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Matina Economopoulou, Maria Troulinaki, Ruben Garcia-Martin, Lutz E Pillunat, Andre Nussenzweig, Triantafyllos Chavakis; The role of DNA repair in the choice between sprouting or proliferating angiogenesis in the context of proliferative retinopathies. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3656.
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© ARVO (1962-2015); The Authors (2016-present)
In proliferative retinopathies, the hypoxia response is sustained by the failure of the retina to revascularize its ischemic areas. Promoting revascularization of the retinal avascular area could interfere with this vicious cycle and lead to vessel normalization. One cardinal difference between tip endothelial cells (ECs) that lead sprouting /revascularizing vessels and prolifrerating tuft cells, is the cell cycle phase. Tip ECs are constantly at G0/G1, whereas tuft ECs are constantly cycling and are very often at S phase. Hypoxia is one of the noxious factors that can activate the DNA repair cascade in the ECs that eventually leads to cell cycle arrest and DNA repair or cell death. Here we examine the role of two distinct DNA Repair mechanisms (Homologous Recombination HR and non homologous end joining NHEJ) and the proteins that orchestrate them: BRCA1 for HR and 53bp1 for NHEJ, in the function of tip vs tuft cells. NHEJ is only functional during G0/G1 whereas HR is active during S/G2 phases.
We subjected C57bl/6, 53bp1 +/+ and -/- mice as well as 53bp1 +/+ and -/- mice combined with endothelial specific deletion of BRCA1 (EC-BRCA1-WT, EC-BRCA1-KO) to the murine ROP model. The eyes were processed for retina lectin IB4 staining for quantification of avascular area and tip cell number or for paraffin sections for the quantification of neovascular tufts. Wholemounts were also processed for BrdU staining.
In wholemounts of WT mice we could verify that the tip cells were Brdu- , whereas tuft cells were Brdu+. After subjection to the ROP model, the 53bp1 -/- mice showed more BrdU+ ECs, more neovascular tufts and less tip cell formation compared to their wildtype littermates. On the other hand, when 53bp1 -/- was combined with EC BRCA1 deficiency, the tufts decresded and the tip cells increased. Furthermore, when we injected a HR inhibitor (RI-1) intravitrealy in 53bp1 +/+ and -/- mice, the inhibitor decreased the proliferation in 53bp1 -/- mice.
Our results show that in the model of ROP, the tip cell formation is dependent on an intact NHEJ. Furthermore, in the absence of 53bp1, HR is increased, leading to more tuft cells in a BRCA1 dependent manner. We conclude that by influencing survival factors that operate in different cell cycle phases one can selectively influence the function of either tip or tuft ECs.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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