expansion and remodelling of the cerebral vasculature via the induction of pro‐angiogenic genes is a crucial issue in many pathologies. Urrutia et al 1 demonstrate how NG2‐derived cells in the brain sense oxygen to promote neurovascular angiogenesis.…”

“…In the meantime, it's worth taking a closer look at PHD inhibition in basic science, as it likely will help us understand upcoming clinical data. In this regard, Urrutia et al 1 present a meticulous assessment of brain angiogenesis, a highly relevant topic in ischemic and degenerative central nervous disorders 13 . With help of knockout technology ranging from single to quadruple, they dissect the contributions of PHD‐1 to ‐3 and EPO in this process.…”