Myeloid-derived VEGF and HIF are dispensable for ocular neovascularisation

Abstract

Purpose : Ocular neovascularisation (ONV) is a pathological feature of diseases such as diabetic retinopathy and age-related macular degeneration. Myeloid cells are considered an important source of the vascular endothelial growth factor A (VEGF) in mouse models of ONV. By inducing neovascularization in transgenic mouse models using oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), we investigated the hypothesis that myeloid-derived VEGF and its upstream regulators hypoxia-inducible factor (HIF)-1α and HIF2α are key drivers of ONV.

Methods : The Vegfa^+/LacZ reporter and in situ hybridisation (ISH) were used to analyse Vegfa expression. Cre-lox systems used Lysm^+/Cre or Tie2-Cre to target myeloid cells for reporter expression with Rosa26^Yfp and Rosa26^mT/mG or deletion of Hif1a^fl/fl, Hif2a^fl/fl orVegfa^fl/fl. Established OIR and CNV protocols were performed with littermate controls. Anti-isolectin B4 staining on P17 retinal flatmounts was used to identify avascular (AV) areas and retinal neovascularization (RNV) in OIR. The area of resultant CNV post-laser rupture of Bruch’s membrane was analysed using fluorescein angiography at day D7 and D14. Vegfa gene locus recombination, VEGF protein levels and mRNA levels of Vegfa, Hif1a and Hif2a were analysed using genomic PCR, ELISA and quantitative RT-PCR respectively. Flow cytometry was used to isolate and analyse myeloid subsets. The t test and ANOVA were used for statistical analysis.

Results : Immunofluorescence and flow cytometry on reporter lines confirmed that Cre-targeted myeloid cells accumulate at sites of RNV and CNV. The Vegfa^+/LacZ reporter and ISH showed that Vegfa is highly expressed by several cell types, but not by myeloid cells. The Lysm^+/Cre allele did not affect the extent of AV, RNV or CNV. Conditional deletion of Vegfa, Hif1a and Hif2a was confirmed at genomic and transcript level (P<0.05). Myeloid-specific VEGF ablation did not reduce total ocular VEGF during CNV and RNV (P>0.05). In agreement, the deletion of Vegfa, Hif1a or Hif2a in recruited and resident myeloid cells that accumulated at sites of neovascularisation did not significantly reduce CNV or RNV (P>0.05).

Conclusions : These findings show that myeloid-derived HIF1α, HIF2α and VEGF are not significant in these mouse models of ONV, suggesting that myeloid function and VEGF production in neovascular eye disease differs from wound healing and other neovascular pathologies.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.