Abstract
Purpose::
Hypoxia induces replication arrest in cells. Nevertheless, endothelial cells need to actively proliferate during hypoxia-induced angiogenesis. To understand this we studied the DNA damage response to hypoxia in endothelial cells in vitro and in hypoxia-driven retinal neovascularisation in vivo.
Methods::
The DNA damage response in endothelial cells in vitro and in mouse retinas was studied by immunofluorescence and biochemically by western blot analysis. Endothelial cell proliferation and apoptosis was assessed in vitro and in mouse retinas in vivo. The retinopathy of prematurity (ROP)-model was used to assess hypoxia-driven neovascularisation.
Results::
Hypoxia induced a rapid ataxia-telangiectasia mutated and Rad3-related (ATR)-dependent response (chk1 phosphorylation) in endothelial cells accompanied by replication stress associated foci that were positive for replication protein A (RPA) and proliferating cell nuclear antigen (PCNA). Phosphorylated H2AX (γ-H2AX) and 53BP1 also localized to the hypoxia-induced foci. To functionally define this hypoxia-induced response, we performed siRNA-mediated knockdown of H2AX in endothelial cells. H2AX knockdown marginally affected VEGF- or bFGF-stimulated endothelial cell proliferation under normoxia but it inhibited endothelial proliferation under hypoxia almost completely. In vivo, γ-H2AX was detected in newly formed retina vessels in mice that were subjected to the hypoxia-induced retina neovascularisation model, as opposed to normoxic mice. Whereas physiologic vessel formation in the retina was unaffected by H2AX deficiency, H2AX-/- mice displayed a 50% reduction in hypoxia-induced retina neovascularisation, associated with decreased endothelial cell proliferation and increased endothelial cell apoptosis in the retina.
Conclusions::
Together, H2AX functions to help endothelial cells overcome the hypoxia induced replication arrest, thus, identifying a novel crosstalk between the DNA repair response and hypoxia-driven angiogenesis.
Keywords: retinal neovascularization • hypoxia • vascular cells