Increased expression and activity of 12-lipoxygenase in oxygen-induced ischemic retinopathy and proliferative diabetic retinopathy: implications in retinal neovascularization

Abstract

Objective: Arachidonic acid is metabolized by 12-lipoxygenase (12-LOX) to 12-hydroxyeicosatetraenoic acid (12-HETE) and has an important role in the regulation of angiogenesis and endothelial cell proliferation and migration. The goal of this study was to investigate whether 12-LOX plays a role in retinal neovascularization (NV).

Research design and methods: Experiments were performed using retinas from a murine model of oxygen-induced ischemic retinopathy (OIR) that was treated with and without the LOX pathway inhibitor, baicalein, or lacking 12-LOX. We also analyzed vitreous samples from patients with and without proliferative diabetic retinopathy (PDR). Western blotting and RT-PCR were used to assess the expression of 12-LOX, vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor (PEDF). Liquid chromatography-mass spectrometry was used to assess the amounts of HETEs in the murine retina and human vitreous samples. The effects of 12-HETE on VEGF and PEDF expression were evaluated in Müller cells (rMCs), primary mouse retinal pigment epithelial cells, and astrocytes.

Results: Retinal NV during OIR was associated with increased 12-LOX expression and 12-, 15-, and 5-HETE production. The amounts of HETEs also were significantly higher in the vitreous of diabetic patients with PDR. Retinal NV was markedly abrogated in mice treated with baicalein or mice lacking 12-LOX. This was associated with decreased VEGF expression and restoration of PEDF levels. PEDF expression was reduced in 12-HETE-treated rMCs, astrocytes, and the retinal pigment epithelium. Only rMCs and astrocytes showed increased VEGF expression by 12-HETE.

Conclusions: 12-LOX and its product HETE are important regulators of retinal NV through modulation of VEGF and PEDF expression and could provide a new therapeutic target to prevent and treat ischemic retinopathy.

FIG. 1.

Analysis of 12-LOX expression in the retina. Western blot analysis of leukocyte (A) and platelet (B) 12-LOX showed significant increases in OIR compared with the control (C). *P < 0.05 vs. control. C: Immunolocalization using vascular marker (isolectin-B4, red) and anti–12-LOX (green) showed a marked increase in 12-LOX immunoreactivity in retinal vessels (arrows) and RPE cells (arrowhead) of OIR compared with the control (C). D: Western blot analysis of 12-LOX expression showed a significant increase in retina of diabetic mice (D) compared with the control (C). *P < 0.05 vs. control. E: Immunolocalization of 12-LOX (green) indicated that it is upregulated in retinal vessels (red) of diabetic mice (D). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

LC/MS assay of HETEs production in mouse retina. There was a significant increase in the amount of 12-HETE (A), 5-HETE (B), and 15-HETE (C) in OIR compared with the control (C). The amount of 12-HETE was reduced in animals treated with baicalein or lacking 12-LOX (LOX^−/−). *P < 0.05 vs. control; #P < 0.05 vs. OIR.

FIG. 3.

LC/MS assay of HETE production in human vitreous samples. The amounts of 12-HETE (A), 15-HETE (B), and 5-HETE (C) were increased in diabetic subjects with PDR compared with the control (C). *P < 0.05.

FIG. 4.

Whole-mount retinas labeled with vascular marker (isolectin-B4). The total area of new capillary tufts (arrows, circled area) was reduced in baicalein-treated (OIR+baicalein) and 12-LOX knockout mice (OIR-12LOX^−/−) compared with the nontreated group (OIR-WT). *P < 0.05 vs. the nontreated group. (A high-quality color representation of this figure is available in the online issue.)

FIG. 5.

Western blot analysis of VEGF and PEDF in the retina. A and B: OIR in wild-type (WT) mice was associated with significantly higher VEGF expression compared with the control. The change in VEGF expression was prevented by baicalein (OIR+baicalein) or deletion of 12-LOX (OIR-12LOX^−/−). C: PEDF expression was reduced in the nontreated group (OIR-WT) and was restored by baicalein. *P < 0.05 vs. control; #P < 0.05 vs. the nontreated group.

FIG. 6.

Effect of 12-HETE on the level of VEGF and PEDF expression in rMCs. A: VEGF level in rMC–conditioned medium was determined by ELISA. There was a marked increase in the amounts of VEGF by 12-HETE (0.5 and 1 μmol/L) compared with the control (C). *P < 0.05 vs. control. B: Western blot analysis of PEDF expression in rMCs. Note the decreased PEDF expression by 12-HETE (0.5 or 1.0 μmol/L) compared with the control (C). *P < 0.05 vs. control.

FIG. 7.

Effect of HETEs on VEGF and PEDF expression in primary mouse retinal astrocytes and RPE cells. ELISA assay of VEGF in cultured mouse retinal astrocytes (A) and RPE cells (B). 12-HETE induced a significant increase in VEGF expression of retinal astrocytes compared with the control and 5-HETE– and 15-HETE–treated cells (*P < 0.05 vs. control, 5-, and 15-HETE). There was no effect on VEGF expression in RPE cells incubated with any of the HETEs compared with the control. C: Western blot analysis of PEDF expression in RPE cells showed marked inhibition by 12- and 5-HETE compared with the control and 15-HETE.

FIG. 8.

Quantitative PCR analysis of VEGF (A) and PEDF (B) in murine RPE cells and retinal astrocytes. There was no significant change in VEGF or PEDF mRNA level of RPE cells or astrocytes incubated with different HETEs.