Abstract
The impact of α-tocopherol on atherosclerosis is unclear and controversial. While some studies suggest potential benefits, such as antioxidant properties that may reduce oxidative stress, other studies indicate no significant preventive effects. The intricate interplay of various factors, including dosage, individual differences, and study methodologies, contributes to the ongoing uncertainty surrounding α-tocopherol’s role in atherosclerosis. Further research is needed to clarify its impact and establish clearer guidelines. Therefore, we aimed to evaluate the impact of α-tocopherol on atherogenesis in ApoE−/− fibrillin (Fbn)1C1039G/+ mice, which is a unique mouse model of advanced atherosclerosis with typical features, such as large necrotic cores, high levels of inflammation, and intraplaque neovascularization, that resemble the unstable phenotype of human plaques. ApoE−/− Fbn1C1039G+/− mice were fed a western-type diet (WD) supplemented with a high dose of α-tocopherol (500 mg/kg diet), while control mice were fed a WD containing a low dose of α-tocopherol (50 mg/kg diet). The high dose of α-tocopherol reduced plaque thickness and necrotic core area in the right common carotid artery (RCCA) after 24 weeks WD. Moreover, α-tocopherol decreased plaque formation and intraplaque neovascularization in the RCCA. In addition to its antiatherogenic effect, chronic supplementation of α-tocopherol improved cardiac function in ApoE−/− Fbn1C1039G/+ mice. However, chronic supplementation of α-tocopherol did not decrease lipid peroxidation. On the contrary, α-tocopherol acted as a prooxidant by increasing plasma levels of oxidized LDL and plaque malondialdehyde, an end product of lipid peroxidation. Our data indicate that α-tocopherol inhibits atherogenesis and improves cardiac function independent of its antioxidant properties.
Introduction
Atherosclerosis is a progressive inflammatory disease of large- and medium-sized arteries characterized by the formation of plaques in the arterial wall (1). Lipid oxidation plays a critical role in atherogenesis (2). When lipids, especially low-density lipoprotein (LDL) cholesterol, undergo oxidation, they become more prone to uptake by immune cells, triggering the inflammatory response within arterial walls (3). Oxidized LDL (oxLDL) contributes to the recruitment of macrophages, leading to the formation of foam cells, a hallmark of early atherosclerotic lesions. The oxidative modification of lipids also induces endothelial dysfunction, disrupting the protective lining of blood vessels and promoting the adhesion of immune cells (4). This process enhances the permeability of the arterial wall and facilitates the entry of lipids, exacerbating plaque formation. Additionally, oxidized lipids contribute to the activation of inflammatory signaling pathways, further promoting atherosclerosis (5). Over time, the accumulated oxidized lipids contribute to the progression of atherosclerotic plaques, making them more vulnerable to rupture, which can lead to acute cardiovascular events such as heart attacks and strokes. Managing lipid oxidation is therefore crucial in preventing and mitigating atherosclerosis and its associated cardiovascular complications.
Vitamin E is a lipophilic antioxidant that includes both tocopherols and tocotrienols. It is found in vegetable oils and nuts. The most active isomer of vitamin E in the human diet is α-tocopherol (6). As an antioxidant, vitamin E is able to inhibit the formation of lipid peroxidation by two different mechanisms. Vitamin E acts as a radical-trapping antioxidant, thereby interrupting the cascade of chain reactions during lipid peroxidation of membranes (7). Furthermore, vitamin E inhibits ferroptosis by blocking lipoxygenases, which catalyze the formation of lipid peroxides (8, 9). In addition to its antioxidant function, vitamin E exerts different non-antioxidant activities such as inhibiting protein kinase C and enhancing immune responses (6, 10). To date, the effect of vitamin E supplementation on atherosclerosis development is still unclear. Numerous animal studies and clinical trials have been carried out to investigate the effect of vitamin E supplementation in atherosclerosis prevention. Although most animal studies report an anti-atherogenic effect for vitamin E, the results of large clinical trials are conflicting (10, 11, 12).
In the present study, our aim was to evaluate the impact of α-tocopherol on atherogenesis in ApoE−/− fibrillin (Fbn)1C1039G/+ mice, which is a unique mouse model of advanced atherosclerosis (13). The heterozygous mutation C1039G/+ in the Fbn1 gene results in fragmentation of elastic fibers in the media of the vessel wall, leading to increased arterial stiffness (14). As a result, ApoE−/− Fbn1C1039G/+ mice show highly unstable plaques with typical features of human unstable lesions such as large necrotic cores, high levels of inflammation, intraplaque neovascularization, and intraplaque hemorrhages (15). Moreover, these mice show myocardial infarction, stroke, and sudden death without surgical intervention (15). Therefore, this mouse model is an adequate tool to investigate the effect of α-tocopherol on the development of advanced atherosclerotic plaques.
Materials and methods
Mice
Female ApoE−/− Fbn1C1039G/+ mice were fed a western-type diet (WD; C1000 diet supplemented with 20% milk fat and 0.15% cholesterol, Altromin) containing 500 mg α-tocopherol per kg diet (#100207, Altromin) starting at the age of 6–8 weeks. WD supplemented with 50 mg α-tocopherol per kg diet (#100206, Altromin) was used for the control group. Female ApoE−/− Fbn1C1039G/+ mice were chosen because the Fbn1 mutation frequently causes aortic dissection in male ApoE−/− Fbn1C1039G/+ mice (15). 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 24 weeks of WD, blood samples were collected via the retro-orbital plexus of anesthetized mice (sodium pentobarbital 75 mg/kg, i.p.). Subsequently, mice were sacrificed using an overdose of sodium pentobarbital (250 mg/kg, i.p.). All experiments were conducted according to the ARRIVE guidelines and approved by the Ethical Committee of the University of Antwerp.
Plasma analysis
Plasma levels of total cholesterol and oxLDL were measured using a commercially available kit (Randox Laboratories, Crumlin, UK) or an ELISA (MyBioSource, MBS263208), respectively. α-Tocopherol was analyzed based on a validated HPLC method after a three times extraction with hexane (16). Briefly, plasma samples (50 µL) were diluted with ethanol (100 µL), and α-tocopherol was extracted with 500 µL hexane by vortexing for 5 min and centrifugation (4000 g , 4°C, 10 min). After evaporation of the combined extracts by vacuum centrifugation, the residue was dissolved in 100 µL ethanol. Seven standard solutions of α-tocopherol (range: 31.625 µg/mL to 0.494 µg/mL) and blanks for the calibration curve were extracted and prepared in ethanol. Dissolved residues were analyzed by an HPLC system from Agilent (type 1260 quaternary pump, 1260 auto-injector and 1290 temperature controller), linked to an ESA-5600A CoulArray eight-channel electrochemical detector (Thermo Fisher Scientific). Mobile phase A (methanol/H2O/ammonium acetate 1 M (pH 4 with acetic acid); 90:8:2 v:v:v) and B (methanol/1-propanol/ammonium acetate 1 M (pH 4 with acetic acid); 78:20:2 v:v:v) were used at a flow of 0.6 mL/min. The elution profile was set at a 21-min linear gradient from 0% to 80% B, followed by a 10 min linear gradient from 80% to 100% B, and a 14 min isocratic elution at 100% B before returning to initial conditions (run time 50 min). Samples were cooled at 4°C. The electrochemical cells were set at 290, 540, and 600 mV. A Hypersil octadecyl silane (ODS, C18) RP analytical column (150 × 3 mm, 3 µm) from Thermo Scientific, was used, and heated to 37°C. The resulting chromatograms were analyzed with CoulArrayWin software. The retention time of α-tocopherol was 16.9 min.
Leukocyte subsets
EDTA-treated blood (500 µL) was lysed using red blood cell lysis buffer (Sigma-Aldrich). Remaining leukocytes were counted using a hemocytometer and labeled with the following anti-mouse monoclonal antibodies (BioLegend, San Diego, CA, USA): PerCP or FITC anti-CD45 (clone 30-F11), APC anti-CD3ε (clone 145-2C11), PE anti-CD19 (clone 6D5), FITC anti-NK1.1 (clone PK136), APC anti-Ly6C (clone HK1.4), PE anti-Gr-1 (clone RB6-8C5), and PerCP anti-CD11b (clone M1/70). Staining was performed in darkness at 4°C in FACS buffer (PBS supplemented with 0.1% BSA (Sigma-Aldrich) and 0.05% NaN3 (Merck)) in the presence of CD16/32 Fc-receptor blocker (BioLegend). Cells were analyzed using a BD Accuri C6 cytometer equipped with a blue and red laser (Becton Dickinson, Franklin Lakes, NJ, USA). Dead cells and debris were excluded based on forward scatter, side scatter, and positive staining for propidium iodide (Life Technologies). The following antibody combinations were used for the identification of the different leukocyte subsets: CD45+CD3+NK1.1− (T cells), CD45+CD19+ (B cells), CD45+Ly6Clow or CD45+Ly6Chigh (monocytes), and CD45+CD11b+Gr-1high (neutrophils).
Echocardiography
Transthoracic echocardiograms were performed on anesthetized mice (isoflurane, 4% for induction and 2% for maintenance) at the start of the experiment (0 weeks WD), at 12 weeks WD, at 18 weeks WD, and at the end of the experiment (24 weeks WD) using a VEVO2100 (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) and ejection fraction (EF) were calculated from these parameters.
To study arterial stiffness, pulse wave velocity (PWV) was determined in the abdominal aorta using a 24-MHz transducer, as previously described (17). Briefly, the aortic diameter (D) was measured on 700 frames-per-second B-mode images of the abdominal aorta in EKV mode. Subsequently, the aortic flow velocity (V) was determined by pulse wave Doppler tracing. PWV was then calculated via the ln(D)-V loop method using MathLab v2014 software (MathWorks).
Histological analysis
After euthanasia, the right common carotid artery (RCCA) and the heart were collected. Tissues were fixed in 4% formalin for 24 h, dehydrated overnight in 60% isopropanol, and subsequently embedded in paraffin. Serial cross sections (5 µm) of the heart were prepared for histological analyses. Serial longitudinal sections (5 µm thick) were cut from the RCCA. 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. The plaque thickness was assessed by taking the median value of 10 random measurements in the respective area. The plaque formation index was calculated on longitudinal sections of the carotid artery using the following formula: (∑ total plaque length/∑ total vessel length) × 100 (18). The presence of microvessels was analyzed on H&E stained sections. Collagen content was determined on Sirius red-stained sections. Macrophages and vascular smooth muscle cells were detected by immunohistochemistry using an anti-Mac3 (BD Pharmingen, 550292) and anti-α-smooth muscle actin (α-SMA, Sigma, A2547) staining, respectively. Endothelial cells were measured via immunohistochemical staining using an anti-CD31 antibody (Abcam, ab56299). The expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular adhesion molecule 1 (VCAM-1) on endothelial cells was analyzed using anti-ICAM-1 staining (Abcam, ab179707) and anti-VCAM-1 staining (Abcam, ab134047), respectively. ICAM-1 and VCAM-1 positivity were expressed as a percentage of CD31 positivity. The occurrence of myocardial infarction (defined as large fibrotic areas with infiltration of inflammatory cells) and coronary arteries were analyzed on Masson’s trichome-stained transversal sections (cut from the middle of the heart to the apex). Images were acquired with an Olympus SC 50 microscope and quantified with Image J software (NIH).
Malondialdehyde analysis
The aortic arch was collected and immediately snap frozen. Tissues were homogenized in phosphate buffer, and protein content was determined via the BCA method. MDA content in atherosclerotic plaques of the aortic arch was determined using a commercially available MDA ELISA kit (Mybiosource, MBS741034).
Statistical analysis
Statistical analyses were performed using SPSS software (version 29, IBM SPSS). All data were expressed as mean ± s.e.m. Statistical tests are specified in the figure legends. Differences were considered significant at P < 0.05.
Results
Chronic supplementation of α-tocopherol increases plasma oxLDL levels in ApoE−/− Fbn1C1039G/+ mice
ApoE−/− Fbn1C1039G/+ mice were fed a western-type diet (WD) supplemented with 50 mg or 500 mg α-tocopherol per kg diet for 24 weeks to study the effect of α-tocopherol on plaque formation. After 24 weeks of WD, α-tocopherol levels in plasma were not different between both groups (12 ± 2 µg/mL vs 15 ± 2 µg/mL; P = 0.114, n = 11–17, independent samples t-test). Further analysis of plasma samples revealed a modest reduction of total plasma cholesterol in mice treated with a high dose of α-tocopherol as compared to the control mice (585 ± 42 mg/dL vs 707 ± 42 mg/dL; P < 0.05, n = 23–24, independent samples t-test). Unexpectedly, plasma levels of oxLDL were significantly increased in mice treated with a high dose of α-tocopherol (Fig. 1A), albeit without any changes in % survival (Fig. 1B). Body, lung, and kidney weight did not differ between both groups of mice (Table 1). However, spleen weight was significantly increased in mice treated with a high dose of α-tocopherol as compared to the control group (Table 1). Given the regulatory role of α-tocopherol in immunity and inflammation (19, 20), an analysis of the circulating blood immune cells was performed. The percentage of circulating T and B cells, Ly6high and Ly6low monocytes as well as neutrophils remained unchanged after treatment with a high dose of α‑tocopherol (Table 1).
Chronic treatment with a high dose of α-tocopherol increases plasma oxLDL levels in ApoE−/− Fbn1C1039G/+ mice. ApoE−/− Fbn1C1039G/+ mice were fed a western-type diet supplemented with α-tocopherol in a low concentration (50 mg/kg diet) or high concentration (500 mg/kg diet). (A) Analysis of oxLDL levels in plasma. **P < 0.01 vs 50 mg/kg diet (n = 14 for both groups; Independent samples t-test). (B) Analysis of survival (n = 41–45 for both groups; log-rank test).
Citation: Vascular Biology 6, 1; 10.1530/VB-24-0002
Characteristics of ApoE− /− Fbn1C1039G/+ mouse fed a western-type diet supplemented with 50 mg α-tocopherol or 500 mg α-tocopherol per kg diet. Data presented as mean ± s.e.m.
| 50 mg/kg diet | 500 mg/kg diet | |
|---|---|---|
| General (n = 26–31) | ||
| Body weight (g) | 22 ± 1 | 21 ± 1 |
| Spleen weight (mg) | 210 ± 17 | 275 ± 24a |
| Lung weight (mg) | 241 ± 7 | 253 ± 10 |
| Kidney weight (mg) | 163 ± 4 | 172 ± 7 |
| Circulating leukocyte subsets (n = 11–17) | ||
| T cells (%) | 22 ± 2 | 24 ± 4 |
| B cells (%) | 51 ± 3 | 49 ± 5 |
| Ly6low monocytes (%) | 10 ± 1 | 10 ± 2 |
| Ly6high monocytes (%) | 7.8 ± 0.6 | 7.0 ± 0.5 |
| Neutrophils (%) | 12 ± 1 | 15 ± 3 |
| Plaque analysis in RCCA (n = 10–16) | ||
| Plaque formation index (%) | 55 ± 7 | 21 ± 5c |
| Plaque thickness (µm) | 686 ± 54 | 381 ± 47c |
| Necrotic core area (%) | 4.0 ± 1.0 | 1.1 ± 0.5a |
| Macrophages (%) | 18 ± 4 | 26 ± 5 |
| Vascular SMCs (%) | 4 ± 1 | 5 ± 1 |
| Total collagen (%) | 23 ± 3 | 25 ± 3 |
| ICAM-1-positive ECs (%) | 81 ± 4 | 94 ± 4b |
| VCAM-1-positive ECs (%) | 44 ± 7 | 58 ± 10 |
| Cleaved caspase 3 (%) | 0.10 ± 0.03 | 0.03 ± 0.01a |
Independent samples t-test.
aP < 0.05, bP < 0.01, cP < 0.001 vs 50 mg/kg diet.
RCCA, right common carotid artery; SMCs, smooth muscle cells.
Chronic supplementation of α-tocopherol decreases atherosclerotic plaque formation in the right common carotid artery of ApoE−/− Fbn1C1039G/+ mice
Despite increased levels of plasma oxLDL in mice treated with a high dose of α-tocopherol, no difference in plaque development in the thoracic aorta was observed between both groups (46 ± 8 vs 47 ± 6 %, Independent samples t-test). However, plaque formation in the right common carotid artery (RCCA), as shown by the plaque formation index, was significantly decreased in mice treated with a high dose of α-tocopherol (Table 1). Furthermore, plaque thickness in the RCCA was significantly reduced in mice treated with a high dose of α-tocopherol (Table 1). In addition, a WD supplemented with 500 mg α-tocopherol per kg diet reduced the necrotic core area (Table 1). Further analysis of plaque composition in the RCCA showed that total collagen as well as the macrophage and vascular smooth muscle content were not different between the animals treated with a high dose of α-tocopherol and the control group (Table 1). Surprisingly, analyses of endothelial adhesion molecules ICAM-1 and VCAM1 revealed that the percentage of ICAM-1-positive endothelial cells was significantly increased after treatment with a high dose of α-tocopherol, although the percentage of VCAM-1-positive endothelial cells was not significantly different between both groups (Table 1). Furthermore, immunostaining for cleaved caspase 3 revealed that plaque apoptosis was significantly inhibited in atherosclerotic plaques of mice treated with a high dose of α-tocopherol (Table 1).
Because α-tocopherol acts as a radical-trapping antioxidant and limits lipid peroxidation (7), MDA content in plaque lysates was analyzed. MDA content was significantly higher in plaques of mice treated with a high dose of α-tocopherol as compared to the mice treated with a low dose of α-tocopherol (Fig. 2A). In addition to α-tocopherol, GPX4 protects the cells against lipid peroxidation (21). To evaluate whether the increased MDA content was caused by decreased expression of GPX4, an immunostaining for GPX4 was performed. However, the expression of GPX4 did not change between both groups (Fig. 2B). Overall, these data demonstrate that a chronic supplementation of α-tocopherol does not protect against lipid peroxidation.
Chronic treatment with a high dose of α-tocopherol increases lipid peroxidation in atherosclerotic plaques of ApoE−/− Fbn1C1039G/+ mice. ApoE−/− Fbn1C1039G/+ mice were fed a western-type diet supplemented with α-tocopherol in a low concentration (50 mg/kg diet) or high concentration (500 mg/kg diet). (A) MDA analysis was performed on plaque lysates of the aortic arch. (B) Longitudinal sections of the right common carotid artery were stained with anti-GPX4 antibody. *P < 0.05 vs 50 mg/kg diet (n = 10–16 for both groups; independent samples t-test).
Citation: Vascular Biology 6, 1; 10.1530/VB-24-0002
Finally, we analyzed the occurrence of intraplaque neovascularization. Because red blood cells are a major source of iron, intraplaque hemorrhages due to leakage of fragile intraplaque neovessels cause iron deposition in the plaque, which may induce ferroptotic cell death (22). Ten out of 17 (59%) control mice treated with a low dose of α-tocopherol developed microvessels in the RCCA, whereas only 3 out of 12 (25%) mice treated with a high dose of α-tocopherol showed signs of intraplaque neovascularization. However, this trend in the reduction of intraplaque microvessels was statistically not significant (Fisher’s exact test, P = 0.130).
Chronic supplementation of α-tocopherol reduces cardiac mass and improves cardiac function and left ventricular dilatation
Echocardiography revealed a significant decrease in LVIDd and LVIDs as early as 18 weeks of treatment with a high dose of α-tocopherol, albeit without changing IVSd, IVSs, and LVPWs (Table 2). In contrast, LVPWd increased in mice treated for 24 weeks with a high dose of α-tocopherol as compared to the control mice. The decrease in LVID resulted in a significantly increased fractional shortening and ejection fraction after 24 weeks of WD (Table 2). Moreover, a significant reduction in heart weight over body weight was observed in the group of mice that were fed a WD supplemented with a high dose of α-tocopherol (Table 2).
Cardiac parameters of ApoE−/− Fbn1C1039G/+ mouse treated with a western-type diet (WD) supplemented with 50 mg or 500 mg α-tocopherol per kg diet. Data are presented as mean ± s.e.m.
| Cardiac parameters | 0 weeks WD | 12 weeks WD | 18 weeks WD | 24 weeks WD | ||||
|---|---|---|---|---|---|---|---|---|
| 50 | 500 | 50 | 500 | 50 | 500 | 50 | 500 | |
| HW/BW (10−3)a | NA | NA | NA | NA | NA | NA | 9.6 ± 0.6 | 8.4 ± 0.3c |
| PWV (m/s)b | 2.2 ± 0.2 | 2.0 ± 0.1 | 2.5 ± 0.3 | 2.6 ± 0.5 | 2.6 ± 0.3 | 3.0 ± 0.4 | 2.5 ± 0.3 | 2.3 ± 0.3 |
| EF (%)b | 75 ± 2 | 71 ± 3 | 68 ± 3 | 76 ± 3 | 64 ± 4 | 71 ± 2 | 58 ± 3 | 68 ± 2c |
| FS (%)b | 43 ± 2 | 39 ± 2 | 38 ± 2 | 45 ± 3d | 36 ± 3 | 40 ± 2 | 31 ± 2 | 37 ± 2c |
| IVSd (mm)b | 0.93 ± 0.03 | 0.97 ± 0.04 | 0.99 ± 0.04 | 1.00 ± 0.05 | 1.04 ± 0.05 | 1.00 ± 0.03 | 1.04 ± 0.04 | 1.00 ± 0.04 |
| IVSs (mm)b | 1.5 ± 0.1 | 1.4 ± 0.1 | 1.4 ± 0.1 | 1.5 ± 0.1 | 1.5 ± 0.1 | 1.5 ± 0.1 | 1.5 ± 0.1 | 1.5 ± 0.1 |
| LVIDd (mm)b | 3.0 ± 0.1 | 3.0 ± 0.1 | 3.3 ± 0.1 | 3.1 ± 0.2 | 3.7 ± 0.2 | 3.2 ± 0.1c | 3.7 ± 0.2 | 3.3 ± 0.2c |
| LVIDs (mm)b | 1.7 ± 0.1 | 1.7 ± 0.1 | 2.1 ± 0.1 | 1.7 ± 0.2 | 2.5 ± 0.2 | 2.0 ± 0.1c | 2.6 ± 0.2 | 2.2 ± 0.1c |
| LVPWd (mm)b | 1.4 ± 0.1 | 1.4 ± 0.1 | 1.4 ± 0.1 | 1.6 ± 0.1 | 1.4 ± 0.1 | 1.4 ± 0.1 | 1.4 ± 0.1 | 1.6 ± 0.1c |
| LVPWs (mm)b | 1.0 ± 0.1 | 1.0 ± 0.1 | 1.1 ± 0.1 | 1.1 ± 0.1 | 1.1 ± 0.1 | 1.1 ± 0.1 | 1.1 ± 0.1 | 1.2 ± 0.1 |
aIndependent samples t-test; bTwo-way ANOVA followed by post hoc Bonferroni’s test; cP < 0.05; dP < 0.01 vs 50 mg/kg diet, n = 21–29 for each group.
BW, body weight; EF, ejection fraction; FS, fractional shortening; HW, heart weight; IVSs, IVS during systole; IVSd, interventricular septum during diastole; LVIDd, left ventricular internal diameter during diastole; LVIDs, LVID during systole; LVPWd, left ventricular posterior wall thickness during diastole; LVPWs, LVPW during systole; NA, not applicable; PWV, pulse wave velocity.
Cross-sectional histological evaluation of the heart revealed a significant decrease in the occurrence of myocardial infarction in ApoE−/− Fbn1c1039G/+ mice that were treated with a high dose of α-tocopherol (Fig. 3A). Concomitant with the decrease in myocardial infarctions, the occurrence of coronary plaques tended to decrease in mice treated with a high dose of α-tocopherol (Fig. 3B, P = 0.098). Arterial stiffness measured by pulse wave velocity was not affected by treatment with a high dose of α-tocopherol as compared to treatment with a low dose of α-tocopherol (Table 2).
Chronic treatment with a high dose of α-tocopherol decreases the incidence of myocardial infarctions in ApoE–/– Fbn1C1039G/+ mice. ApoE−/− Fbn1C1039G/+ mice were fed a western-type diet supplemented with α-tocopherol in a low concentration (50 mg/kg diet) or high concentration (500 mg/kg diet). Masson’s trichrome staining of heart tissue was performed to evaluate the (A) incidence of myocardial infarction (blue, indicated by the arrow) or (B) presence of coronary plaques. *P < 0.05 vs 50 mg/kg diet (n = 17–22; Fisher’s exact test). Representative images are shown. P, plaque; L, lumen. Scale bar = 1 mm (A) or 50 µm (B).
Citation: Vascular Biology 6, 1; 10.1530/VB-24-0002
Discussion
The oxidation of LDL in the subendothelial space is thought to play a causative role in the development of atherosclerotic plaques (1, 23). Hence, antioxidants such as vitamin E gained a lot of attention as potential therapeutic compounds to treat atherosclerosis. Different animal studies have shown a beneficial effect of vitamin E on atherosclerosis. However, several clinical studies revealed variable results, the majority being negative (11, 12, 24). The reasons for these conflicting findings between animal and clinical studies might be attributed to differences in dose, duration of therapy as well as the stage of atherosclerosis (25). Therefore, the role of vitamin E (most notably α-tocopherol) in atherosclerosis has to be further clarified. Animal studies have already demonstrated the atheroprotective role of α-tocopherol as an antioxidant (26, 27). Moreover, in addition to its antioxidant properties, α-tocopherol might reduce atherosclerosis by suppressing the expression of adhesion molecules, reducing the expression of pro-inflammatory cytokines, inhibiting macrophage activation, reducing proliferation of vascular smooth muscle cells, and suppressing thromboxane production (28, 29, 30, 31, 32, 33).
In the present study, we documented the impact of α-tocopherol in ApoE−/− Fbn1C1039G/+ mice, which, in contrast to standard ApoE−/− mice, develop atherosclerotic plaques with features that resemble the highly unstable phenotype of human plaques, including presence of a large necrotic core, high levels of inflammation, intraplaque neovascularization, and hemorrhages (15). Chronic administration of a WD supplemented with a high dose of α-tocopherol (500 mg/kg WD) to ApoE−/− Fbn1C1039G/+ mice did not result in significantly elevated α-tocopherol plasma levels, when compared to ApoE−/− Fbn1C1039G/+ mice fed a WD supplemented with a low dose of α-tocopherol (50 mg/kg WD). Given the high lipophilicity of α-tocopherol, it is most likely that α-tocopherol is rapidly removed from the bloodstream and redistributed into various tissues. Interestingly, plaques in the right common carotid artery (RCCA) of ApoE−/− Fbn1C1039G/+ mice that were fed a WD containing 500 mg α-tocopherol per kg diet showed a decreased plaque thickness as compared to mice treated with a low dose of α-tocopherol. Moreover, the necrotic core was significantly reduced in RCCA plaques of ApoE−/− Fbn1C1039G+/− mice treated with a high dose of α-tocopherol, which could be the result of cell death inhibition. However, neither the number of vascular smooth muscle cells and macrophages in the plaque nor the total collagen content changed in mice treated with a high dose of α-tocopherol vs control mice. Atherogenesis, as indicated by the plaque formation index, was significantly inhibited in the RCCA of ApoE−/− Fbn1C1039G+/− mice treated with a high dose of α-tocopherol, suggesting that α-tocopherol slows down plaque progression in an early stage. In addition, only 3 out of 12 mice treated with a high dose of α-tocopherol showed atherosclerotic plaques with neovascularization, which demonstrates that the atherosclerotic plaques located in the RCCA of mice treated with a high dose of α-tocopherol are less advanced as compared to the atherosclerotic plaques of the control mice. However, it should be noted that treatment of ApoE−/− Fbn1C1039G/+ mice with a WD supplemented with 500 mg α-tocopherol per kg diet did not inhibit lipid peroxidation in atherosclerotic plaques. On the contrary, MDA analysis showed that lipid peroxidation was increased. Furthermore, plasma oxLDL levels were significantly increased after chronic treatment with a high dose of α-tocopherol. Overall, these data demonstrate that chronic administration of WD supplemented with 500 mg α-tocopherol per kg diet does not inhibit lipid peroxidation but promotes a prooxidant activity of α-tocopherol in ApoE−/− Fbn1C1039G/+ mouse. This observation is in line with previous reports demonstrating that α-tocopherol is not merely an antioxidant. On the contrary, α-tocopherol exhibits antioxidant and prooxidant activities depending on the presence of other co-antioxidants. The prooxidant effect of α-tocopherol on LDL is related to the production of α-tocopheroxyl radical formation, which can be reduced by co-antioxidants such as vitamin C and ubiquinol-10 (34, 35, 36). Possibly, the WD supplemented with 500 mg α-tocopherol per kg diet did not contain the correct concentration of co-antioxidants necessary for the reduction of α-tocopheroxyl radicals. Even though α-tocopherol acted as a prooxidant in the present study, the progression of atherosclerosis was decreased in mice treated with a high dose of α-tocopherol, and the antiatherogenic effects of α-tocopherol in the present study seem to be the result of an antioxidant-independent role of α-tocopherol.
Previous reports have demonstrated that α-tocopherol inhibits the expression of adhesion molecules such as ICAM-1 and VCAM-1 (28, 29, 37). Upregulation of adhesion molecules by endothelial cells is an important event in early lesions, as they stimulate monocyte infiltration in the lesion (1, 23). Surprisingly, the present study showed a significant upregulation of endothelial ICAM-1 in the plaques of mice fed a WD supplemented with 500 mg α-tocopherol per kg diet, while endothelial VCAM-1 expression was not affected. A possible explanation for this finding is that the increased expression of endothelial ICAM-1 is caused by the elevated plasma levels of oxLDL in mice treated with a high dose of α-tocopherol (38). It is also conceivable that the reduced plaque progression in the RCCA of mice fed a WD supplemented with a high dose of α-tocopherol is due to a moderate (18%) decrease in total plasma cholesterol levels. Even though plasma cholesterol levels were still very high (> 500 mg/dL) and plaque development was not different in the thoracic aorta, we cannot rule out that the decrease in total plasma cholesterol contributes partially to the observed changes in plaque area and plaque formation of the RCCA. In addition, we observed a decrease in apoptosis in RCCA plaques of mice treated with a high dose of α-tocopherol, as previously reported by other groups (39, 40). Apart from these effects of α-tocopherol on atherogenesis, α-tocopherol inhibits different cellular enzymes such as protein kinase C, cyclooxygenase 2, and 5-lipoxygenase (31, 41, 42). Moreover, α-tocopherol inhibits the gene transcription of scavenger receptors such as SR-A and CD36 in both vascular smooth muscle cells and macrophages, which in turn leads to less foam cell formation (43, 44, 45). It is plausible that the decrease in plaque formation and plaque thickness in the RCCA after chronic treatment with high dose α-tocopherol is a result of a combination of the mechanisms described above. Finally, the present study showed that α-tocopherol has a cardioprotective role in addition to its antiatherogenic effect. Chronic supplementation of α-tocopherol not only tended to reduce the formation of coronary plaques, resulting in fewer myocardial but also improved heart function.
Conclusion
The present study demonstrates a protective role of α-tocopherol in atherosclerosis and cardiac function. Despite these positive effects, it is important to note that α-tocopherol supplementation may also cause adverse effects. Indeed, α-tocopherol is not merely an antioxidant, but may also exhibit prooxidant activities as plasma levels of oxLDL were significantly increased in mice treated with a high dose of α-tocopherol. Along these lines, another group reported that vitamin E can stimulate DNA damage and cell transformation (46), thereby providing a biological rationale for an increased incidence of prostate cancer among healthy men supplemented with vitamin E (SELECT trial) (47). Moreover, given the anticoagulant properties of α-tocopherol, previous studies reported that chronic supplementation with α-tocopherol increases bleeding risk (48, 49). In addition, a meta-analysis of several clinical trials demonstrated that high doses of α-tocopherol may increase all-cause mortality (24). Most clinical trials with high doses of α-tocopherol were performed in a small group of patients with chronic diseases (24), indicating that more studies are needed to determine the optimal dosage of α-tocopherol supplementation. Only then it can be safely used as an add-on therapy in the prevention and treatment of cardiovascular diseases. As suggested above, α-tocopherol, but also other natural antioxidants such as resveratrol (50), display hormetic effects whereby low-dose amounts have a beneficial effect and high-dose amounts are either inhibitory to function or even toxic. Given the increased levels of plasma oxLDL and plaque MDA, it is conceivable that the α-tocopherol dosage used in the present study was too high so that lower doses should be used in follow-up studies to confirm that α-tocopherol inhibits atherogenesis and improves cardiac function independently of its antioxidant properties.
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 Research Foundation – Flanders (grant nos. EOS 30826052 and G026723N) and the Hercules Foundation (grant no. AUHA/13/03).
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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