CITED2 regulates hypoxia-induced retinal neovascularization in a mouse model of oxygen-induced retinopathy

Abstract

Purpose : CITED2 forms a negative feedback loop competing with hypoxia inducible factor 1(HIF1) α for binding of CBP/p300. A short peptide derived from the native CITED2 sequence functions allosterically to displace HIF1α from CBP/p300 (Berlow et al. Nature 2017). In this study we investigate the effect of CITED2 peptide in a murine model of ischemic retinopathy [the oxygen-induced retinopathy (OIR) mouse] to determine if this peptide could be useful as an anti-angiogenic agent for neovascular retinal diseases.

Methods : OIR was induced in C57BL/6J mice via exposure to 75% oxygen from postnatal day 7 (P7) to P12 and subsequent transfer to room air. A CITED2 peptide and a negative control peptide (CITED2APAA) were injected intravitreally into P12 OIR eyes. Retinal vaso-obliteration (VO) and neovascularization (NV) were evaluated at P17. Using Alexa488-conjugated peptides, the distribution of injected peptides within the retina was confirmed by confocal microscopy. Retinal cells containing the CITED2 peptides were isolated from the total dissociated cell population by flow cytometry and the expression of HIF downstream genes evaluated by qPCR. Aflibercept was also injected into OIR; NV and VO were quantified after treatment with aflibercept alone or in combination with CITED2. Transcriptional changes of the retina after injections were evaluated by RNA sequencing.

Results : Both NV and VO in OIR at P17 were significantly inhibited in retinas injected with 20nM, 100nM, and 1uM of CITED2 peptide compared to the CITED2APAA control (p<0.01). 12 hours after Alexa488-conjugated CITED2 peptide injections, it was widely distributed on the inner surface, as well as within, the retina. VEGF expression in CITED2-containing retinal cells was significantly suppressed compared to that in CITED2APAA-containing cells (p<0.001). Aflibercept rescued only NV, but not VO, in OIR. However, the combination therapy of CITED2 peptide and Aflibercept significantly decreased both NV and VO (p<0.0001). RNA sequencing showed that several HIF target genes are down-regulated in CITED2-injected retinas relative to Aflibercept treatment and injection of the CITED2 peptide enriched Gene Ontology (GO) categories based on response to hypoxia rather than angiogenesis.

Conclusions : We conclude that the CITED2 peptide can rescue hypoxia-induced retinal NV by modulating the hypoxic response through direct competition with HIF1.

This is a 2020 ARVO Annual Meeting abstract.