Abstract
Ferroptosis is a type of regulated necrosis that is associated with iron-dependent accumulation of lipid hydroperoxides. Given that iron deposition and lipid peroxidation initiate ferroptosis in atherosclerosis and contribute to further plaque development, we hypothesized that inhibition of ferroptosis could be of value in the treatment of atherosclerosis. Glutathione peroxidase 4 (GPX4) is the only enzyme known capable of reducing lipid hydroperoxides. Previous studies have demonstrated that inactivation of GPX4 results in ferroptosis, while overexpression of GPX4 confers resistance to ferroptosis. In the present study, we examined the impact of GPX4 overexpression on the development of atherosclerotic plaques. GPX4-overexpressing mice (GPX4Tg/+) were crossbred with ApoE−/− mice and fed a western-type diet for 16 weeks. Atherosclerotic plaques of GPX4Tg/+ ApoE−/− mice showed increased GPX4 expression and a reduced amount of lipid hydroperoxides. However, plaque size and composition were not different as compared to control animals. Similarly, GPX4-overexpressing vascular smooth muscle cells and bone marrow-derived macrophages were not protected against lipid peroxidation and cell death triggered by the ferroptosis inducers erastin and 1S,3R-RSL3. We concluded that GPX4 overexpression reduces lipid peroxidation in plaques of ApoE−/− mice, yet GPX4 overexpression is not sufficiently powerful to change plaque size or composition.
Introduction
Ferroptosis has recently been identified in atherosclerotic plaques as an iron-dependent and nonapoptotic form of cell death (1, 2, 3, 4). This type of cell death is characterized by the accumulation of lipid peroxides and promotes atherogenesis. Glutathione peroxidase 4 (GPX4) limits the formation of lipid hydroperoxides by reducing hydroperoxide groups on phospholipids, lipoproteins and cholesterol esters to alcohols using glutathione (GSH) as a reductant. It is the only known enzyme that is able to reduce lipid hydroperoxides within biological membranes (5). Deletion of GPX4 in cells results in the rapid accumulation of lipid hydroperoxides followed by ferroptosis, which can be suppressed by iron chelators and the lipid antioxidant α-tocopherol (vitamin E) (6, 7, 8). Moreover, systemic deletion of GPX4 in mice causes embryonic lethality (6, 9), while overexpression of GPX4 blocks cell death mediated by lipid reactive oxygen species (10). These data underline the importance of GPX4 in preventing lipid peroxidation and associated cell death. Because lipid peroxidation and iron deposition contribute significantly to the pathogenesis of atherosclerosis (11, 12), targeting of cell death mediated by excessive lipid peroxidation could be an interesting therapeutic strategy to stabilize atherosclerotic plaques. Indeed, it has previously been reported that GPX4 overexpression in ApoE−/− mice reduces atherosclerosis by limiting the level of oxidized lipids in the aorta (13). However, a link between GPX4 overexpression and ferroptosis inhibition has not yet been studied in atherosclerosis. Here, we aim to investigate whether GPX4 overexpression results in atherosclerotic plaque stabilization by inhibition of ferroptosis.
Materials and methods
Mice
Transgenic mice overexpressing GPX4 (GPX4Tg/+) (10) were crossbred with apolipoprotein E-deficient (ApoE−/−) mice (Jackson Laboratory, stock number 002052) to obtain GPX4Tg/+ ApoE−/− mice as well as GPX4+/+ ApoE−/− controls. Because vitamin E suppresses ferroptosis by inhibiting excess lipid peroxidation (14), mice were maintained on standard chow containing a 2.5-fold lower vitamin E concentration (50 mg vitamin E/kg diet; standard diet modified, #132088, Altromin) as compared to the standard chow. At the age of 6 weeks, female GPX4Tg/+ ApoE−/− and GPX4+/+ ApoE−/− mice (n = 12 per group) were fed a western-type diet (WD; C1000 diet supplemented with 20% milkfat and 0.15% cholesterol and 50 mg vitamin E per kg diet, #100206, Altromin). Only female mice were used because atherosclerotic plaque formation is more severe in females, both in ApoE−/− and LDLR−/− models (15, 16). While both sexes display a similar degree of atherosclerosis after 8 weeks on WD, females develop significantly more atherosclerosis after >14 weeks WD (16). The animals were housed in a temperature-controlled room with a 12 h light:12 h darkness cycle and had free access to water and food. After 16 weeks WD, blood samples were taken via the retro-orbital plexus of anesthetized mice (sodium pentobarbital 75 mg/kg, i.p.). Subsequently, mice were sacrificed with sodium pentobarbital (250 mg/kg, i.p.). Analysis of total plasma cholesterol was performed via a commercially available kit (Randox laboratories, Crumlin, UK). All experiments were conducted according to the ARRIVE guidelines and approved by the Ethical Committee of the University of Antwerp.
Echocardiography
Transthoracic echocardiograms were performed on anesthetized mice (isoflurane, 4% for induction and 2% for maintenance) at the start (0 weeks WD) and the end (16 weeks WD) of the experiment using a VEVO2100 (FujiFilm VisualSonics), equipped with a 25 MHz transducer. Body temperature was maintained at 36–38°C and heart rate at 500 ± 50 beats/min. Left ventricular (LV) wall thickness and cavity diameters at the end of diastole and systole were measured. Fractional shortening (FS), ejection fraction (EF) and stroke volume were calculated from these parameters.
Histological analysis
After sacrificing GPX4Tg/+ ApoE−/− and GPX4+/+ ApoE−/− mice, the brachiocephalic artery and proximal ascending aorta were collected. Tissues were fixed in 4% formaldehyde (pH 7.4) for 24 h, dehydrated overnight in 60% isopropanol and subsequently embedded in paraffin. Serial cross sections (5 µm) of the brachiocephalic artery and proximal ascending aorta were prepared for histological analysis. Atherosclerotic plaque size and necrotic core area (defined as acellular areas with a threshold of 3000 µm²) were analyzed on hematoxylin–eosin (H&E) stained sections. Collagen content was analyzed with Sirius red. Macrophages were detected by immunohistochemistry using an anti-Mac3 (BD Pharmingen, 550292, San Diego, CA, USA) staining. The fibrous cap thickness was assessed by taking the median value of 10 random measurements on α-smooth muscle actin (α-SMA, Sigma, A2547) stained sections. The expression of GPX4 was analyzed using anti-GPX4 (Abcam, ab125066). Images were acquired with an Olympus SC50 microscope and quantified with ImageJ software (National Institutes of Health). For tissue analysis of malondialdehyde (MDA) and GSH, the aortic arch was collected and immediately snap frozen. Tissues were homogenized in phosphate buffer and protein content was determined via the bicinchoninic acid assay (BCA) method. MDA content was determined using a commercially available ELISA kit (Mybiosource, MBS741034, San Diego, CA, USA). For GSH analysis, tissue samples were filtered over a 3 kDa filter by centrifugation (14,000 g , 4°C, 20 min). The filtrate was 1:2 diluted with mobile phase A, acidified with 5% H3PO4 and GSH levels were analyzed by an Agilent 1260 Infinity HPLC with electrochemical detection (ESA-5600 Coularray 8-channel detector) as described by Magielse et al. (17). Cell potentials were set at 720, 910 and 950 mV.
Cell culture
Bone marrow-derived macrophages (BMDMs) were harvested by flushing bone marrow of the femur with a 25G needle and heparinized (10 IU/mL) RPMI 1640 medium (Gibco, Life Technologies). After washing and filtration, cells were cultured in RPMI 1640 medium supplemented with GlutaMAX (Gibco, Life Technologies) and 15% L929-cell conditioned medium containing monocyte colony stimulating factor for 7 days at 37°C in 95% air/5% CO2 until 80–90% confluency was reached. Vascular smooth muscle cells (VSMCs) were isolated as previously described (18, 19). Briefly, aortas were incubated in an enzyme solution containing 1 mg/mL collagenase type II (Worthington), 1 mg/mL soybean trypsin inhibitor (Worthington) and 0.744 U/mL elastase (Worthington) for 15 min at 37°C to remove the adventitia. Subsequently, aortas were placed in a fresh enzyme solution for 75 min at 37°C. Isolated cells were collected, washed and resuspended in DMEM/F12 medium (Gibco, Life Technologies) supplemented with 20% heat-inactivated fetal bovine serum (Gibco, Life Technologies). Purity of VSMC cultures was evaluated by staining cells for selective VSMC markers such as Myh11 and Cnn1 (20). Cells were used from passage 4 till 10 and cultured in DMEM/F12 medium supplemented with 10% heat-inactivated fetal bovine serum. To induce ferroptosis, BMDMs and VSMCs were treated with 0.5–5 µM 1S,3R-RSL3 (Tocris, 6618) or 0–30 µM erastin (Tocris, 5449, Bristol, UK) for the indicated times. Cell viability was evaluated via a neutral red assay as previously described (21). Briefly, cells were incubated with 0.1% neutral red solution at 37°C in 95% air/5% CO2. Subsequently, cells were washed with PBS and neutral red was extracted by addition of 0.05 M NaH2PO4 in 50% ethanol. After 3 min, optical density was read at 540 nm using a microtiter plate reader. To study lipid peroxidation, cells were incubated with 5 µM C11-BODIPY 581/591 (ThermoFisher, D3861) for 1 h at 37°C. After incubation, cells were harvested with 0.05% trypsin solution (Invitrogen), resuspended in FACS buffer (PBS with 0.1% BSA and 0.05% NaN3) and immediately analyzed with a BD Accuri C6 flow cytometer, equipped with a 488 nm laser for excitation. Data were collected from the FL-1 (oxidized lipids) and FL-2 channel (reduced lipids). A minimum of 10,000 cells were analyzed per condition.
Western blot
Thoracic aorta, heart, spleen, kidney and liver were isolated and immediately snap frozen. Tissues were homogenized in RIPA buffer supplemented with protease and phosphate inhibitors. Subsequently, protein concentration was determined using the BCA method. Tissue homogenates were diluted to a concentration of 1 mg/mL with Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol (Sigma-Aldrich) and heat-denatured (5 min in boiling water). Thereafter, samples were loaded on Bolt 4–12% Bis-Tris gels (Invitrogen) and after electrophoresis transferred to Immobilon-FL polyvinylidene difluoride membranes (Millipore) according to standard procedures. Next, membranes were blocked for 1 h in Odyssey Li-COR blocking buffer. After blocking, membranes were probed overnight at 4°C with mouse anti-β-actin (Abcam, Ab8226) and rabbit anti-GPX4 (Abcam, ab125066) diluted in Odyssey Li-COR blocking buffer followed by a 1-h incubation with IRDye-labeled secondary antibodies (Li-COR Biosciences (Lincoln, NE, USA): IgG926-32211 (goat anti-rabbit); IgG926-68070 (goat anti-mouse)) at room temperature. Signals were visualized with an Odyssey SA infrared imaging system (Li-COR Biosciences).
Statistical analysis
Statistical analyses were performed using SPSS software (version 29, IBM SPSS). All data are expressed as mean ± s.e.m. Statistical tests are specified in the figure legends. Differences were considered significant at P < 0.05.
Results
GPX4 is overexpressed in different tissues of GPX4Tg/+ ApoE−/− mice
Transgenic mice overexpressing human GPX4 (GPX4Tg/+) were crossbred with apolipoprotein E-deficient (ApoE−/−) mice to study the effect of GPX4 overexpression on the development of atherosclerosis. To induce atherosclerotic plaque formation, GPX4Tg/+ ApoE−/− and GPX4+/+ ApoE−/− mice were fed a WD for 16 weeks. Because GPX4 and vitamin E can cooperatively protect cells from lipid peroxidation and ferroptosis, WD contained reduced levels of vitamin E (50 mg/kg diet). No significant difference in body weight was observed between both genotypes at the end of the experiment (GPX4+/+ ApoE−/−: 27 ± 2 g, GPX4Tg/+ ApoE−/−: 23 ± 2 g; independent samples t-test). Next, we validated the overexpression of GPX4 in GPX4Tg/+ ApoE−/− mice via western blot analysis on tissue lysates of different organs including aorta, heart, spleen, kidney and liver. GPX4 expression was approximately two-fold higher in the heart, spleen, liver and kidney of GPX4Tg/+ ApoE−/− mice as compared to control mice (Fig. 1A). However, GPX4 was only moderately overexpressed (1.3–1.8-fold) in the thoracic aorta (Fig. 1A) and in isolated VSMCs or BMDMs of GPX4Tg/+ ApoE−/− mice (Fig. 1B). To evaluate GPX4 expression within atherosclerotic plaques, an immunostaining for GPX4 was performed. GPX4 expression was significantly increased in plaques of GPX4Tg/+ ApoE−/− mice as compared to plaques of GPX4+/+ ApoE−/− mice (Fig. 1C).
GPX4 is overexpressed in different organs and cell types isolated from GPX4Tg/+ ApoE−/− mice. (A) GPX4+/+ ApoE−/− (+/+) and GPX4Tg/+ ApoE−/− (Tg/+) mice were fed a western-type diet for 16 weeks. Thoracic aorta, heart, spleen, kidney and liver were isolated and snap frozen. Western blot analysis for GPX4 expression (β-actin was used as loading control). (B) Vascular smooth muscle cells (VSMCs) and bone-marrow-derived macrophages (BMDMs) were isolated from GPX4+/+ ApoE−/− (+/+) and GPX4Tg/+ ApoE−/− (Tg/+) mice. Western blot analysis for GPX4 expression (β-actin was used as loading control). (C) Sections of atherosclerotic plaques in the brachiocephalic artery of GPX4+/+ ApoE−/− (+/+) and GPX4Tg/+ ApoE−/− (Tg/+) mice were stained with anti-GPX4. Representative images are shown. Scale bar = 100 µm. (*P < 0.05, **P < 0.01, ***P < 0.001 vs. +/+; n = 6–8 for both groups; independent samples t-test).
Citation: Vascular Biology 6, 1; 10.1530/VB-23-0020
GPX4 overexpression does not affect cardiac function in ApoE−/− mice
Left ventricle function was monitored at the start of the WD and at the end of the experiment (16 weeks of WD). GPX4 expression did not influence heart weight (Table 1). Furthermore, no significant differences were observed in either EF and FS between GPX4+/+ ApoE−/− mice and GPX4Tg/+ ApoE−/− mice (Table 1). The left ventricular internal diameter, LV posterior wall thickness and interventricular septum thickness during diastole and systole were also similar between GPX4Tg/+ ApoE−/− and GPX4+/+ ApoE−/− mice at the start of the experiment and after 16 weeks WD (Table 1).
Cardiac parameters of GPX4+/+ ApoE−/− and GPX4Tg/+ ApoE−/− mice at the start of the experiment (0 weeks of western-type diet (WD)) and after 16 weeks WD.
| 0 weeks WD | 16 weeks WD | |||
|---|---|---|---|---|
| GPX4+/+ApoE−/− | GPX4Tg/+ApoE−/− | GPX4+/+ ApoE−/− | GPX4Tg/+ApoE−/− | |
| n | 7 | 9 | 11 | 12 |
| HW (mg)a | ND | ND | 129 ± 8 | 133 ± 5 |
| EF (%)b | 77 ± 5 | 75 ± 4 | 75 ± 3 | 70 ± 4 |
| FS (%)b | 46 ± 5 | 43 ± 3 | 44 ± 2 | 39 ± 3 |
| IVSd (mm)b | 0.97 ± 0.07 | 0.86 ± 0.05 | 1.3 ± 0.2 | 1.0 ± 0.1 |
| IVSs (mm)b | 1.4 ± 0.1 | 1.3 ± 0.1 | 1.7 ± 0.1 | 1.9 ± 0.3 |
| LVIDd (mm)b | 2.9 ± 0.3 | 2.7 ± 0.1 | 2.9 ± 0.2 | 3.0 ± 0.2 |
| LVIDs (mm)b | 1.6 ± 0.3 | 1.6 ± 0.1 | 1.6 ± 0.2 | 1.9 ± 0.2 |
| LVPWd (mm)b | 1.2 ± 0.2 | 1.2 ± 0.2 | 1.5 ± 0.2 | 1.2 ± 0.2 |
| LVPWs (mm)b | 1.6 ± 0.2 | 1.6 ± 0.2 | 1.9 ± 0.2 | 1.5 ± 0.2 |
aIndependent samples t-test showed no significance (P > 0.05); bTwo-way ANOVA followed by Bonferroni’s post hoc test showed no significance (P > 0.05).
EF, ejection fraction; FS, fractional shortening; HW, heart weight; IVSd, interventricular septum during diastole; IVSs, IVS during systole; LVIDd, left ventricular internal diameter during diastole; LVIDs, LVID during systole; LVPWd, left ventricular posterior wall thickness during diastole; LVPWs, LVPW during systole; ND, not determined; WD, western diet.
Atherosclerotic plaque size and composition are not altered by GPX4 overexpression in ApoE−/− mice
Analysis of total plasma cholesterol revealed no difference between GPX4-overexpressing mice and control mice after 16 weeks WD (GPX4+/+ ApoE−/−: 638 ± 90 mg/dL, GPX4Tg/+ ApoE−/−: 653 ± 57 mg/dL; independent samples t-test). To evaluate atherosclerotic plaque size and composition, the proximal ascending aorta and brachiocephalic artery were collected at 16 weeks WD. Atherosclerotic plaque size and percentage of necrosis were similar between GPX4Tg/+ ApoE−/− and GPX4+/+ ApoE−/− mice (Table 2 and Supplementary Fig. 1, see section on supplementary materials given at the end of this article). Furthermore, no significant differences were observed in the collagen content and number of macrophages and VSMCs (Table 2). The thickness of the fibrous cap and percentage of apoptosis (cleaved caspase-3 positive cells) were not different in atherosclerotic plaques of the proximal ascending aorta and brachiocephalic artery of GPX4Tg/+ ApoE−/− and GPX4+/+ ApoE−/− mice (Table 2).
Plaque characteristics of the proximal ascending aorta and brachiocephalic artery from GPX4+/+ ApoE−/− and GPX4Tg/+ ApoE−/− mice after 16 weeks of western-type diet.
| Proximal ascending aorta | Brachiocephalic artery | |||
|---|---|---|---|---|
| GPX4+/+ApoE−/− | GPX4Tg/+ApoE−/− | GPX4+/+ApoE−/− | GPX4Tg/+ApoE−/− | |
| n | 9–10 | 11–12 | 7–10 | 6–9 |
| Plaque area (× 103 µm2) | 130 ± 28 | 154 ± 21 | 117 ± 27 | 154 ± 30 |
| Necrotic core (%) | 6 ± 3 | 6 ± 2 | 8 ± 3 | 14 ± 2 |
| Total collagen (%) | 23 ± 3 | 30 ± 4 | 30 ± 5 | 36 ± 3 |
| Macrophages (%) | 5 ± 3 | 5 ± 2 | 4 ± 2 | 10 ± 4 |
| VSMCs (%) | 6 ± 2 | 7 ± 2 | 0.6 ± 0.2 | 0.5 ± 0.2 |
| Fibrous cap (µm) | 4.4 ± 1.0 | 3.1 ± 0.5 | 2.1 ± 0.6 | 2.5 ± 0.6 |
| Cleaved caspase-3 (%) | 0.6 ± 0.3 | 1.0 ± 0.3 | 4 ± 3 | 2 ± 1 |
Independent samples t-test showed no significance (P > 0.05).
VSMCs, vascular smooth muscle cells.
To evaluate whether GPX4 overexpression affected lipid peroxidation in atherosclerotic plaques, MDA levels were analyzed. MDA is one of the end products of polyunsaturated fatty acid peroxidation in cells. Atherosclerotic plaques of GPX4Tg/+ ApoE−/− mice contained less MDA as compared to atherosclerotic plaques of GPX4+/+ ApoE−/− mice, suggesting that lipid peroxidation was inhibited in plaques of GPX4-overexpressing mice (Fig. 2A). Because GPX4 uses GSH as a cofactor to reduce lipid peroxides to lipid alcohols, the level of GSH was also examined in atherosclerotic plaques. However, no significant difference in GSH level was observed between GPX4 overexpressing and control mice (Fig. 2B).
Lipid peroxidation is inhibited in atherosclerotic plaques of GPX4Tg/+ ApoE−/− mice. GPX4+/+ ApoE−/− (+/+) and GPX4Tg/+ ApoE−/− (Tg/+) mice were fed a western-type diet for 16 weeks. The aortic arch was isolated and snap frozen. (A) MDA analysis on the whole plaque lysate. (B) GSH analysis on the whole plaque lysate. (*P < 0.05, NS, not significant vs. GPX4+/+; n = 9–10 aortic arches per group; independent samples t-test).
Citation: Vascular Biology 6, 1; 10.1530/VB-23-0020
GPX4 overexpression does not protect VSMCs and BMDMs against cell death
To induce ferroptotic cell death, VSMCs and BMDMs were isolated from GPX4+/+ ApoE−/− and GPX4Tg/+ ApoE−/− mice and treated with two different ferroptosis inducers, namely erastin and 1S,3R-RSL3. Both compounds induce ferroptosis via inhibition of GPX4. Erastin inhibits GPX4 indirectly by decreasing the synthesis of the cofactor GSH through inhibition of the cystine–glutamate antiporter, whereas 1S,3R-RSL3 inhibits GPX4 directly. 1S,3R-RSL3 as well as erastin treatment resulted in a concentration-dependent decrease of cell viability in both GPX4+/+ ApoE−/− and GPX4Tg/+ ApoE−/− VSMCs (Table 3). BMDMs were more sensitive to cell death induced by 1S,3R-RSL3 and erastin compared to VSMCs, especially after treatment with 1S,3R-RSL3. However, GPX4Tg/+ ApoE−/− VSMCs were not resistant to cell death. Consistent with VSMCs, no difference in cell viability was observed between GPX4+/+ ApoE−/− and GPX4Tg/+ ApoE−/− BMDMs after treatment with the ferroptosis inducers (Table 3).
Percentage viability of vascular smooth muscle cells (VSMCs) and bone-marrow-derived macrophages (BMDMs) isolated from GPX4+/+ ApoE−/− (+/+) and GPX4Tg/+ ApoE−/− (Tg/+) mice and exposed to 1S,3R-RSL3 (6 h) or erastin (8 h).
| VSMCs | BMDMs | |||
|---|---|---|---|---|
| +/+ | Tg/+ | +/+ | Tg/+ | |
| 1S,3R-RSL3 (µM) | ||||
| 0.5 | 54 ± 10 | 45 ± 16 | 7 ± 2 | 9 ± 2 |
| 1 | 47 ± 5 | 40 ± 13 | 3 ± 1 | 3 ± 1 |
| 5 | 38 ± 4 | 30 ± 4 | 3 ± 1 | 2 ± 1 |
| Erastin (µM) | ||||
| 3 | 105 ± 7 | 99 ± 2 | 73 ± 5 | 68 ± 16 |
| 10 | 89 ± 11 | 88 ± 7 | 66 ± 1 | 60 ± 12 |
| 30 | 83 ± 6 | 77 ± 5 | 56 ± 8 | 49 ± 14 |
Two-way ANOVA followed by Bonferroni’s post hoc test showed no significance (P > 0.05) between +/+ and Tg/+ groups (n = 3 experiments in duplicate).
GPX4 overexpression does not protect VSMCs and BMDMs against lipid peroxidation
Because ferroptosis is caused by an excess of lipid peroxidation, we evaluated lipid peroxidation in GPX4+/+ ApoE−/− and GPX4Tg/+ ApoE−/− VSMCs and BMDMs treated with the ferroptosis inducers erastin and 1S,3R-RSL3. Lipid peroxidation, expressed by the ratio of oxidized lipids to reduced lipids, was significantly increased in both GPX4+/+ ApoE−/− VSMCs and BMDMs after treatment with 1S,3R-RSL3 (Table 4). However, VSMCs and BMDMs that overexpress GPX4 did not reveal improved protection against lipid peroxidation after 1S,3R-RSL3 treatment (Table 4). In contrast to 1S,3R-RSL3, erastin treatment did not significantly change lipid peroxidation in VSMCs and BMDMs. Overexpression of GPX4 in both VSMCs and BMDMs did not reduce lipid peroxidation after erastin treatment (Table 4).
Lipid peroxidation in VSMCs and BMDMs isolated from GPX4+/+ ApoE−/− (+/+) and GPX4Tg/+ ApoE−/− (Tg/+) mice and exposed to 1S,3R-RSL3 or erastin. Lipid peroxidation was monitored by flow cytometry using C11-BODIPY staining. Data were collected from the FL-1 channel (oxidized lipids) and FL-2 channel (reduced lipids). Data are expressed as mean fluorescence FL1/mean fluorescence FL2.
| VSMCs | BMDMs | |||
|---|---|---|---|---|
| +/+ | Tg/+ | +/+ | Tg/+ | |
| 1S,3R-RSL3 (1 µM) | ||||
| Untreated | 0.16 ± 0.06 | 0.23 ± 0.03 | 0.79 ± 0.11 | 0.58 ± 0.10 |
| 1 h | 0.66 ± 0.05 | 0.69 ± 0.05 | 3.18 ± 0.76 | 3.87 ± 1.62 |
| 2 h | 0.85 ± 0.07 | 0.94 ± 0.05 | 3.63 ± 0.78 | 4.62 ± 2.05 |
| Erastin (10 µM) | ||||
| Untreated | 0.18 ± 0.07 | 0.25 ± 0.04 | 0.65 ± 0.07 | 0.52 ± 0.03 |
| 3 h | 0.22 ± 0.09 | 0.26 ± 0.04 | 0.84 ± 0.12 | 0.62 ± 0.06 |
| 6 h | 0.40 ± 0.18 | 0.59 ± 0.19 | 0.84 ± 0.12 | 0.66 ± 0.05 |
Two-way ANOVA followed by Bonferroni’s post hoc test showed no significance (P > 0.05) between +/+ and Tg/+ groups (VSMCs n = 3, BMDMs n = 5).
BMDM, bone-marrow-derived macrophages; VSMC, vascular smooth muscle cells.
Discussion
Ferroptosis is a type of regulated necrosis characterized by iron-dependent accumulation of lipid peroxides and has already been linked to different diseases including atherosclerosis (1, 2, 3, 4). GPX4 plays a protective role against ferroptosis by reducing lipid peroxidation, a common feature of advanced atherosclerotic plaques (22). However, expression of GPX4 is significantly reduced in atherosclerotic ApoE−/− mice (1, 2). Pharmacological inhibition of ferroptosis with ferrostatin-1 (Fer-1) or structurally improved Fer-1 analogs such as UAMC-3203 reduces plaque burden in ApoE−/− mice (1, 4). Fer-1 is a potent radical trapping agent (RTA), especially in lipid bilayers, and prevents lipid peroxidation (23, 24). Moreover, Fer-1 attenuates the decline in GPX4 levels (1, 2), and this effect may also contribute, at least partially, to the observed atheroprotective effects. Next to inhibition of ferroptosis, some studies indicate that GPX4 protects against apoptotic cell death (10). In addition, GPX4 inhibits the uptake of low density lipoproteins and thus protects against foam cell formation, as macrophages of GPX4 knockout mice upregulate scavenger receptor type A and LOX-1 expression and downregulate ABCA1 and ABCG1 expression (25). Finally, it is worth mentioning that elevated GPX4 activity in several experimental models of inflammatory disease has been associated with suppression of tissue inflammation, in part due to suppression of NF-κB activation and local or systemic production of cytokines and chemokines (26, 27, 28). Therefore, we hypothesized in the present study that overexpression of GPX4 is a promising strategy to halt the progression and destabilization of atherosclerotic plaques. Indeed, Guo et al. (13) already reported that overexpression of GPX4 in ApoE−/− mice slows down plaque progression by lowering lipid peroxidation in blood plasma. Likewise, we could demonstrate in the present study that MDA, a marker for lipid peroxidation, is decreased in atherosclerotic plaques, yet the decrease in MDA was not associated with a reduction in plaque area. One factor that might explain this discrepancy is the expression level of GPX4 in aortic tissue. In the present study, protein levels of GPX4 were only increased by 60% in the thoracic aorta of GPX4Tg/+ ApoE−/− mice. In other tissues such as heart, kidney, liver and spleen, we detected approximately two-fold higher GPX4 protein levels, which is in line with previous studies overexpressing GPX4 in mice (10, 29). Guo et al. (13) demonstrated a 2.5-fold higher GPX4 expression in the aorta of GPX4Tg/+ ApoE−/− mice versus controls. If we evaluate the expression of GPX4 exclusively in atherosclerotic plaques using immunohistochemistry, plaques of GPX4Tg/+ ApoE−/− mice showed a 3-fold increase of GPX4 expression as compared to plaques of GPX4+/+ ApoE−/− mice, which is in the range of the data reported by Guo et al. (13). According to the human protein atlas, GPX4 mRNA is widely present in all tissues. It is most abundantly expressed in testis, but undetectable at the protein level in several cell types including smooth muscle cells. Also noteworthy is that GPX4 protein expression in bone marrow and lymphoid tissues is rather low. These findings correlate with the poor GPX4 overexpression in aorta versus other tissues. However, GPX4 activity can be modulated by lipids, cytokines and antioxidants in the presence of adequate selenium (30), which might explain the higher fold change in GPX4 positive cells in plaques of GPX4tg/+ ApoE−/− mice versus plaques of GPX4+/+ ApoE−/− control mice.
In addition to GPX4 expression levels, the vitamin E concentration in the western diet can be a reason for the conflicting results. Vitamin E is a lipophilic antioxidant and suppresses ferroptosis cooperatively with GPX4 (14). To study the effect of GPX4 overexpression as a ferroptosis inhibitor, we decreased the vitamin E concentration from 130 mg per kg of diet (vitamin E concentration in a standard western-type diet) to 50 mg per kg of diet in the present study, yet this concentration still meets the daily nutrient requirements of mice (approximately 22 mg/kg of diet) (31). Guo et al. (13) did not mention the vitamin E levels in the diet. Possibly, they fed their mice a diet that contained much higher levels of vitamin E (standard diets contain 130–180 mg vitamin E/kg of diet), thereby inhibiting phospholipid hydroperoxide formation and ferroptosis induction much more efficiently in plaques of GPX4Tg/+ ApoE−/− mice. If true, GPX4 should be considered a weak protector against ferroptosis induction, and requires additional actions such as the supplementation of sufficiently high levels of antioxidants including vitamin E or ferroptosis inhibitors to obtain significant effects.
A third reason for the conflicting results may be the gender of the animals used in the study. The present study included female mice, whereas Guo et al. (13) used male mice to investigate the effect of GPX4 overexpression on atherosclerosis. It was recently reported that GPX4 expression in rats does not display differences between sexes (32), making this third option unlikely.
Together with the plaque area, the present study also showed no effect of GPX4 overexpression on plaque composition in ApoE−/− mice. Furthermore, no difference in cardiac function was observed. Overall, although GPX4 was overexpressed in different organs, we could not observe any changes in the development of atherosclerotic plaques. It is important to note that the present study was performed in ApoE−/− mice, which do not develop unstable atherosclerotic plaques characterized by the presence of neovessels and are likely to contain only a limited amount of ferrous iron (33). However, it is well established that intraplaque neovascularization and subsequent intraplaque hemorrhages are critical events in the pathology of atherosclerosis (34). During intraplaque hemorrhages, hemolysis occurs along with the deposition of free cholesterol into the plaque. Furthermore, free iron is released from hemoglobin, which further contributes to oxidative reactions including lipid peroxidation (35). Therefore, studying the effect of GPX4 overexpression in a mouse model that develops atherosclerotic plaques with neovascularization and intraplaque hemorrhages such as ApoE−/− Fbn1C1039G/+ mice (36, 37) would be more conclusive. In addition, because GPX4 main function involves reduction of lipid peroxidation that plays an essential role in the early stages of atherosclerosis (38), it would be worthwhile to examine also early time points of plaque development where effects of GPX4 expression might be more pronounced than in the more advanced stages.
Despite overexpression of GPX4, our in vitro results showed that both VSMCs and BMDMs isolated from GPX4Tg/+ ApoE−/− mice were not protected against cell death induced by 1S,3R-RSL3 and erastin. Moreover, lipid peroxidation was not reduced in GPX4-overexpressing VSMCs and BMDMs after treatment with ferroptosis inducers. One possible explanation for these unexpected results might be that GPX4 expression is only moderately increased in VSMCs and BMDMs of GPX4Tg/+ ApoE−/− mice. The relatively limited overexpression of GPX4 might be too low to reduce the lipid peroxides that are produced in the cells after 1S,3R-RSL3 or erastin treatment.
Conclusion
The present study could not demonstrate that GPX4 overexpression reduces atherosclerosis through the inhibition of lipid peroxidation. Although GPX4 is a multifunctional protein capable of reducing peroxidized lipids and cell death, we conclude that overexpressing this protein represents a strategy that is not sufficiently powerful to counteract atherosclerosis, despite previous studies showing that GPX4 overexpression exerts a protective role in other disease models, for example by retarding motor neuron disease in SOD1G93A mice (29). Importantly, we reduced the vitamin E concentration in the diet to better study the protective effects of GPX4 overexpression, as vitamin E and GPX4 cooperatively protect cells from lipid peroxidation and ferroptosis. However, supplementation of abundantly high levels of vitamin E, as present in standard diets for mice, combined with GPX4 overexpression may be required to obtain significant effects. Along these lines, it should be noted that another anti-ferroptosis pathway acting via ferroptosis suppressor protein (FSP-1) has recently been identified that could serve as a better protection. FSP-1 is a key component of a non-mitochondrial CoQ antioxidant system and acts independently of GPX4 to inhibit lipid peroxidation and ferroptosis (39). Apart from dietary antioxidants, overexpression of FSP-1 or simultaneous regulation of both FSP-1 and GPX4 may be a promising alternative strategy to target lipid peroxidation and ferroptosis in atherosclerosis, as reported recently for other human diseases (40).
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/VB-23-0020.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the study reported.
Funding
This work was supported by the Fund for Scientific Research (FWO)-Flanders (grant numbers EOS 30826052 and G026723N).
Author contribution statement
Conceptualization: IC, GRYDM, WM; data collection and analysis: IC, AB, NH; writing original draft: IC, WM; revision and further editing: all authors; funding acquisition: GRYDM, WM.
Acknowledgements
The authors thank Hermine Fret and Mandy Vermont for their excellent technical support.
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