Developmental vascular pruning in neonatal mouse retinas is programmed in the astrocytic oxygen sensing mechanism

Abstract

Vascular pruning is crucial for normal development, but its underlying mechanisms are poorly understood. Here, we report that retinal vascular pruning is controlled by the oxygen-sensing mechanism in local astrocytes. Oxygen sensing is mediated by prolyl hydroxylase domain proteins (PHDs), which use O 2 as a substrate to hydroxylate specific prolyl residues on hypoxia inducible factor (HIF)-α proteins, labeling them for polyubiquitylation and proteasomal degradation. In neonatal mice, astrocytic PHD2 deficiency led to elevated HIF-2α protein levels, expanded retinal astrocyte population and defective vascular pruning. Although astrocytic VEGF-A was also increased, anti-VEGF failed to rescue vascular pruning. However, stimulation of retinal astrocytic growth by intravitreal delivery of PDGF-A was sufficient to block retinal vascular pruning in wild-type mice. We propose that in normal development, oxygen from nascent retinal vasculature triggers PHD2-dependent HIF-2α degradation in nearby astrocytic precursors, thus limiting their further growth by driving them to differentiate into non-proliferative mature astrocytes. The physiological limit of retinal capillary density may be set by astrocytes available to support their survival, with excess capillaries destined for regression. This article has an associated 'The people behind the papers' interview.

Environmental oxygen regulates astrocyte proliferation to guide angiogenesis during retinal development

“…Because astrocyte phenotypes precede vessel growth and occur in peripheral retina prior to the arrival of vessels, we favor a model in which relative hypoxia affects astrocytes, and astrocytes in turn contribute to pathologic retinal vascularization. This would be consistent with other manipulations of astrocyte abundance during development, each of which demonstrate that vascular development is highly sensitive to the number of astrocytes within the angiogenic template (Duan and Fong, 2019;Fruttiger et al, 1996;O'Sullivan et al, 2017;Puñal et al, 2019;Tao and Zhang, 2016). However, our experiments cannot exclude the possibility that additional factors, including astrocyte responsiveness to vascular signals (Mi et al, 2001;Sakimoto et al, 2012;Selvam et al, 2018), may also contribute to the correlation between astrocytic and vascular phenotypes.…”

“…relative hypoxia, was delivered via the NOIR protocol. Conversely, when the HIF pathway becomes hyperactivated in astrocytes, astrocyte numbers are increased ( Duan and Fong, 2019 ). Together, these observations support our model whereby astrocyte proliferation rates are proportional to the amount of HIF signaling.…”

“…These migrations are coordinated through a sequential series of cell-cell interactions: RGC axons guide astrocyte migration, and astrocytes in turn guide endothelial cell growth ( Dorrell et al, 2002 ; Fruttiger et al, 1996 ; Gerhardt et al, 2003 ; O'Sullivan et al, 2017 ). Perturbation of these interactions leads to disruption or arrest of angiogenesis ( Duan and Fong, 2019 ; Fruttiger et al, 1996 ; O'Sullivan et al, 2017 ; Tao and Zhang, 2016 ), which is a hallmark of human retinal developmental vascular disorders, such as retinopathy of prematurity (ROP) ( Foos, 1987 ; Hellström et al, 2013 ). It is therefore of great interest to understand the developmental mechanisms that enable orderly progression of retinal angiogenesis and how these mechanisms might become perturbed in the context of ROP pathology.…”

Irisin Attenuates Pathological Neovascularization in Oxygen-Induced Retinopathy Mice

“…Astrocytes in the retina were isolated from neonatal pups of C57BL/6J mice following the previous protocol. 33 The astrocyte cultures were seeded in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Waltham, MA) containing 10% fetal bovine serum (Gibco) and maintained in a 95% humidified incubator at 37°C with 5% CO 2 .…”

Tailless and hypoxia inducible factor-2α cooperate to sustain proangiogenic states of retinal astrocytes in neonatal mice

“…At the molecular level, retinal astrocyte differentiation is controlled, at least in part, by oxygen sensing mechanisms ( Bruick and McKnight, 2001 ; Carmeliet et al, 1998 ; Duan and Fong, 2019 ; Duan et al, 2017 , 2014 ; Epstein et al, 2001 ; Ivan et al, 2002 ; Maxwell et al, 1999 ; Perelli et al, 2021 ; Wang and Semenza, 1995 ; West et al, 2005 ). Oxygen sensing and hypoxia responses are ubiquitous processes in different types of cells, and are mediated by prolyl hydroxylase domain proteins (PHDs, also known as Egg Laying Nine or EGLN) and hypoxia inducible factor (HIF)-α proteins ( Bruick and McKnight, 2001 ; Carmeliet et al, 1998 ; Epstein et al, 2001 ; Ivan et al, 2002 ; Jiang et al, 2021 ; Maltepe et al, 1997 ; Peng et al, 2000 ; Tian et al, 1998 , 1997 ; Wang et al, 1995 ).…”