Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA329

Poster Communications

Hematopoietic Hypoxia-inducible factor 2α deficiency ameliorates pathological retinal neovascularization via modulation of endothelial cell apoptosis.

A. Klotzsche - von Ameln1, I. Korovina2, A. Neuwirth2, D. Sprott2, B. Wielockx2, T. Chavakis2, A. Deussen1

1. Institute of Physiology, Technische Universität Dresden, Dresden, Saxony, Germany. 2. Institute for Clinical Chemistry and Laboratory Medicine, Technische Universität Dresden, Dresden, Germany.

A hallmark of proliferative retinopathies, such as retinopathy of prematurity (ROP), is pathological neovascularization triggered by hypoxia. Synergisms between hypoxia, angiogenesis and the innate immune response have been suggested, which could be integrated at the level of the hypoxia-inducible factors (HIFs). Interestingly, HIF2α is highly expressed in endothelial and hematopoietic cells, e.g. macrophages and microglia. Here we studied the role of HIF2α in hematopoietic cells for pathological retina neovascularization in the murine model of ROP (oxygen-induced retinal neovascularization). Briefly, seven-days old mice were exposed to 75% O2 for 5 days, and then returned to normoxic conditions. Eyes were collected at postnatal day 17 (P17) and neovascularization was quantified by counting of epiretinal nuclei in PAS stained histological sections (Smith et al., 1994). In order to study hematopoietic specific deletion of Hif2α, Vav-cre (Stadtfeld et al., 2005) mice were crossed with Hif2αf/f (Gruber et al., 2007) mice. Littermates from Vav-Cre- Hif2αf/f (Hif2α-HWT) and hematopoietic Hif2α deficient Vav-Cre+ Hif2αf/f (Hif2α-HKO) mice were compared in the animal experiments (approved by the Landesdirektion Sachsen, Germany). We found that hematopoietic-specific deficiency of HIF2α ameliorated the pathological neovascularization in the ROP-model (n=21-26; HWT 37.57±3.21, Hif2α-HKO 23.7±3.74%; P<0.01; 2-tailed t-test), which was associated with enhanced endothelial cell apoptosis. Enhanced apoptosis was concluded from increased cleaved caspase-3 staining in the pathological endothelial cells in retinal whole mounts (n=8; HWT 100±15.18%, Hif2α-HKO 245.7±23.17%; P<0.001; 2-tailed t-test). The latter finding resulted from an up-regulation of the apoptosis inducer FasL in Hif2α-deficient microglia (assessed by qPCR and Western blot). Consistently, pharmacological inhibition of FasL by an intravitreal injection of a FasL-blocking antibody (at P14 of the ROP: 1 µg in the right eye and isotype control antibody in the left eye; mice anaesthetized with 0.03 mg/g BW Ketamin and 1µg/g BW Dormitor) reversed the reduced pathological neovascularization due to hematopoietic-specific Hif2α deficiency (n=6-12 mice; Hif2α-HWT vs. Hif2α-HKO plus FasL-blocking antibody not significant; 2 tailed t-test). Taken together, our experiments identified that hematopoietic cell HIF2α contributes to pathological retina angiogenesis. Our findings not only provide novel insights regarding the complex interplay between immune cells and endothelial cells in hypoxia-driven retina neovascularization but may also have implications for novel therapeutic approaches in proliferative retinopathies.

Where applicable, experiments conform with Society ethical requirements