SOCS3 in retinal neurons and glial cells suppresses VEGF signaling to prevent pathological neovascular growth

Abstract

Neurons and glial cells in the retina contribute to neovascularization, or the formation of abnormal new blood vessels, in proliferative retinopathy, a condition that can lead to vision loss or blindness. We identified a mechanism by which suppressor of cytokine signaling 3 (SOCS3) in neurons and glial cells prevents neovascularization. We found that Socs3 expression was increased in the retinal ganglion cell and inner nuclear layers after oxygen-induced retinopathy. Mice with Socs3 deficiency in neuronal and glial cells had substantially reduced vaso-obliterated retinal areas and increased pathological retinal neovascularization in response to oxygen-induced retinopathy, suggesting that loss of neuronal/glial SOCS3 increased both retinal vascular regrowth and pathological neovascularization. Furthermore, retinal expression of Vegfa (which encodes vascular endothelial growth factor A) was higher in these mice than in Socs3 flox/flox controls, indicating that neuronal and glial SOCS3 suppressed Vegfa expression during pathological conditions. Lack of neuronal and glial SOCS3 resulted in greater phosphorylation and activation of STAT3, which led to increased expression of its gene target Vegfa, and increased endothelial cell proliferation. In summary, SOCS3 in neurons and glial cells inhibited the STAT3-mediated secretion of VEGF from these cells, which suppresses endothelial cell activation, resulting in decreased endothelial cell proliferation and angiogenesis. These results suggest that neuronal and glial cell SOCS3 limits pathological retinal angiogenesis by suppressing VEGF signaling.

Figure 1. Socs3 mRNA expression was induced in neuronal layers in the OIR model

(A) Schematic diagram of OIR. Neonatal mice were exposed to 75% oxygen from postnatal day (P) 7–12 to induce vessel loss and returned to room air from P12–P17 to induce maximum pathological neovascularization at P17. (B) Socs3 mRNA expression in P17 retinal layers from laser-capture-micro-dissection from retinas with OIR compared to age-matched normoxia (Norm) control retinas (n=6 retinas per group). Socs3 mRNA was increased in RGC and INL, but not in ONL in retinas with OIR compared with normoxia control retinas. Images on the left show representative retinal cross sections from normoxia and retinas with OIR stained with isolectin B4 (Lectin, red) for endothelial cells and DAPI (blue), with dotted lines highlighting the areas for laser-capture-micro-dissection. (C) Socs3 was decreased by 80% in the retinas of mice that were generated by crossing Socs3 flox/flox (Socs3 f/f) mice with nestin-Cre driven mice (Socs3 cKO). Decreased SOCS3 abundance at the protein level was confirmed in whole retinas by Western blot (n= 6 retinas per group). (D) P17 retinal cross sections from Socs3 cKO;mTmG retinas with OIR show that Nestin-driven Cre recombinase present in all the neuronal layers (labeled with GFP) (n= 3 mice with 3 retinas per group). Scale bar: 100 µm.

Figure 2. Neuronal/glial Socs3 attenuated pathological neovascularization in mouse model with OIR

(A) Representative retinal flat-mounts (A, left panel) from P17 Socs3 f/f and Socs3 cKO retinas with OIR stained with isolectin B4 (red). The areas of neovascularization and vaso-obliteration were highlighted for quantification (white). Insets show enlarged pathological neovessels. Quantification of pathological neovascularization (A, right panel) showed that Socs3 attenuated pathological neovascularization (n=14–27 retinas per group) and decreased the area of central vaso-obliteration in P17 retinas with OIR (n=10–27 retinas per group). (B) Between Socs3 f/f and Socs3 Nes-ko retinas, the retinal vaso-obliteration areas were comparable at P12 (n=6–10 retinas per group). (C and D) There was no significant difference in normal developmental retina areas at P7 (n=5–6 retinas per group) (C), or in vessel layers in adults (D), or in layer thicknesses of entire retina, INL and nerve fiber layer (NFL)+IPL layer in live Socs3 f/f and Socs3 Nes-ko mice (n=6 retinas per group) (E) Black arrows in fundus images indicate the position of OCT. Scale bar for flat mounts in A–C: 1000 µm; for inset in A: 100 µm; for cross section in D: 50 µm; for layers in D: 25 µm; for OCT in E: 50 µm. NV, neovascularization; VO, vaso-obliteration.

Figure 3. Neuronal/glial Socs3 deficiency enhanced Vegfa expression

(A) Vegfa mRNA expression in retinal layers from laser-capture-micro-dissection from P17 retinas with OIR compared to age-matched normoxia (Norm) control retinas. Vegfa mRNA expression was increased in RGC and INL, but not in ONL in retinas layers from P17 retinas with OIR compared with age-matched normoxic control retinas (n=6 retinas per group). Socs3 suppression in retinal neurons and glia increased Vegfa mRNA expression (n= 6 retinas per group) (B) and Vegfa isoform 120 and 164, but not 188 (C), and VEGFA-164 protein abundance (~3.5 fold, n= 6 retinas per group) (D) were increased in P17 Socs3 cKO retinas with OIR compared with control retinas. (E) Immunohistochemistry staining showed increased VEGFA (VEGFA-164) mainly in the INL and RGC layers. Representative images were from 3 mice per group. Scale bar: 50 µm.

Figure 4. Knocking out Socs3 in neurons and glia increased STAT3 activity

(A, B) In P17 retinas with OIR, the mRNA expression of HIF-1α target genes Epo, Angptl4, Ang1, PDGFα and TGFβ and HIF-1α protein abundance were comparable between Socs3 cKO retinas and littermate Socs3 f/f retinas (n=6 retinas per group). (C) In P17 retinas with OIR, representative Western blots show that phosphorylated STAT3 (pSTAT3) was highly increased in Socs3 cKO retinas compared Socs3 f/f retinas. Band densities for Western blots were quantified using Image J in the bottom panel (n=4 retinas per group). (D) In P17 retinas with OIR, phosphorylated STAT3 (pSTAT3) was increased mainly in the INL and RGC layers. Cross-sections were stained with pSTAT3 (green) and DAPI (blue). Representative images were from 3 mice per group. Scale bar: 50 µm.

Figure 5. Neuronal/glial Socs3 deficiency increased endothelial cell proliferation and ERK phosphorylation

(A and B) In P17 retinas with OIR, Ki67 positive (which denotes proliferation) cells were increased in Socs3 cKO retinas compared to Socs3 f/f control retinas. Cross-sections from P17 retinas with OIR were stained for proliferative cells with Ki67 (green, proliferative cells), isolectin B4 (magenta, endothelial cells) and cell nuclei with DAPI (blue) (A). Scale bar: 25 µm. Quantification of Ki67 pixels is shown in (B) (n=6 retinas per group). (C) In P17 retinas with OIR, representative Western blots and quantification show that phosphorylated ERK (pERK) was increased in Socs3 cKO retinas compared to Socs3 f/f OIR retinas (n=4 retinas per group).

Figure 6. Neuronal/glial deficiency of Socs3 results in activated Müller glial cells and astrocytes

(A) In P17 Socs3 cKO;mTmG reporter retinas with OIR, CralBP, labeled Müller cells and astrocytes colocalized with GFP. Cross-sections were stained for Müller cells and astrocytes with anti-CralBP (magenta) and cell nuclei with DAPI (blue). White arrow: astrocytes or end-feet of activated Müller cells. Open arrowheads: activated Müller cells. Scale bar: 10 µm. Images are representative of 3 mice per group. (B) Cross-sections from P17 Socs3 cKO retinas with OIR were stained with CralBP (magenta), VEGF (green) and DAPI (blue). VEGF colocalized with CralBP positive cells. Scale bar: 10 µm. (C) A diagram showing VEGF in the RGC and INL layers including activated astrocytes and Müller cells. (D) In P17 retinas with OIR, GFAP-labeled activated Müller cells in Socs3 cKO retinas were increased compared to Socs3 f/f control retinas. Cross-sections from P17 Socs3 cKO and Socs3 f/f control retinas with OIR were stained for endothelial cells with isolectin B4 (magenta), activated Müller cells and astrocytes with anti-GFAP (green) and cell nuclei with DAPI (blue). Scale bar: 25 µm. (E) GFAP-labeled astrocytes and end-feet of activated Müller cells in flat-mounted P17 Socs3 cKO and Socs3 f/f retinas with OIR. Representative images in A, B, D, and E were from 6 mice per group. White arrow: astrocytes or end-feet of activated Müller cells. Open arrowheads: activated Müller cells. Scale bar: 50µm.