Myeloid-Derived Vascular Endothelial Growth Factor and Hypoxia-Inducible Factor Are Dispensable for Ocular Neovascularization—Brief Report

Supplemental Digital Content is available in the text.

M yeloid-derived vascular endothelial growth factor (VEGF) has been proposed to drive ocular neovascularization (ONV), 1-4 a pathological feature common to leading causes of blindness, including retinopathy of prematurity in infants, proliferative diabetic retinopathy in the working population, and age-related macular degeneration in the elderly. 5 In mice with oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity, VEGF-expressing macrophages are recruited to sites of retinal neovascularization (RNV), and clodronate-induced or genetic macrophage depletion reduces RNV, raising the possibility that myeloid-derived VEGF promotes RNV. 3,6,7 In laser-induced choroidal neovascularization (CNV), a mouse model of age-related macular degeneration-associated neovascularization, peak VEGF expression correlates with maximal myeloid infiltration, and clodronate-induced macrophage depletion reduces both See cover image VEGF levels and CNV area. 1 The absence of VEGF-producing CCR2 + macrophages also reduces CNV area. 2 Human CNV lesions have also been reported to contain VEGF-expressing macrophages, which were suggested to cooperate with VEGF-expressing retinal pigment epithelium (RPE) to drive angiogenesis. 8 These findings raised the possibility that myeloid-derived VEGF also promotes CNV. However, others contested that myeloid-derived VEGF enhances CNV. 9 The significance of myeloid-derived VEGF in ONV, therefore, remains controversial. Moreover, the importance of myeloidderived hypoxia-inducible factors, HIF1α and HIF2α, has not yet been defined for ONV, even though they regulate VEGF expression, 10 have been implicated in myeloid-mediated angiogenesis in various tissues 11 and are expressed in OIR and CNV models. 12,13 To test the prevailing idea in the current literature that myeloid VEGF is nonredundant with other VEGF sources in ONV, we used conditional mouse knockout models to target Vegfa and its upstream regulators, Hif1a and Epas1 (Hif2a), in myeloid cells, and analyzed the effects of their deletion on RNV and CNV. Unexpectedly, we found that myeloid-derived HIFs and VEGF are dispensable ONV, suggesting that they do not present useful targets for therapy of ocular disease.

Materials and Methods
Materials and Methods are available in the online-only Data Supplement.

Results
We induced OIR or CNV in Vegfa +/LacZ mice, previously shown to faithfully report Vegfa gene expression in macrophages and other cells types. 17,22 X-gal staining of eye sections indicated prominent Vegfa expression in the RPE, inner nuclear layer, and retinal ganglion cell layer on postnatal day (P) 17 in the OIR and on day (D) 3 postlasering in the CNV model, when VEGF levels and myeloid infiltration peak, 1,23 but Vegfa expression was below the detection limit in IBA1 + F4/80 + microglia/ macrophages ( Figure 1A and 1B). Vegfa expression was also undetectable in YFP + IB4 + myeloid cells by in situ hybridization on D3 after lasering Lysm +/Cre eyes carrying the Rosa26 Yfp reporter to identify myeloid cells, even though other cell types strongly expressed Vegfa ( Figure 1C). These findings suggest that, compared with other ocular cell types, myeloid cells are unlikely a significant local source of VEGF for ONV.
Next, we examined Lysm +/Cre ;Vegfa fl/fl mice, which are deficient in myeloid cell-derived Vegfa and were previously shown to have reduced pathological angiogenesis in wound healing and cancer models. 22,24 Lysm +/Cre ;Vegfa fl/fl mice appeared healthy as previously reported and had normal retinal angiogenesis ( Figure IA in the online-only Data Supplement). YFP-expressing splenic myeloid cells showed efficient Vegfa gene targeting and, accordingly, Vegfa mRNA was reduced in mutant compared with control YFP + splenic myeloid cells (Figure 2A and 2B). Nevertheless, myeloid VEGF deletion did not alter overall VEGF protein or mRNA levels in the P17 OIR retina or D3 postlasering RPE/choroid ( Figure 2C and 2D). In agreement, the size of the central avascular and neovascular areas in P17 OIR retina and D7 and D14 CNV lesions was similar in Lysm +/Cre ;Vegfa fl/fl mice and controls ( Figure 2E-2F′). Moreover, myeloid VEGF depletion did not affect CD11b + cell recruitment to the RPE/ choroid on D3 postlasering ( Figure 2G).
We also examined Tie2-Cre;Vegfa fl/fl mice because Tie2-Cre targets yolk sac-derived tissue-resident macrophages more efficiently than Lysm Cre/+ , including microglia in the brain 25,26 and retina ( Figure 2H and 2I). Tie2-Cre;Vegfa fl/fl mutant mice are healthy, and despite targeting of Vegfa in hematopoietic and endothelial cells, have no obvious vascular defects and only develop vascular dysfunction in old age. 16,27 In agreement, angiogenesis and the density of resident myeloid cells were similar in mutant and control postnatal retinas ( Figure  IB and IC in the online-only Data Supplement). Moreover, the size of the central avascular and neovascular areas in P17 OIR retina and CNV lesions was not significantly different between mutants and controls ( Figure 2J-2K′). These data suggest that VEGF expression by resident microglia/macrophages does not explain the lack of angiogenesis defects in mice with Lysm Cre -mediated targeting of VEGF in myeloid cells. Myeloid cell-derived VEGF is therefore dispensable for retinal angiogenesis and pathological ONV.
Because HIFs promote the expression of Vegfa and other hypoxia-induced proangiogenic molecules, 10

Discussion
Nonmyeloid VEGF is thought to promote RNV because retinal ganglion cells 28   neural or glial sources previously implicated in ONV. Myeloid cells have also been found to influence angiogenesis by VEGFindependent mechanisms, for example, by acting as cellular chaperones to promote endothelial tip cell fusion during vascular development 26 or by producing proangiogenic factors different from VEGF during tumor vascularization. 38 The molecular mechanisms of inflammatory cell modulation of neovascular eye disease, therefore, differs significantly from nonocular disease models, in which myeloid-derived VEGF is nonredundant with other VEGF sources to promote pathological angiogenesis, even when nonmyeloid VEGF is abundant, for example, during tumor vascularization or in skin wound healing. 22,24