The role of supplemental oxygen and JAK/STAT signaling in intravitreous neovascularization in a ROP rat model

Abstract

Purpose: To investigate whether oxygen stresses experienced in retinopathy of prematurity (ROP) trigger signaling through reactive oxygen species (ROS) and whether the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway lead to intravitreous neovascularization (IVNV) in an oxygen-induced retinopathy (OIR) rat model.

Methods: Newborn rat pups exposed to repeated fluctuations in oxygen and rescued in supplemental oxygen (28% O(2), 50/10 OIR+SO) were treated with apocynin, an NADPH oxidase and ROS inhibitor (10 mg/kg/d), AG490, a JAK2 inhibitor (5 mg/kg/d), or phosphate-buffered saline. Intraperitoneal injections were given from postnatal day (P)12 to P17 (apocynin), or from P3 to P17 (AG490). Outcomes were intravitreous neovascularization and avascular/total retinal areas, vascular endothelial growth factor, phosphorylated JAK2, and phosphorylated STAT3.

Results: Apocynin significantly reduced phosphorylated STAT3 in 50/10 OIR+SO (P = 0.04), in association with previously reported inhibition of the IVNV area. Inhibition of JAK with AG490 significantly reduced phosphorylated JAK2 (P < 0.001), phosphorylated STAT3 (P = 0.002), and IVNV area (P = 0.033) in the 50/10 OIR+SO model compared with control.

Conclusions: Activation of NADPH oxidase from supplemental oxygen works through activated STAT3 to lead to IVNV. In addition, inhibition of the JAK/STAT pathway reduces IVNV. Further studies are needed to determine the effects and relationships of oxygen stresses on JAK/STAT and NAPDH oxidase signaling.

Figure 1

STAT3 mRNA expression measured at time points in retinas of pups from room air development and under repeated oxygen fluctuations (rep oxy fluc) through post-natal day (p)14. STAT3 mRNA expression was analyzed by real-time PCR in 4 to 5 retinas at each time point and data expressed as fold change over p0. Beta-actin was run as the internal control gene. There was only a modest increase in expression of STAT3 mRNA under the condition of oxygen fluctuations compared to room air at several time points.

Figure 2

Densitometry results (A, C) and representative gel images (B, D) of phosphorylated/total JAK2 protein (A, B) and phosphorylated/total STAT3 protein (C, D) in 50/10 OIR retinas from AG490 treated and controls analyzed by western blot at postnatal days (p)7 and p14. 50ug total protein separated by SDS-PAGE and probed with anti- phospho JAK2 and JAK2 antibodies or anti-phospho STAT3 and STAT3 antibodies. Beta-actin was used as a loading control and to normalize values. Odd numbered lanes are from AG490 treated retinas (p7) and even numbered lanes show representative samples of PBS treated retinas (p7). Data represents mean±SE of n= 5 retinas for each analysis. *p=0.04, **p=0.02, ^∓p=0.03, and ^∓∓p=0.01. There were significant decreases in JAK2 and STAT3 phosphorylation following treatment with AG490 compared to controls.

Figure 3

Areas of intravitreous neovascularization (IVNV) (indicated by white arrows) at postnatal day (p)18 in retinas from the 50/10 OIR+SO model treated with daily intraperitoneal (IP) injections (5mg/kg) of AG490 or PBS starting at p3 and continuing through p17. (A) Mean±SE pixel density in 10 or more retinas analyzed per group, *p=0.03 (B) Flat mount images representative of each treatment group. AG490 treatment significantly reduced intravitreous neovascularization in the 50/10 OIR+SO model compared to control.

Figure 4

Densitometry results of JAK/STAT activation in 50/10 OIR+SO retinas from AG490 treated and control animals analyzed by western blot at postnatal day (p)18. 50ug total protein separated by SDS-PAGE and probed with respective antibodies. (A) anti- phospho JAK2, and total JAK2, (B) anti-phospho STAT3, and total STAT3. Beta-actin was used as a loading control and to normalize values. Data represents mean±SE of n= 5 retinas each repeated from several litters. *p<0.001, and **p=0.002. Both STAT3 and JAK2 activations were significantly reduced in retinas from AG490 treated animals compared to controls in the 50/10 OIR+SO model.

Figure 5

JAK/STAT activation in retinas from 50/10 OIR+SO model. Western blot of (A) phospho/total STAT3 and (B) phospho/total JAK2 after treatment with daily intraperitoneal (IP) injections of apocynin (10 mg/kg) or PBS from postnatal day (p)12 to p17 and analyzed at p18. A total of 6 retinas were analyzed for each group and data represent values following normalization to beta-actin. *p=0.04, n.s. – not significant. In the 50/10 OIR+SO model, apocynin significantly reduced STAT3 but not JAK2 phosphorylaton compared to control.

Figure 6

Proposed steps in signaling pathways in the 50/10 OIR+SO model that may be triggered to cause intravitreous neonascularization.