Activated NAD(P)H oxidase from supplemental oxygen induces neovascularization independent of VEGF in retinopathy of prematurity model

Abstract

Purpose: To study NAD(P)H oxidase-dependent outcomes after oxygen stresses that are similar to those experienced by preterm infants today using a rat model of retinopathy of prematurity.

Methods: Within 4 hours of birth, pups and their mothers were cycled between 50% and 10% oxygen daily for 14 days and were returned to room air (21% O2, 50/10 oxygen-induced retinopathy [OIR]) or supplemental oxygen (28% O2, 50/10 OIR+SO) for 4 days. Pups received intraperitoneal injections of the specific NAD(P)H oxidase inhibitor apocynin (10 mg/kg/d) or of PBS from postnatal day (P)12 to P17, and some received intraperitoneal injections of hypoxyprobe before kill. Intravitreous neovascularization (IVNV), avascular/total retinal areas, vascular endothelial growth factor (VEGF), NAD(P)H oxidase activity, or hypoxic retina (conjugated hypoxyprobe) were determined in neurosensory retinas. Human retinal microvascular endothelial cells (RMVECs) treated with apocynin or control were exposed to 1% or 21% O2 and assayed for phosphorylated (p-)Janus kinase (JNK) and NAD(P)H oxidase activity.

Results: Retinas from 50/10 OIR+SO had increased NAD(P)H oxidase activity and lower VEGF than did retinas from 50/10 OIR. Apocynin treatment reduced the IVNV area and hypoxic retina in 50/10 OIR+SO. RMVECs treated with 1% O2 had increased p-JNK compared with RMVECs exposed to room air.

Conclusions: Different oxygen stresses activate NAD(P)H oxidase to varying degrees to trigger disparate pathways (angiogenesis or apoptosis). The oxygen stresses and outcomes used in this study are relevant to human ROP and may explain some of the complexity in the pathophysiology of ROP resulting from oxygen exposure.

Figure 1

Lectin-stained (red) flatmounts and cryosections of retinas from 50/10 OIR and room air-raised pups given IP injections of pimonidazole (HP; green) 90 minutes before humane killing. (A) P18 50/10 OIR flatmount demonstrating hypoxic (green) retina in avascular zone and in regions surrounding lectin-stained blood vessels (red) in vascularized retina. (B) P4 room air-raised pup demonstrating little staining with HP, even though avascular retina is present approximately to the same extent as for P18 50/10 OIR. (C) Cryosection of eye from P18 50/10 OIR pup demonstrating inner retinal hypoxia (green) within avascular zone and some staining in ganglion cell layer in vascularized retina. Representative of eyes from three litters.

Figure 2

Western blot of retinas from P18 50/10 OIR+SO and P18 50/10 OIR immunoprecipitated for NAD(P)H oxidase cytoplasmic subunit p47^phox blotted for phosphoserine to determine phosphorylated p47^phox/total p47^phox. Total p47^phox was determined after membranes were stripped and reprobed with p47^phox antibody (*P ≤ 0.02; Student’s t-test). Retinas taken from at least four separate litters; at least five retinas were analyzed for each treatment group.

Figure 3

Avascular/total retina areas from pups in 50/10 OIR+SO and 50/10 OIR treated with IP injections of apocynin (10 mg/kg/d P12–P17) or an equivalent volume of PBS and assayed at P18 (P = NS). Retinas taken from at least four separate litters; at least five retinas were analyzed for each treatment group.

Figure 4

IVNV area from pups in 50/10 OIR+SO and 50/10 OIR treated with IP injections of apocynin (10 mg/kg/d P12–P17) or an equivalent volume of PBS and assayed at P18 (*P = 0.047; Student’s t-test). Retinas taken from at least four separate litters; at least five retinas were analyzed for each treatment group.

Figure 5

Ratio of hypoxyprobe/actin Western blot in 50/10 OIR+SO and 50/10 OIR treated with IP injections of apocynin (10 mg/kg/d P12–P17) or an equivalent volume of PBS and assayed at P18 (*P = 0.002; Student’s t-test). Retinas taken from two separate litters; at least five retinas were analyzed for each treatment group.

Figure 6

ELISA of VEGF from pups in 50/10 OIR or in 50/10 OIR+SO treated with either IP injections of apocynin (10 mg/kg/d) or an equivalent volume of PBS from P12–P17 and assayed at P18 (*P < 0.001; Student’s t-test). Retinas taken from at least four separate litters; at least five retinas were analyzed for each treatment group.

Figure 7

Western blot of human RMVECs treated with either apocynin (30 μg/mL final concentration) or PBS control and exposed to 1% O₂ or 21% O₂ for 6 hours, then immunoprecipitated for NAD(P)H oxidase cytoplasmic subunit p47^phox blotted for phosphoserine to determine phosphorylated p47^phox/total p47^phox. Total p47^phox was determined after membranes were stripped and reprobed with p47^phox antibody. Individual Student’s t-tests for 1% O₂ PBS versus 21% O₂ PBS (*P = 0.02) and for 1%O₂ PBS vs. 21% O₂ apocynin (**P = 0.006). n = 4 for each treatment group.

Figure 8

Human RMVECs treated with either apocynin (30 μg/mL final concentration) or PBS control and exposed to 1% O₂ or 21% O₂ for 6 hours, then undergoing ELISA testing for phospho-JNK. Individual Student’s t-tests (*P ≤ 0.04 for 1% O₂ PBS vs. 21% O₂ PBS and 1% O₂ PBS vs. 21% O₂ apocynin). ELISA was performed in duplicate for each treatment group.