Irisin Attenuates Pathological Neovascularization in Oxygen-Induced Retinopathy Mice

Abstract

Purpose: Abnormal angiogenesis is a defining feature in a couple of ocular neovascular diseases. The application of anti-VEGFA therapy has achieved certain benefits in the clinic, accompanying side effects and poor responsiveness in many patients. The present study investigated the role of irisin in retinal neovascularization.

Methods: Western blot and quantitative PCR were used to determine irisin expression in the oxygen-induced retinopathy mice model. The pathological angiogenesis and inflammation index were examined after irisin administration. Primary retinal astrocytes were cultured and analyzed for VEGFA expression in vitro. Astrocyte-conditioned medium was collected for transwell assay and tube formation assay in human microvascular endothelial cells-1.

Results: Irisin was downregulated in the oxygen-induced retinopathy mice retinae. Additional irisin attenuated pathological angiogenesis, inflammation, and apoptosis in vivo. In vitro, irisin decreased astrocyte VEGFA production, and the conditioned medium suppressed human microvascular endothelial cells-1 migration. Last, irisin inhibited hypoxia-inducible factor-2α, nuclear factor-κB, and pNF-κB (Phospho-Nuclear Factor-κB) expression.

Conclusions: Irisin mitigates retinal pathological angiogenesis.

Chinese Abstract.

Figure 1.

Irisin is decreased in the retinae of the OIR mice. (A) Diagrammatic illustration of the OIR mice model. (B) A qPCR analysis of irisin mRNA expression in normoxic and OIR retinae at P14 and P17. Data were normalized to gene expression of the normoxia group, and β-actin was used as an internal control (n = 3-4). (C) Western blot analysis and quantification of irisin in normoxic and OIR retinae at P14 (n = 4). (D) Representative immunofluorescence images of irisin (red) and GFAP (green) in P14 normoxic and OIR mice retina cryosections. Scale bar, 100 µm.*P < 0.05. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NFL, nerve fiber layer; ONL, outer nuclear layer.

Figure 2.

Supplementation of irisin alleviates aberrant angiogenesis in OIR mice. (A) Irisin or saline was administered to the OIR mice via vitreous injection at P13, and retinae were analyzed at P17. (B) IB4 staining of whole-mount retinae from OIR + saline and OIR + irisin mice at P17. The yellow area indicates the avascular area in the insets, and the white area indicates the neovascular area. (C) Quantification of the avascular area and neovascular area at P17 in OIR + saline and OIR + irisin mice retinae, related to (B). (D and E) Representative images of CD31 (green) in superficial, intermittent, and deep retinal layers in OIR + saline and OIR + irisin mice retinae at P17, with quantification of the vessel length and branching points of superficial, intermediate, and deep vascular network (n = 7–8). (F) Western blot analysis and quantification of PCNA protein in the retinae of OIR + saline and OIR + irisin mice. (G and H) Representative images and the corresponding quantification of PCNA⁺/IB4⁺ cells in OIR + saline and OIR + irisin mice (n = 3–4). *P < 0.05.

Figure 3.

VEGFA is downregulated by irisin administration in OIR mice. (A) Western blot analysis and quantification of VEGFA in OIR + saline and OIR + irisin retinae at P17 (n = 7). (B) Representative images of retinal cryosection staining with VEGFA (green) and GFAP (red) in OIR + saline and OIR + irisin mice at P17. (C) Fluorescence signal intensities of VEGFA and GFAP costaining quantified from OIR + saline and OIR + irisin group, related to (B) (n = 4–5). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NFL, nerve fiber layer; ONL, outer nuclear layer.

Figure 4.

Irisin impairs hypoxia induced VEGFA production in astrocytes in vitro. (A) Schematic illustration of astrocytic preparations for ELISA, Western blot, and qPCR. Astrocytes were exposed to 250 µM CoCl₂ in the absence or presence of irisin (10 nM) and/or RGD peptide (100 ng/mL) for 24 hours. The NC group was treated with the same volume of the solvent PBS (1% DMSO). (B) Free VEGFA concentrations in conditioned media from primary astrocytes were determined by ELISA. (C) A qPCR analysis of VEGFA mRNA expression in astrocytes from NC, irisin, CoCl₂ and CoCl₂ + irisin groups. (D) Western blot analysis and quantification of VEGFA and HIF-2α protein level in astrocytes from CoCl₂, CoCl₂ + irisin, CoCl₂ + irisin + RGD, and CoCl₂ + RGD groups (n = 4). *P < 0.05.

Figure 5.

Effects of ACM from different groups on HMECs-1 migration and tube formation. (A) Schematic illustration of cell experiments. (B) Representative images of HMECs-1 transwell assay in fresh medium or ACM. Both fresh medium and ACM include the NC and irisin groups. (C) The quantification of migrated cells in HMECs-1, related to (B) (n = 3). *P < 0.05. (D) Representative images of tube formation in HMECs-1 in fresh medium or ACM. Fresh medium was divided into CoCl₂ and CoCl₂ + irisin groups. ACM included four groups: CoCl₂ ACM, CoCl₂ + irisin ACM (CoCl₂ + irisin), ACM + VEGFA, and CoCl₂ ACM + VEGFA. (E) The quantification of tube formation in HMECs-1, related to (D) (n = 3–4). *P < 0.05.

Figure 6.

HIF-2α expression is reduced in the OIR mice retinae after irisin administration. (A) Western blot and quantification of HIF-2α in the retinae of OIR + saline and OIR + irisin mice at P17 (n = 3–4). (B and C) Whole-mount immunostaining with GFAP and IB4 in the avascular and neovascular areas in OIR + saline and OIR + irisin retinae. (D) Quantification of the astrocyte density in the avascular and neovascular areas, related to (B) and (C) (n = 3–4). *P < 0.05.

Figure 7.

Irisin prevents cell apoptosis in the OIR retinae. (A) Representative images of TUNEL staining in the retinae of normoxia + saline, normoxia + irisin, OIR + saline, and OIR + irisin mice. (B) The quantitative analysis of TUNEL-positive cells related to A. (C) Western blot analysis and quantification of active caspase 3 expression in normoxia + saline, normoxia + irisin, OIR + saline, and OIR + irisin retinae at P17. (D) qPCR analysis of TNF-α, IL-1β, and monocyte chemoattractant protein-1 (MCP-1) mRNA in normoxia + saline, normoxia + irisin, OIR + saline, and OIR + irisin retinae at P17. Data were normalized to the normoxia + saline and OIR + saline groups. β-Actin was used as an internal control. (E) Western blot analysis and quantification of NF-κB and pNF-κB protein in the retinae from normoxia + saline, normoxia + irisin, OIR + saline, and OIR + irisin mice upon P17 (n = 3–4). *P < 0.05. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.