Increased angiogenic factors associated with peripheral avascular retina and intravitreous neovascularization: a model of retinopathy of prematurity

Abstract

Objectives: To determine expression of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor, and their respective receptors in retinas using a model of retinopathy of prematurity.

Methods: Retinas isolated from a 50/10 oxygen (inspired oxygen cycled between 50% oxygen and 10% oxygen every 24 hours)-induced rat model of retinopathy of prematurity (50/10 OIR model), and from room air-raised rat pups (RA) at birth, age 14 days (persistent peripheral avascular retina in the 50/10 OIR model and complete retinal vascularization in RA) and age 18 days (intravitreous neovascularization in the 50/10 OIR model) were analyzed for messenger RNA of VEGF(164), neuropilin 1, neuropilin 2, VEGF receptor 1, VEGF receptor 2, pigment epithelium-derived factor, and pigment epithelium-derived factor receptor by real-time polymerase chain reaction.

Results: In the 50/10 OIR model compared with RA, fold changes in expression of VEGF(164), neuropilin 1, and neuropilin 2 were significantly increased at ages 14 and 18 days. A trend for increased fold change was noted in expression of VEGF receptor 2 at age 14 days and a significant increase at age 18 days in the 50/10 OIR model compared with RA. Pigment epithelium-derived factor receptor was significantly increased at age 14 days in the 50/10 OIR model compared with RA.

Conclusion: Increased expression of VEGF(164) and angiogenic receptors were found in association with both avascular retina at day 14 and intravitreous neovascularization at day 18 in a relevant model of retinopathy of prematurity.

Clinical relevance: Increased VEGF and angiogenic receptors may have a role in the development of peripheral avascular retina and stage 3 retinopathy of prematurity.

Figure 1

Lectin-stained retinal flatmount from postnatal day 14 rat pup in the 50/10 OIR model following 7 cycles of oxygen fluctuations every 24 hours between 50% and 10% oxygen. Peripheral avascular retina persists, whereas room air counterparts have fully vascularized retina.

Figure 2

Lectin-stained retinal flatmount from postnatal day 18 rat pup in 50/10 OIR (7 cycles of oxygen fluctuations every 24 hours between 50% and 10%, followed by 4 days in room air). Intravitreous neovascularization exists at the border of vascular and avascular retina.

Figure 3

Fold change in VEGF₁₆₄ mRNA expression, relative to β-actin, at p0, p14 and p18 room air (RA) and 50/10 OIR. Significantly increased fold expression in VEGF₁₆₄ mRNA was present in 50/10 OIR compared to RA at p14 (*p=0.0004) and p18 (**p=0.02). Student t-tests.

Figure 4

Fold change in expression of VEGFR2 mRNA, relative to β-actin, at p0, p14 and p18 room air (RA) and 50/10 OIR. Significantly increased fold expression in VEGFR2 mRNA was present in 50/10 OIR compared to RA at p18 (*p=0.004). A trend toward increased fold expression of VEGFR2 in 50/10 OIR was noted at p14 (p=0.06). Student t-tests.

Figure 5

Fold change in expression of VEGFR1 mRNA, relative to β-actin, at p0, p14 and p18 room air (RA) and 50/10 OIR. No significant differences were found at p14 (p=0.47) or p18 (p=0.97). Student t-tests.

Figure 6

Fold change in expression of neuropilin-1 (NP-1) mRNA, relative to β-actin, at p0, p14 and p18 room air (RA) and 50/10 OIR. Significantly increased fold expression in NP-1 mRNA was present in 50/10 OIR compared to RA at p14 (*p=0.042) and p18 (**p=0.027). Student t-tests.

Figure 7

Fold change in expression of neuropilin-2 (NP-2) mRNA, relative to β-actin, at p0, p14 and p18 room air (RA) and 50/10 OIR. Significantly increased fold expression in NP-2 mRNA was present in 50/10 OIR compared to RA at p14 (*p=0.001) and p18 (**p=0.002). Student t-tests.

Figure 8

Fold change in expression of PEDF mRNA, relative to β-actin, at p0, p14 and p18 room air (RA) and 50/10 OIR. No significant difference was found at p14 (p=0.47) or p18 (p=0.39). Student t-tests.

Figure 9

Fold change in expression of PEDFR mRNA, relative to β-actin, at p0, p14 and p18 room air (RA) and 50/10 OIR. Significantly increased fold expression in PEDFR mRNA was present in 50/10 OIR compared to RA at p14 (*p=0.0006). No significant difference was found at p18 (p=0.32). Student t-tests.