Stanniocalcin-1 is a Modifier of Oxygen-Induced Retinopathy Severity

Abstract

Purpose/Aim: Abnormal activation of signaling pathways related to angiogenesis, inflammation, and oxidative stress has been implicated in the pathophysiology of retinopathy of prematurity (ROP), a leading cause of blindness in pre-term infants. Therapies for ROP include laser and anti-vascular endothelial growth factor agents. However, these therapies have side effects, and even with adequate treatment, visual acuity can be impaired. Novel therapeutic options are needed. Stanniocalcin-1 (STC-1) is a neuroprotective protein with anti-inflammatory and anti-oxidative stress properties. Rodent models of oxygen-induced retinopathy (OIR) were selected to determine whether STC-1 plays a role in the development of OIR.Materials and methods: STC-1 gene and protein expression was first evaluated in the Sprague Dawley rat OIR model that is most similar to human ROP. OIR was then induced in wild-type and Stc-1^-/- mice. Retinas were isolated and evaluated for avascular and neovascular area on retinal flat mounts. Quantification of gene expression by quantitative real-time PCR was performed. VEGF was assayed by ELISA in media obtained from induced pluripotent stem-cell-derived retinal pigment epithelial (iPS-RPE) cells following treatment with recombinant STC-1.Results: STC-1 was significantly upregulated in a rat model of OIR compared to room air controls at the gene (P < .05) and protein (P < .001) level. Stc-1^-/- OIR mice showed significantly worse ROP compared to wild-type mice as assessed by avascular (20.2 ± 2.4% vs 15.2 ± 2.5%; P = .02) and neovascular area (14.3 ± 2.7% vs 8.8 ± 3.7%; P < .05). Transcript levels of vascular endothelial growth factor-A were significantly higher in Stc-1^-/- OIR mice compared to wild-type controls (P = .03). STC-1 reduced VEGF production in iPS-RPE cells (P = .01).Conclusions: STC-1 plays a role in the OIR stress response and development of pathologic vascular features in rodent OIR models by regulating VEGF levels.

Keywords: Retinopathy of prematurity; STC-1; Stanniocalcin-1; oxygen induced retinopathy.

Figure 1.. Stanniocalcin-1 is upregulated in a rat model of oxygen induced retinopathy.

(A) Quantitative real-time PCR shows a significant increase in STC-1 expression at P17 (P<0.01) and P20 (P=0.02) in wild-type oxygen induced retinopathy (OIR) rats compared to room air (RA) controls. Note there was no significant difference at P14. (B) Representative western blot reveals that STC-1 is upregulated in retinal lysates obtained from P18 OIR rats compared to RA. (C) Densitometry of STC-1 normalized to β-actin (Relative Quantification; RQ) revealed significantly increased STC-1 in OIR (n=10, p<0.001)*** compared to P18 RA controls (n=3). Error bars represent SEM.

Figure 2.. Stc-1^−/− mice develop worse retinopathy in a mouse model of oxygen induced retinopathy.

(A) Representative retinal flat mount of mice subjected to OIR shows avascular (asterisk) and neovascularization (arrow head) in wild-type mice (left panel) that is less severe compared to Stc-1^−/− mice (right panel) at P17. (B) Graph showing increased avascular (P=0.02) and neovascular (P<0.05) regions in Stc-1^−/− mice compared to wild-type controls at P17. Error bars represent SEM.

Figure 3. Stc-1^−/− mice show higher levels of VEGFA compared to wild-type in a mouse model of oxygen induced retinopathy

Quantitative real-time PCR shows that Stc-1^−/− mice had a significant increase in expression of VEGF-A (P=0.03)* at P17 compared to wild-type controls. Error bars represent SEM.

Figure 4. VEGF is decreased in cultures of induced pluripotent stem cell derived retinal pigment epithelium cell cultures following addition of STC-1.

ELISA for VEGF in conditioned media from induced pluripotent stem cell derived retinal pigment epithelium (iPS-RPE) shows a decrease in VEGF expression 24 hours after addition of recombinant human STC-1 (500ng/mL) (n=4, P=0.01). Error bars represent SEM.