HO-1-mediated ferroptosis as a target for protection against retinal pigment epithelium degeneration

Abstract

Oxidative stress-mediated retinal pigment epithelium (RPE) degeneration plays a vital role in retinal degeneration with irreversible visual impairment, most notably in age-related macular degeneration (AMD), but a key pathogenic factor and the targeted medical control remain controversial and unclear. In this work, by sophisticated high-throughput sequencing and biochemistry investigations, the major pathologic processes during RPE degeneration in the sodium iodate-induced oxidative stress model has been identified to be heme oxygenase-1 (HO-1)-regulated ferroptosis, which is controlled by the Nrf2-SLC7A11-HO-1 hierarchy, through which ferrous ion accumulation and lethal oxidative stress cause RPE death and subsequently photoreceptor degeneration. By direct knockdown of HO-1 or using HO-1 inhibitor ZnPP, the specific inhibition of HO-1 overexpression has been determined to significantly block RPE ferroptosis. In mice, treatment with ZnPP effectively rescued RPE degeneration and achieved superior therapeutic effects: substantial recovery of the retinal structure and visual function. These findings highlight that targeting HO-1-mediated RPE ferroptosis could serve as an effectively retinal-protective strategy for retinal degenerative diseases prevention, including AMD.

Keywords: Age-related macular degeneration; Ferroptosis; Heme oxygenase-1; Iron chelation; Photoreceptor; Retinal pigment epithelium.

Scheme 1

HO-1-mediated ferroptosis as a target for protection against retinal pigment epithelium degeneration. Left panel: RPE ferroptosis is a major pathological process responsible for NaIO₃–induced retinal oxidative stress, which is mainly mediated by a vicious cycle between HO-1 upregulation and iron overload with lethal oxidative stresses of ROS/LOS accumulation. Green ball: ferrous ions; red ball: ROS; purple ball: LOS. Right panel: HO-1 inhibitor ZnPP inhibits RPE ferroptosis by specifically inhibiting HO-1 overexpression, and hampering the detrimental circulative effects between HO-1 and ferrous ion to decrease LOS and ROS overload, eventually suppressing RPE death and subsequent photoreceptor degeneration.

Fig. 1

Ferroptosis is a major pathological process in oxidative stress-mediated RPE degeneration. (A) Ultrastructure of mitochondria in ARPE-19 cells treated with Erastin (5 μM) and NaIO₃ (30 mM) for 24 h was imaged by TEM. Red arrowheads: shrunken mitochondria. Scale bars, 0.5 μm. ARPE-19 cells were pretreated with ferroptosis inhibitors (Fer-1, 10 μM and DFO, 75 μM) for 1 h followed by NaIO₃ and Erastin treatment, 24 h later, (B) cell viability detected by CCK-8, (C) ferrous ion levels by FerroOrange staining and (D) corresponding quantification by Image J were evaluated. Scale bars, 50 μm n = 3, means ± SD; one-way ANOVA with Bonferroni correction; **P < 0.01. Flow cytometry plots and bar graphs of lipid ROS (LOS) production in (E, F) NaIO₃- and (G, H) Erastin-treated ARPE-19 cells using C11-BODIPY were showed. n = 3, means ± SD; one-way ANOVA with Bonferroni correction; *P < 0.05, **P < 0.01. In vivo mRPE cells were pretreated with Fer-1 or DFO followed by NaIO₃ and Erastin treatment, 2 weeks later, (I) MDA analysis for evaluation of LOS levels, (J) protein blots and (K) semi-quantitation of the protein expression levels of ferroptosis biomarkers PTGS2 and GPX4 were determined. n = 3, means ± SD; one-way ANOVA with Bonferroni correction; *P < 0.05, **P < 0.01. (L–N) RNA-sequencing analysis of PBS (control) and NaIO₃-treated ARPE-19 cells for 24 h. (L) The number of genes in differential expressions (≥1.5-fold difference: upregulated (yellow) and downregulated genes (blue)) was presented in volcano plot. (M) KEGG enrichment analysis identified the most significantly affected pathways in response to NaIO₃ treatment in differential expressed genes. (N) FPKM values of main regulators in ferroptosis and apoptosis after NaIO₃ incubation. n = 3, mean ± SD; two-tailed t-test; *p < 0.05 and **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Excessive activation of HO-1 via Nrf2–SLC7A11–HO-1 hierarchy contributes to RPE ferroptosis. (A) Differentially expressed genes that are closely associated with RPE ferroptosis in NaIO₃-treated cells were determined by RNA-sequencing analysis. Blue: low expression levels. Yellow: high expression levels. (B) Protein blots and (C) semi-quantitation of the protein expression levels of HO-1 and SLC7A11 in ARPE-19 in response to NaIO₃ were examined. n = 3, means ± SD; two-tailed t-test; **P < 0.01. (D) Protein expression levels and (E) semi-quantitation of HO-1 upstream, including Nrf2, YY1, AP-1, HIF-1α, and STAT3 were analyzed by western blot. n = 3, means ± SD; two-tailed t-test; **P < 0.01, N.S., not significant. (F) IF staining and (G) corresponding quantification of the relative expression of Nrf2, SLC7A11 and HO-1 were performed in mRPE after NaIO₃ treatment for 2 weeks. Scale bars, 20 μm, n = 3, means ± SD; two-tailed t-test; *P < 0.05, **P < 0.01. By western blot and further semi-quantitation analysis, relationship of Nrf2, SLC7A11 and HO-1 in RPE ferroptosis was identified by knockdown of (H, I) Nrf2 (siNrf2), (J, K) SLC7A11 (siSLC7A11), and (L, M) HO-1 (siHO-1) in ARPE-19 cells followed by NaIO₃ treatment. n = 3, means ± SD; one-way ANOVA with Bonferroni correction; **P < 0.01, N.S., not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

HO-1 upregulation elicits cellular ferrous accumulation to boost RPE ferroptosis. ARPE-19 cells were pretreated with HO-1 inhibitor zinc protoporphyrin-9 (ZnPP, 10 μM) for 1 h followed by transfection of the hHO-1-expressed plasmid. After 24 h, (A) cell survival/death by live/dead staining (Calcein AM: live cells, PI: dead cells), (B) corresponding quantification of cell viability by Image J, (C) ferrous ion levels by FerroOrange staining and (D) corresponding quantification were determined. Scale bars, 100 μm n = 3, mean ± SD, one-way ANOVA with Bonferroni correction; **p < 0.01. (E) The mRNA expression levels by qPCR, (F) protein expression levels by western blot analysis and (G) semi-quantification of iron homeostasis-related makers, FTH1, SLC40A1 and TFR in NaIO₃-treated ARPE-19 cells were performed. n = 3, mean ± SD, two-tailed t-test, **p < 0.01, N.S., not significant. (H) The mRNA expression levels of FTH1, SLC40A1 and TFR by qPCR, (I) protein expression levels by IF analysis and (J) relative quantification of SLC40A1 and TFR were evaluated in mRPE from NaIO₃-treated retinae. Scale bars, 20 μm n = 3, mean ± SD, two-tailed t-test, **p < 0.01, N.S., not significant. (K) The mRNA expression levels by qPCR, (L) protein expression levels by western blot analysis and (M) semi-quantification of FTH1, SLC40A1 and TFR in ARPE-19 cells transfected with the hHO-1-expressed plasmid were performed. n = 3, mean ± SD, two-tailed t-test, **p < 0.01, N.S., not significant. (N) Western blot analysis and (O) semi-quantification of HO-1 in protein levels were evaluated in ARPE-19 after FAC incubation (250 umol/L) for 24 h. (P) Scheme of detailed mechanism in NaIO₃-induced RPE ferroptosis.

Fig. 4

Inhibiting HO-1 overexpression protects against RPE ferroptosis in vitro. HO-1 knockdown ARPE-19 cells were treated with NaIO₃ for 24 h, followed by evaluation of (A) protein expression of ZO-1 by ICC staining and (B) relative quantification, (C) cell survival/death by live/dead staining (Calcein AM: live cells, PI: dead cells), (D) corresponding cell viability quantification, and (E) further quantification of cell viability by CCK-8 analysis. Scale bars, 50 μm in (A), 100 μm in (C). n = 3, mean ± SD, one-way ANOVA with Bonferroni correction; **p < 0.01. (F) Intracellular LOS levels by C11-BODIPY staining, (G) ferrous staining by FerroOrange probe and (H) corresponding quantification were carried out in siHO-1-infected ARPE-19 cells with NaIO₃ treatment for 24 h. Scale bars, 100 μm n = 3, means ± SD; one-way ANOVA with Bonferroni correction; **P < 0.01.

Fig. 5

Inhibiting HO-1 overexpression protects against RPE ferroptosis and photoreceptor degeneration in vivo. Retinae were intraperitoneally pre-treated with ZnPP (50 mg/kg) for 15 min followed by treatment with NaIO₃, 2 weeks later, (A) protein expression levels and (B) relative quantification of HO-1 and RPE visual recycle biomarkers RPE65 and RLBP1 were determined in mRPE. (C) H&E staining for RPE pigmentation and morphological changes in mRPE pretreated with ZnPP followed by NaIO₃ treatment for 2 weeks. White arrows: mRPE cells. Scale bars, 200 μm. (D) RPE flat mounts for ZO-1 staining and and (E) relative quantification were evaluated. Scale bars, 25 μm n = 3, means ± SD; one-way ANOVA with Bonferroni correction; **P < 0.01. Protein expression levels by (F) western blot analysis and (G) relative quantification, as well as further by (H) IF staining and (I) relative quantification of Arrestin and Rhodopsin were analyzed. OS: outer segment, ONL: outer nuclear layer, INL: inner nuclear layer. Scale bars, 50 μm n = 3, means ± SD; one-way ANOVA with Bonferroni correction; **P < 0.01. (J–M) Full field ERG was used to detect visual function under scotopic conditions traced from mice in (J) PBS, (K) NaIO₃, (L) NaIO₃ + ZnPP groups. (M) Scotopic-ERG a- and b-wave amplitude values by full-field ERG were measured. n = 7, mean ± SD, one-way ANOVA with Bonferroni correction, **p < 0.01.

Fig. 6

Inhibiting HO-1 overexpression suppresses RPE ferroptosis by decreasing cellular ferrous accumulation. The mRPE was intraperitoneally pre-treated with ZnPP (50 mg/kg) for 15 min followed by treatment with NaIO₃, 2 weeks later, effects of ZnPP on (A) mRNA expression levels by qPCR analysis, (B) protein expression levels by western-blot analysis and (C) relative quantification of RPE ferroptosis-related markers, HO-1, SLC40A1 and TFR were evaluated in NaIO₃-treated ARPE-19 cells. n = 3, mean ± SD, one-way ANOVA with Bonferroni correction, *p < 0.05, **p < 0.01. Effects of ZnPP on (D) mRNA expression levels by qPCR analysis, (E) protein expression levels by IF staining and (F) relative quantification of HO-1, SLC40A1 and TFR were evaluated in NaIO₃-treated mRPE. Scale bars, 20 μm n = 3, mean ± SD, one-way ANOVA with Bonferroni correction, *p < 0.05, **p < 0.01. (G) ROS levels by DHE staining and (H) quantification were evaluated in NaIO₃-treated mRPE with ZnPP treatment for 2 weeks. White arrows: mRPE monolayer cells. Scale bars, 20 μm n = 3, means ± SD; one-way ANOVA with Bonferroni correction; **P < 0.01. By western blot, (I) protein blots and (J) semi-quantification of the protein expression levels of Nrf2, SLC7A11 and FTH1 were assessed in NaIO₃-treated ARPE-19 cells with or without ZnPP treatment. n = 3, means ± SD; one-way ANOVA with Bonferroni correction; **P < 0.01, N.S., not significant. (K) Scheme of protective mechanism of inhibiting HO-1 overexpression by ZnPP treatment against RPE ferroptosis.